W097/0480I PCT/US96/12251 3/ 10

84 = ·-....~ 8 ...e ~

~ ....~ z·-= 78 ~

76

0 20 40 60 80 100 Time (days) FIG. 5

= ·-....~ =... ~

~ ....~ 7 ·-= z 76 ~

74

72 O 20 40 60 80 100

Time (days) Coherus BioScience Inc. EXHIBIT 1328 FIG. 6 Part 5 of 6 IPR Petition for U.S. Patent No. 9,085,619 suasmurE SHEET (RULE 28}

IPR Page 1683 of 2482 W097/04801 PCT/US96/12251

4 / 10 83..------.

75 ~0___ _.2_0 ___ 4.,_0----',""""o ___ .,_80----1100 Time (days) FIG. 7

84

=Cl> ·-~ 8 C... ~ Cl> ~

~ ·-z= ~

76

0 20 40 60 80 100 Time (days) FIG. 8

SUBSTITUTE SHEET (RULE 26)

IPR Page 1684 of 2482 PCT/US96/12251 W097/04801 5/ 10

5.0

-~ 4.0 -cu ~ 3.0

=~ ....cu 2.0 ~ ~ < 1.0

0.0 6 time FIG. 9

30 ,,.-.... 25 ,._,~ 20 .....Cl) cu 15 bl) Cl) s. 10 bl) bl) < 5 0 10 20 30 40 time (Weeks) FIG. 10

SUBSTITUTE SHEET (RULE 26)

IPR Page 1685 of 2482 W097/04801 PCT/US96/12251 6 / 10

s.o 4.0

3.0 2.0 1.0

0.0 ..1..-t....,...... _~...... '""""""t"-...... _.~~"""-'-:"=---~

FIG. 11

so 40 30

20 10

0 ..1...... a-,-...... o=-=-~:z::3e:::~~=-===4:'7"{ .....,__

FIG. 12

_SUBSTITUTE SHEET (RULE 28)

IPR Page 1686 of 2482 W097/04801 PCT/US96/12251

7 I 10

40°c

2s 0 c

2-8°C

14.0 15.0 16.0 17.0 18.0 time (Minutes) FIG. 13

40°c

2s 0 c

2-8°C

14.0 15.0 16.0 17.0 18.0 time (Minutes) FIG. 14

SUBSTITUTE SHEET (RULE 26)

IPR Page 1687 of 2482 W097/04801 PCT/US96/12251

8 I 10

40°C

25°C

2-8°C

14.0 15.0 16.0 17.0 18.0 time (Minutes) FIG. 15 35.0

30.0

25.0 -~ -....a, 20.0 ~ ...~a, ~ 15.0 <~ 10.0

s.o

0 500 1000 1500 2000 2500 Molar Ratio of Sugar to rhuMAb E25 FIG. 16

SUBSTITUTE SHEET (RULE 26)

IPR Page 1688 of 2482 WO 97/04801 PCT/US96/12251

9 I 10

4.0

,-... 3.S ._..~ 3.0 .....~ 2.S cu Cl) 2.0 ...~ Cl) Cl) 1.S , , ,~-----~------~------< 1.0 ~ ~ -ic:= • o.s 10 20 30 40 time (Weeks) FIG. 17

4.0

,.-._ 3.S ._..~ 3.0 .....~ 2.S cu Cl) 2.0 ...~ _-a-- - -El Cl) I.S -B- - Cl) ------&------_.. -- =--=--=-•-----· < 1.0 o.s 10 20 30 0 time (Weeks) FIG. 18

SUBSTITUTE SHEET (RULE 26)

IPR Page 1689 of 2482 W097/04801 PCT/US96/12251

10/ 10

14.0 ..-.. 12.0 ~.._., 10.0 ....Q,> 8.0 =Oil 6.0 ...Q,> -El Oil Oil 4.0 -a- -- -- < 2.0 s- - a- - - .... -- =-- =-- =--t _... ---·-----....- - 0.0 10 20 0 0 time (Weeks) FIG. 19

SUB(nn-, ,-p..,. v,,,u,c SHEET (RULE 26)

IPR Page 1690 of 2482 INTERNATIONAL SEARCH REPORT Inter '>n3l Application No PCT/US 96/12251 A. CLASSIFICATION OF SUBJECT MATIER IPC 6 A61K39/00 A61K39/395 C07Kl/00

According to International Patent Oassification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) IPC 6 A61K C07K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT Category• Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X EP 0 661 060 A (IMMUNO AG) 5 July 1995 1,3,6, 8-12 see the whole document --- A WO 89 09402 A (TORAY INDUSTRIES INC.) 5 1-3,6, October 1989 10,12 see the whole document --- A JOURNAL OF IMMUNOLOGICAL METHODS, 1-3,6, vol. 181, no. 1, 12 April 1995, AMSTERDAM, 10,12 NL, pages 37-43, XP002019425 P. DRABER ET AL.: "Stability of monoclonal IgM antibodies freeze-dried in the presence of trehalose. 11 see abstract --- -/--

IT] Further documents are listed in the continuation of box C. [] Patent family members are listed in annex. • Special categories of cited documents : ·r later document published after the international filing date or pnonty date and not in conflict with the application but ·A· document defining the general state of the art which is not cited to understand the pnnc1ple or theory underlying the considered to be of particular relevance invention ·E· earlier document but published on or after the international ·x· document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to *L • document which may throw doubts on priority claim(s) or involve an inventive step when the document is talc.en alone which is cited to estabhsh the publication date of another •y• document of parucular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the ·o· document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled •p• document published prior to the international filing date but in the art. later than the pnonty date claimed ·&.· document member of the same patent family 1 Date of the actual completion of the international search Date of mailing of the international search report 25 November 1996 03.12.96

Name and mailing addre:s of the ISA Authorized officer European Patent Office, P.B. 5818 Patcntlaan 2 NL • 22110 HV Rijswijk Tel. (+31-70) 340-2040, Tx. 31 651 eponl, Fax: ( + 31-70) 340-3016 Nooij. F

Form PCT/ISA/llO {seccnd sheet) (July 1992) page 1 of 2

IPR Page 1691 of 2482 INTERNATIONAL SEARCH REPORT Inter ·onal Application No PCT/US 96/12251 C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category • Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

A JOURNAL OF IMMUNOASSAY, vol. 16, no. 2, May 1995, NEW YORK, NY, USA, pages 183-197, XP000611616 K. NI ELSEN ET AL.: 11 Stabil ity of freeze-dried horseradish peroxidase-conjugated monoclonal antibodies used in diagnostic serology." see the whole document

1

Form PCT/ISA/:110 (continuation of semnd sheet) (July 1992) page 2 of 2

IPR Page 1692 of 2482 INTERNATIONAL SEARCH REPORT lnb:I"' · -mal Application No .nfonnalion on pab:nt family members PCT/US 96/12251

Patent document Publication Patent family Publication cited in search report I date I member(s) I date EP-A-661060 05-07-95 DE-C­ 4344824 31-08-95 CA-A­ 2138853 29-06-95 CZ-A­ 9403284 12-07-95 FI-A­ 946104 29-06-95 HU-A­ 70449 30-1(:)-95 JP-A­ 7206709 08-08-95 NO-A- 945045 29-06-95 WO-A-8909402 05-10-89 AT-T­ 117434 15-02-95 DE-D­ 6892(:)693 02-03-95 DE-T­ 6892(:)693 24-(:)5-95 EP-A­ 0365685 02-05-9(:) US-A- 5262296 16-11-93

Form PCT/ISA/210 (patent family annex) (July 1992)

IPR Page 1693 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: December 16, 2010 Electronic Signature for Deborah L. Nagle, Ph.D.: /Deborah L. Nagle/ Docket No.: 117813-26902 (PATENT) IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Application of: Wolfgang Fraunhofer

Attorney Docket No.: 117813-26902

Application No.: 12/325,049 Group Art Unit: 1646

Filed: November 28, 2008 Examiner: Hissong, Bruce D.

For: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

AMENDMENT AND RESPONSE TO RESTRICTION REQUIREMENT

Dear Sir:

This communication is responsive to the Office Action dated October 18, 2010. An appropriate request for an extension of time is included herewith. Responsive to the Office Action, please amend the above-identified U.S. patent application as follows.

Amendment of the Claims begins on page 2 of this paper.

Remarks/Arguments begin on page 12 of this paper.

Response to Restriction Requirement begins on page 12 of this paper

1 MEI 10742870v.1

IPR Page 1694 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

CLAIM AMENDMENTS

Please amend claim 43. Please add claim 102.

1. (Original) An aqueous formulation comprising a protein and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm and the protein has a molecular weight (Mw) greater than about 47 kDa.

2. (Original) The formulation of claim 1, wherein the protein has a Mw greater than about 57 kDa.

3. (Original) The formulation of claim 1, wherein the protein has a Mw greater than about 100 kDa.

4. (Original) The formulation of claim 1, wherein the protein has a Mw greater than about 150 kDa.

5. (Canceled)

6. (Original) The formulation of claim 1, wherein the protein has a Mw greater than about 250 kDa.

7. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 2 mS/cm.

8. (Canceled)

9. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 1 mS/cm.

10. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

2 MEI 10742870v.1

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11. ( Original) An aqueous formulation comprising a protein at a concentration of at least about 10 µg/mL and water, wherein the formulation has a conductivity of less than about 2 mS/cm.

12. (Canceled)

13. (Original) The formulation of claim 11, wherein the concentration of the protein is at least about 10 mg/mL.

14. (Canceled)

15. (Original) The formulation of claim 11, wherein the concentration of the protein is at least about 100 mg/mL.

16. (Original) The formulation of claim 11, wherein the concentration of the protein is at least about 150 mg/mL.

17. ( Original) The formulation of claim 11, wherein the concentration of the protein is at least about 200 mg/mL.

18. (Original) The formulation of claim 11, wherein the concentration of the protein is greater than about 200 mg/mL.

19. (Canceled)

20. (Original) The formulation of claim 11 , wherein the formulation has a conductivity of less than about 0.5 mS/cm.

21. (Original) An aqueous formulation comprising a protein at a concentration of at least about 50 mg/mL and water wherein the formulation has an osmolality of no more than about 30 mOsmol/kg_,.

22. (Original) The formulation of claim 21, wherein the osmolality of the formulation is no more than about 15 mOsmol/kg.

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23. (Canceled)

24. (Original) The formulation of claim 21, wherein the concentration of the protein is at least about 150 mg/mL.

25. (Canceled)

26. (Original) The formulation of claim 21, wherein the concentration of the protein is greater than about 200 mg/mL.

27. (Original) An aqueous formulation comprising water and a given concentration of a protein, wherein the protein has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the protein in a buffered solution at the given concentration.

28. ( Original) The formulation of claim 27, wherein the Dh of the protein is at least about 50% less than the Dh of the protein in phosphate buffered saline (PBS) at the given concentration.

29. (Original) The formulation of claim 28, wherein the Dh of the protein is at least about 60% less than the Dh of the protein in PBS at the given concentration.

30. (Original) The formulation of claim 28, wherein the Dh of the protein is at least about 70% less than the Dh of the protein in PBS at the given concentration.

31. (Previously Presented) The formulation of any one of claims 1, 11, 21, and 27, wherein the protein is an antibody, or an antigen-binding fragment thereof.

32. (Original) The formulation of claim 31, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

33. (Original) The formulation of claim 31, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

4 MEI 10742870v.1

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34. (Original) The formulation of claim 31, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira (adalimumab), Campath (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetuximab), Herceptin (Trastuzumab), Myoscint (lmciromab Pentetate), ProstaScint (Capromab Pendetide), Remicade (lnfliximab), ReoPro (Abciximab), Rituxan (Rituximab), Simulect (Basiliximab), Synagis (Palivizumab), Verluma (Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin (lbritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex (Edrecolomab), Mylotarg (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO 1275 (ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and 1131 tositumomab), and Avastin (bevacizumab).

35. (Previously Presented) The formulation of any one of claims 1, 11, 21, and 27, wherein the protein is a therapeutic protein.

36. (Original) The formulation of claim 35, wherein the therapeutic protein is selected from the group consisting of Pulmozyme (Dornase alfa), Rebif, Regranex (Becaplermin), Activase (Alteplase), Aldurazyme (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetin alfa), Becaplermin Concentrate, Betaseron (Interferon beta-lb), BOTOX (Botulinum Toxin Type A), Elitek (Rasburicase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel (Etanercept), Fabrazyme (Agalsidase beta), lnfergen (Interferon alfacon-1), Intron A (Interferon alfa-2a), Kineret (Anakinra), MYOBLOC (Botulinum Toxin Type B), Neulasta (Pegfilgrastim), Neumega (Oprelvekin), Neupogen (Filgrastim), Ontak (Denileukin diftitox), PEGASYS (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pulmozyme (Dornase alfa), Rebif (Interferon beta-la), Regranex (Becaplermin), Retavase (Reteplase), Roferon-A (Interferon alfa-2), TNKase (Tenecteplase), Xigris (Drotrecogin alfa), Arcalyst (Rilonacept), NPlate (Romiplostim), Mircera (methoxypolyethylene glycol-epoetin beta), Cinryze (Cl esterase inhibitor), Elaprase (idursulfase), Myozyme (alglucosidase alfa), Orencia (abatacept), Naglazyme (galsulfase), Kepivance (palifermin) and Actimmune (interferon gamma-lb).

37. (Original) An aqueous formulation comprising an antibody, or an antigen- binding fragment, at a concentration of at least about 10 mg/mL and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 5 µm. 5 MEI 10742870v.1

IPR Page 1698 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

38. (Original) The formulation of claim 37, wherein the antibody has a Dh of less than about 3 µm.

39. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

40. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

41. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira (adalimumab ), Campath (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetuximab), Herceptin (Trastuzumab), Myoscint (lmciromab Pentetate), ProstaScint (Capromab Pendetide), Remicade (lnfliximab), ReoPro (Abciximab), Rituxan (Rituximab), Simulect (Basiliximab), Synagis (Palivizumab), Verluma (Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin (lbritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex (Edrecolomab), and Mylotarg (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO 1275 (ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and 1131 tositumomab) and A vastin (bevacizumab ).

42. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, further comprising a non-ionizable excipient.

43. (Currently Amended) The formulation of claim 42, wherein the non- ionizable excipient is selected from the group consisting of sugar alcohols and a polyol, polyols such as mannitol or sorbitol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

44. (Original) The formulation of claim 43, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

45. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

6 MEI 10742870v.1

IPR Page 1699 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

46. (Canceled)

47. (Canceled)

48. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

49. (Canceled)

50. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is suitable for in vitro or in vivo use.

51. (Canceled)

52. (Previously Presented) The formulation of claim 50, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

53. (Previously Presented) Use of the formulation of claim 50 in the treatment of a disorder in a subject.

54. (Previously Presented) A device comprising the formulation of any one claims 1, 11, 21, 27, and 37.

55. (Canceled)

56. (Previously Presented) An article of manufacture comprising the formulation of any one of claims 1, 11, 21, 27, and 37.

57. (Original) A method of preparing an aqueous formulation comprising a protein and water, the method comprising:

7 MEI 10742870v.1

IPR Page 1700 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

a) providing the protein in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved to thereby prepare the aqueous formulation.

58. (Original) A method of preparing an aqueous formulation of a protein, the method comprising: a) providing the protein in a first solution; b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five-fold volume exchange with the water has been achieved to thereby prepare a diafiltered protein solution; and c) concentrating the diafiltered protein solution to thereby prepare the aqueous formulation of the protein.

59. (Original) The method of claim 58, wherein the concentration of the diafiltered protein solution is achieved via centrifugation.

60. (Previously Presented) The method of claim 57 or 58, wherein the diafiltration medium consists of water.

61. (Previously Presented) The method of claim 57 or 58, wherein the first solution is subjected to diafiltration with water until at least about a six-fold volume exchange or at least about a seven-fold volume exchange is achieved.

62. (Canceled)

63. (Previously Presented) The method of claim 57 or 58, wherein the aqueous formulation has a final concentration of excipients which is at least about 95% less or 99% less than the first solution.

64. (Canceled)

65. (Previously Presented) The method of claim 57 or 58, wherein the first protein solution is obtained from a mammalian cell expression system and has been purified to remove host cell proteins (HCPs).

8 MEI 10742870v.1

IPR Page 1701 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

66. (Previously Presented) The method of claim 57 or 58, wherein the protein has a Mw greater than about 1 kDa.

67. (Previously Presented) The method of claim 57 or 58, wherein the protein has a molecular weight (Mw) selected from the group consisting of greater than about 10 kDa, greater than about 47 kDa, reater than about 57 kDa, greater than about 100 kDa, greater than about 150 kDa, greater than about 200 kDa, and greater than about 250 kDa.

68.-73. (Canceled)

74. (Previously Presented) The method of claim 57 or 58, further comprising adding an excipient to the aqueous formulation.

75. (Original) The method of claim 7 4, wherein the aqueous formulation is a pharmaceutical formulation.

76. (Previously Presented) The method of claim 57 or 58, wherein the aqueous formulation is a pharmaceutical formulation.

77. (Original) The method of claim 76, further comprising loading the aqueous formulation into a device suitable for administering the aqueous formulation to a subject.

78. (Canceled)

79. (Previously Presented) The method of claim 57 or 58, wherein the protein is an antibody, or an antigen-binding fragment thereof.

80. (Original) The method of claim 79, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

81. (Original) The method of claim 79, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

9 MEI 10742870v.1

IPR Page 1702 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

82. (Original) The method of claim 79, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira (adalimumab), Campath (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetuximab), Herceptin (Trastuzumab), Myoscint (lmciromab Pentetate), ProstaScint (Capromab Pendetide), Remicade (lnfliximab), ReoPro (Abciximab), Rituxan (Rituximab), Simulect (Basiliximab), Synagis (Palivizumab), Verluma (Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin (lbritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex (Edrecolomab), Mylotarg (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO 1275 (ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and 1131 tositumomab), and Avastin (bevacizumab).

83. (Previously Presented) The method of claim 57 or 58, wherein the protein is a therapeutic protein.

84. (Original) The method of claim 83, wherein the therapeutic protein is selected from the group consisting of Pulmozyme (Dornase alfa), Rebif, Regranex (Becaplermin), Activase (Alteplase), Aldurazyme (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetin alfa), Becaplermin Concentrate, Betaseron (Interferon beta-1 b ), BOTOX (Botulinum Toxin Type A), Elitek (Rasburicase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel (Etanercept), Fabrazyme (Agalsidase beta), lnfergen (Interferon alfacon-1), Intron A (Interferon alfa-2a), Kineret (Anakinra), MYOBLOC (Botulinum Toxin Type B), Neulasta (Pegfilgrastim), Neumega (Oprelvekin), Neupogen (Filgrastim), Ontak (Denileukin diftitox), PEGASYS (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pulmozyme (Dornase alfa), Rebif (Interferon beta-la), Regranex (Becaplermin), Retavase (Reteplase), Roferon-A (Interferon alfa-2), TNKase (Tenecteplase), and Xigris (Drotrecogin alfa), Arcalyst (Rilonacept), NPlate (Romiplostim), Mircera (methoxypolyethylene glycol-epoetin beta), Cinryze (Cl esterase inhibitor), Elaprase (idursulfase), Myozyme (alglucosidase alfa), Orencia (abatacept), Naglazyme (galsulfase), Kepivance (palifermin) and Actimmune (interferon gamma-lb).

85. (Canceled)

10 MEI 10742870v.1

IPR Page 1703 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

86. (Previously Presented) An aqueous formulation prepared according to the methods of claim 58 or 59.

87.-101. (Canceled)

102. (New) The formulation of claim 43, wherein the polyol is mannitol or sorbitol.

11 MEI 10742870v.1

IPR Page 1704 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

REMARKS

Claims 1-4, 6, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52-54, 56, 61, 63, 65-67, 74-77, 79-84, and 86 were pending in the application. Claim 43 has been amended, and new claim 102 has been added. Accordingly, following entry of this amendment, claims 1-4, 6, 7, 9-11, 13, 15-18, 20- 22, 24, 26-45, 48, 50, 52-54, 56, 61, 63, 65-67, 74-77, 79-84, 86, and 102 will be pending in the application.

Support for new claim 102 may be found throughout the specification and claims as originally filed. Specifically, support for new claim 102 may be found in, for example, claim 43 as originally filed. No new matter has been added.

Any amendment to the claims has been made solely to expedite the prosecution of the application. Applicants reserve the right to pursue the claims as originally filed in this or a separate application( s ).

Restriction Under 35 USC§ 121

Group Election:

The Examiner has required restriction between the following inventions in the above­ identified application:

I. Claims 1-4, 6-7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, and 86, drawn to an aqueous solution comprising a protein and water, classified in class 530, subclass 350;

II. Claims 57-61, 63, 65-67, 74-77, and 79-84, drawn to a method of preparing an aqueous formulation, classified in class 514, subclass 21; and

III. Claim 53, drawn to the use of a formulation in the treatment of a disorder in a subject, classified in class 424, subclass 130.1 and/or class 424, subclass 184.1.

12 MEI 10742870v.1

IPR Page 1705 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

Applicants hereby elect, without traverse, the invention of Group I ( claims 1-4, 6-7, 9-11, 13, 15-18, 20-22, 24, and 26-45, 48, 50, 52, 54, 56, and 86), drawn to aqueous solutions comprising a protein and water.

The Examiner has indicated that the claims of Group I and Group II are related as product and process claims. Accordingly, it is Applicants understanding that where Applicants elect claims directed to a product, and the product claims are subsequently found allowable, withdrawn process claims that depend from or otherwise require all the limitations of the allowable product claims will be considered for rejoinder. In the event of rejoinder, the requirement for restriction between the product claims and the rejoined process claims will be withdrawn, and the rejoined process claims will be fully examined for patentability in accordance with 37 C.F.R. § 1.104. See M.P.E.P. § 804.01.

The Examiner has also indicated, and it is Applicants' understanding that, the claims of Group I and Group III are related as product and process of use claims. Accordingly, it is Applicants understanding that where Applicants elect claims directed to a product, and the product claims are subsequently found allowable, withdrawn process of use claims that depend from or otherwise require all the limitations of the allowable product claims will be considered for rejoinder. In the event of rejoinder, the requirement for restriction between the product claims and the rejoined process of use claims will be withdrawn, and the rejoined process of use claims will be fully examined for patentability in accordance with 37 C.F.R. § 1.104. See M.P.E.P. § 804.01.

Applicants reserve the right to traverse the restriction between the non-elected groups in this or a separate application.

Species Election:

The Examiner has also required the election of the following species:

1. Antibody or antibody fragments selected from those recited in claims 34, 41, and 82;

2. Therapeutic proteins selected from those recited in claims 36 and 84;

3. Non-ionizable excipients selected from recited in claim 43; and

13 MEI 10742870v.1

IPR Page 1706 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

4. Non-ionic surfactants selected from those recited in claim 44.

Applicants hereby elect, without traverse and for search purposes only, the following species:

1. Humira (adalimumab);

2. Enbrel (Etanercept);

3. Mannitol; and

4. Polysorbate 80.

It is Applicants' understanding that the search will be extended to the remaining species upon a finding of allowability of a generic linking claim. As described in 37 C.F. R. § 1.144, it is Applicant's understanding that, upon the allowance of a generic linking claim, Applicant will be entitled to consideration of claims to additional species, i.e., non-elected species of claims 34, 36, 41, 43, 44, 82, and 84, which depend from or otherwise require all of the limitations of the allowed generic linking claim. If Applicant's understanding is incorrect, Applicant maintains the right to traverse the species restriction.

Claims of Group I that read on the elected species include 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, 86, and 102.

14 MEI 10742870v.1

IPR Page 1707 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

SUMMARY

If a telephone conversation with Applicant's Attorney would expedite the prosecution of the above-identified application, the Examiner is urged to call the undersigned at (617) 449-6500.

The Commissioner is hereby authorized to charge any fees associated with the filing of this communication or any subsequent filing to our Deposit Account No. 50-4876, under Order No. 117813-26902 from which the undersigned is authorized to draw.

Dated: December 16, 2010 Respectfully submitted,

Electronic signature: /Deborah L. Nagle/

Deborah L. Nagle, Ph.D. Registration No.: 59,978 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6500 (617) 607-9200 Attorney/Agent For Applicant

15 MEI 10742870v.1

IPR Page 1708 of 2482 PTO/SB/22 (02-09) Approved for use through 03/31/2009. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARMENT OF COMMERCE Under the paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid 0MB control number.

PETITION FOR EXTENSION OF TIME UNDER 37 CFR 1.136(a) Docket Number (Optional) FY 2009 117813-26902 (Fees pursuant to the Consolidated Appropriations Act, 2005 (H.R. 4818).) Application Number 12/325,049 Filed November 28, 2008

For Protein Formulations and Methods of Making Same

Art Unit 1646 Examiner Hissong, Bruce D.

This is a request under the provisions of 37 CFR 1.136(a) to extend the period for filing a reply in the above identified application. The requested extension and fee are as follows (check time period desired and enter the appropriate fee below): Fee Small Entity Fee 0 One month (37 CFR 1.17(a)(1)) $130 $65 $ 130 D Two months (37 CFR 1.17(a)(2)) $490 $245 $ D Three months (37 CFR 1.17(a)(3)) $1110 $555 $ D Four months (37 CFR 1.17(a)(4 )) $1730 $865 $ D Five months (37 CFR 1.17(a)(5)) $2350 $1175 $ D Applicant claims small entity status. See 37 CFR 1.27. D A check in the amount of the fee is enclosed. D Payment by credit card. Form PT0-2038 is attached. D The Director has already been authorized to charge fees in this application to a Deposit Account. 0 The Director is hereby authorized to charge any fees which may be required, or credit any overpayment, to Deposit Account Number ....;;5"""0--4"'"'8;;..;7....;6'------WARNING: Information on this form may become public. Credit card information should not be included on this form. Provide credit card information and authorization on PT0-2038. I am the D applicant/inventor. assignee of record of the entire interest. See 37 CFR 3.71. D Statement under 37 CFR 3.73(b) is enclosed (Form PTO/SB/96). 0 attorney or agent of record. Registration Number_5_5_,2_8_1______attorney or agent under 37 CFR 1.34. D Registration number if acting under 37 CFR 1.34 ------/DLN/ December 16, 2010 Signature Date Deborah L. Nagle, Ph.D. (617) 449-6500 Typed or printed name Telephone Number

NOTE: Signatures of all the inventors or assignees of record of the entire interest or their representative(s) are required. Submit multiple forms if more than one signature is required, see below. D Total of forms are submitted. This collection of information 1s required by 37 CFR 1.136(a). The information 1s required to obtain or retain a benefit by the public which 1s to file (and by the USPTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1.11 and 1.14. This collection is estimated to take 6 minutes to complete, including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and Trademark Office, U.S. Department of Commerce, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450.

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IPR Page 1709 of 2482 Privacy Act Statement

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IPR Page 1710 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Filer: Deborah L. Nagle

Attorney Docket Number: 117813-26902

Filed as Large Entity

Utility under 35 USC 111 (a) Filing Fees

Sub-Total in Description Fee Code Quantity Amount USO($)

Basic Filing:

Pages:

Claims:

Miscellaneous-Filing:

Petition:

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Post-Allowance-and-Post-Issuance:

Extension-of-Time:

Extension - 1 month with $0 paid 1251 1 130 130

IPR Page 1711 of 2482 Sub-Total in Description Fee Code Quantity Amount USO($)

Miscellaneous:

Total in USD ($) 130

IPR Page 1712 of 2482 Electronic Acknowledgement Receipt

EFSID: 9048029

Application Number: 12325049

International Application Number:

Confirmation Number: 1766

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Customer Number: 87501

Filer: Deborah L. Nagle

Filer Authorized By:

Attorney Docket Number: 117813-26902

Receipt Date: 16-DEC-2010

Filing Date: 28-NOV-2008

Time Stamp: 09:51:03

Application Type: Utility under 35 USC 111 (a)

Payment information:

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Payment was successfully received in RAM $130

RAM confirmation Number 8566

Deposit Account 504876

Authorized User

The Director of the USPTO is hereby authorized to charge indicated fees and credit any overpayment as follows: Charge any Additional Fees required under 37 C.F.R. Section 1.16 (National application filing, search, and examination fees) Charge any Additional Fees required under 37 C.F.R. Section 1.17 (Patent application and reexamination processing fees)

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File Listing:

Document File Size(Bytes)/ Multi Pages Document Description File Name Number Message Digest Part /.zip (if appl.)

85022 1 Response to Election/ Restriction Filed response.pdf no 15

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Warnings:

Information:

317435 2 Extension of Time PetitionEOT.pdf no 2

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Information:

30493 3 Fee Worksheet (PT0-875) fee-info.pdf no 2

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Warnings:

Information:

Total Files Size (in bytes) 432950

This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents, characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a Post Card, as described in MPEP 503.

New A~~lications Under 35 U.S.C. 111 If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR 1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this Acknowledgement Receipt will establish the filing date of the application.

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IPR Page 1714 of 2482 PTO/SB/06 (07-06) Approved for use through 1/31/2007. 0MB 0651-0032 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid 0MB control number. PATENT APPLICATION FEE DETERMINATION RECORD Application or Docket Number Filing Date Substitute for Form PT0-875 12/325,049 11/28/2008 D To be Mailed

APPLICATION AS FILED - PART I OTHER THAN (Column 1) (Column 2) SMALL ENTITY D OR SMALL ENTITY FOR NUMBER FILED NUMBER EXTRA RATE($) FEE($) RATE($) FEE($)

D BASIC FEE N/A N/A N/A N/A (37 CFR 1.16(a), (b), or (c))

D SEARCH FEE N/A N/A N/A N/A (37 CFR 1.16(k), (i), or (m))

D EXAMINATION FEE N/A N/A N/A N/A (37 CFR 1.16(0), (p), or (q)) TOTAL CLAIMS X $ = OR X $ = (37 CFR 1.16(i)) minus 20 = * INDEPENDENT CLAIMS X $ = X $ = (37 CFR 1.16(h)) minus 3 = * If the specification and drawings exceed 100 sheets of paper, the application size fee due 0APPLICATION SIZE FEE is $250 ($125 for small entity) for each (37 CFR 1.16(s)) additional 50 sheets or fraction thereof. See 35 U.S.C. 41 (a)(1)(G) and 37 CFR 1.16(s).

D MULTIPLE DEPENDENT CLAIM PRESENT (37 CFR 1.16U)) * If the difference in column 1 is less than zero, enter "O" in column 2. TOTAL TOTAL

APPLICATION AS AMENDED- PART II OTHER THAN (Column 1) (Column 2) (Column 3) SMALL ENTITY OR SMALL ENTITY CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) I- 12/16/2010 AFTER PREVIOUSLY EXTRA FEE($) FEE($) z AMENDMENT PAID FOR w Total (37 CFR Minus X $ = OR X $52= ~ 1.16(i)) • 134 ** 839 = 0 0 0 Independent z * 7 Minus ***7 = X $ = OR X $220= w 137 CFR 1.16/h\\ 0 0 ~ D Application Size Fee (37 CFR 1.16(s)) <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L 0 FEE FEE (Column 1) (Column 2) (Column 3) CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) AFTER PREVIOUSLY EXTRA FEE($) FEE($) AMENDMENT PAID FOR I- Total (37 CFR z * Minus ** = X $ = OR X $ = w 1.16(i\\ ~ Independent Minus X $ = OR X $ = 0 (37 CFR 1.16(hll * *** = z w D Application Size Fee (37 CFR 1.16(s)) ~ <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L FEE FEE * If the entry in column 1 is less than the entry in column 2, write "O" in column 3. Legal Instrument Examiner: ** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 20, enter "20". /CAROL BARNES/ *** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 3, enter "3". The "Highest Number Previously Paid For" (Total or Independent) is the highest number found in the appropriate box in column 1. This collection of information 1s required by 37 CFR 1.16. The information 1s required to obtain or retain a benefit by the public which 1s to file (and by the USPTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1.14. This collection is estimated to take 12 minutes to complete, including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and Trademark Office, U.S. Department of Commerce, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450. If you need assistance in completing the form, call 1-800-PT0-9199 and select option 2.

IPR Page 1715 of 2482 UNITED STA1ES p A IBNT AND TRADEMARK OFFICE UNITED STA TES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O. Box 1450 Alexandria., Virginia 22313-1450 www.uspto.gov

APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.

12/325,049 11/28/2008 Wolfgang FRAUNHOFER 117813-26902 1766

87501 7590 03/02/2011 EXAMINER McCarter & English, LLP / Abbott Laboratories Ltd. 265 Franklin Street HISSONG, BRUCE D Boston, MA 02110 ART UNIT PAPER NUMBER

1646

MAIL DATE DELIVERY MODE

03/02/2011 PAPER

Please find below and/or attached an Office communication concerning this application or proceeding.

The time period for reply, if any, is set in the attached communication.

PTOL-90A (Rev. 04/07) IPR Page 1716 of 2482 Application No. Applicant(s)

12/325,049 FRAUNHOFER ET AL.

Office Action Summary Examiner Art Unit Bruce D. Hissong, Ph.D. 1646 -- The MAILING DA TE of this communication appears on the cover sheet with the correspondence address -­ Period for Reply A SHORTENED STATUTORY PERIOD FOR REPLY IS SET TO EXPIRE ;J. MONTH(S) OR THIRTY (30) DAYS, WHICHEVER IS LONGER, FROM THE MAILING DATE OF THIS COMMUNICATION. Extensions of time may be available under the provisions of 37 CFR 1.136(a). In no event, however, may a reply be timely filed after SIX (6) MONTHS from the mailing date of this communication. If NO period for reply is specified above, the maximum statutory period will apply and will expire SIX (6) MONTHS from the mailing date of this communication. Failure to reply within the set or extended period for reply will, by statute, cause the application to become ABANDONED (35 U.S.C. § 133). Any reply received by the Office later than three months after the mailing date of this communication, even if timely filed, may reduce any earned patent term adjustment. See 37 CFR 1.704(b). Status

1 )IZI Responsive to communication(s) filed on 16 December 2010. 2a)0 This action is FINAL. 2b)[8J This action is non-final. 3)0 Since this application is in condition for allowance except for formal matters, prosecution as to the merits is closed in accordance with the practice under Ex parte Quayle, 1935 C.D. 11, 453 O.G. 213.

Disposition of Claims

4)1Zl Claim(s) 1-4. 6-7. 9-11. 13. 15-18. 20-22. 24. 26-45. 48. 50. 52. 54. 56. 61. 63. 65-67. 74-77. 79-84. 86. and 102 is/are pending in the application. 4a) Of the above claim(s) 6. 53. 57-61. 63. 65-67. 74-77. and 79-84 is/are withdrawn from consideration. 5)0 Claim(s) __ is/are allowed. 6)1Zl Claim(s) 1-4.7.9-11. 13. 15-18.20-22.24.26-45.48.50.52.54.56.86 and 102 is/are rejected. 7)0 Claim(s) __ is/are objected to. 8)0 Claim(s) __ are subject to restriction and/or election requirement.

Application Papers

9)0 The specification is objected to by the Examiner. 1 O)IZI The drawing(s) filed on 28 November 2008 and 05 August 2009 is/are: a)IZI accepted or b)O objected to by the Examiner. Applicant may not request that any objection to the drawing(s) be held in abeyance. See 37 CFR 1.85(a). Replacement drawing sheet(s) including the correction is required if the drawing(s) is objected to. See 37 CFR 1.121 (d). 11 )0 The oath or declaration is objected to by the Examiner. Note the attached Office Action or form PT0-152.

Priority under 35 U.S.C. § 119

12)0 Acknowledgment is made of a claim for foreign priority under 35 U.S.C. § 119(a)-(d) or (f). a)O All b)O Some * c)O None of: 1.0 Certified copies of the priority documents have been received. 2.0 Certified copies of the priority documents have been received in Application No. __. 3.0 Copies of the certified copies of the priority documents have been received in this National Stage application from the International Bureau (PCT Rule 17.2(a)). *Seethe attached detailed Office action for a list of the certified copies not received.

Attachment{s) 1) [8J Notice of References Cited (PT0-892) 4) 0 Interview Summary (PT0-413) 2) 0 Notice of Draftsperson's Patent Drawing Review (PT0-948) Paper No(s)/Mail Date. __ . 3) [8J Information Disclosure Statement(s) (PTO/SB/08) 5) 0 Notice of Informal Patent Application Paper No(s)/Mail Date 10/19/2010. 6) 0 Other: __.

U.S. Patent and Trademark Office PTOL-326 (Rev. 08-06) Office Action Summary Part of Paper No./Mail Date 20110225

IPR Page 1717 of 2482 Application/Control Number: 12/325,049 Page 2 Art Unit: 1646

DETAILED ACTION

Election/Restrictions 1. Applicant's election without traverse of Group I, claims 1-4, 6-7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, and 86, and the species elections of Humira (adalimumab), Enbrel (Etanercept), mannitol, and polysorbate 80, in the reply filed on 12/16/2010 is acknowledged.

2. Claims 1-4, 6-7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, 61, 63, 65-67, 74-77, 79-84, 86, and new claim 102 are pending, with claims 53, 57-61, 63, 65-67, 74-77, and 79-84 withdrawn as non-elected subject matter. Additionally, claim 6, drawn to a formulation comprising a protein with a molecular weight greater than 250 kDa, is also withdrawn in view of Applicants' election of Humira and Enbrel.

3. Claims 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, 86 and 102 are presently under examination.

Information Disclosure Statement The information disclosure statement received on 10/19/2010 has been considered. Citations B6, B 15, and B22 have been considered in view of their abstracts only, as these references are not in English.

Specification The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicants are reminded that embedded hyperlinks and/or other forms of browser-executable code are not permitted (MPEP § 608.01), and all trademarks must be properly identified (i.e. be capitalized) and accompanied by generic terminology. Applicants' cooperation is requested in correcting any errors of which Applicants may become aware in the specification. It is also noted that all sequences must be identified by appropriate sequence identifier ("SEQ ID NO: X"). See MPEP '2422.02. Applicants' cooperation in ensuring that all sequences are thus identified is therefore requested.

IPR Page 1718 of 2482 Application/Control Number: 12/325,049 Page 3 Art Unit: 1646

Claim Objections 1. The Examiner suggests amending claims 27-30 to recite an aqueous solution comprising water and a given concentration of a protein, wherein the protein has a hydrodynamic diameter which is at least about 50% less (or 60% or 70% less) than the hydrodynamic diameter of the protein "in a buffered solution at the same concentration."

2. Claim 86 is objected to for depending from a non-elected invention. Specifically, claim 86 depends from withdrawn claims 58 or 59. It is suggested to amend claim 86 to incorporate the limitations of the withdrawn claims.

Claim Rejections - 35 USC § 112, first paragraph - written description The following is a quotation of the first paragraph of 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.

Claims 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, 86, and 102 are rejected under 35 U.S.C. 112, first paragraph, containing subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention. The claims are drawn to aqueous formulations comprising a protein and water, wherein said formulations have a conductivity of less than about 2.5 mS/cm, an osmolality of no more than about 30 mOSmol/kg, or wherein the protein has a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the protein in PBS at the given concentration. As written, the claims encompass formulations comprising any protein. The specification has described formulations in which antibodies, such as anti-IL-12 (adalimumab ), exhibit a conductivity of less than about 2.5 mS/cm and/or an osmolality of no more than about 30 mOSmol/kg. Additionally, the specification describes formulations comprising adalimumab wherein the antibody molecules have a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of adalimumab in PBS. However, the claims read on formulations comprising any protein, and the specification has not described any other formulations, or any other proteins, which can be formulated in such a way as to produce a formulation with a conductivity of less than about 2.5 mS/cm and/or an osmolality of no more than about 30

IPR Page 1719 of 2482 Application/Control Number: 12/325,049 Page 4 Art Unit: 1646 mOSmol/kg. The specification has also not described any proteins, other than antibodies, which would be expected to have a hydrodynamic diameter that is at least about 50% less than the hydrodynamic diameter of the protein in PBS at the given concentration when formulated in a manner which is commensurate with the instant invention. Furthermore, the claims do not require the formulated proteins of the instant invention to have any biological activity, nor any particular structure. Thus, the claims are drawn to a genus of proteins which have not been described in the specification in such a way as to convey to a person of ordinary skill in the art that the inventors, at the time of invention, had possession of the full genus. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. In this case, the only factor present in the claims is a requirement that a formulation exhibiting various physical characteristics (conductivity and/or osmolality) comprise any protein. Accordingly, in the absence of sufficient distinguishing characteristics, the specification does not provide adequate written description of the claimed genus.

Claim Rejections - 35 USC § 112, second paragraph The following is a quotation of the second paragraph of 35 U.S.C. 112: The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.

Claims 34, 36, and 41 recite numerous antibodies identified only by trademark/trade name. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112, second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe the recited antibodies and, accordingly, the identification/description is indefinite.

IPR Page 1720 of 2482 Application/Control Number: 12/325,049 Page 5 Art Unit: 1646

Claim Rejections - 35 USC § 103 The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action: (a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.

1. Claims 1-4, 7, 9-11, 13, 15-18, 20, 31-35, 42-43, 45, 48, 50, 52, 54, 56, 86, and 102 are rejected under 35 U.S.C. 103(a) as being unpatentable over Konstantinov et al ("Konstantinov" - US 20060149042) in view of Matheus et al ("Matheus" - US 20070172475). The claims of the instant invention are drawn to an aqueous formulation comprising a protein and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and the protein has a molecular weight greater than 47 kDa. The claims further recite formulations comprising proteins having molecular weights greater than 57 kDa, 100 kDa, or 150 kDa, and wherein the conductivity of the formulation is less than about 2 mS/cm, 1 mS/cm, or 0.5 mS/cm, and wherein the concentration of the protein is at least about 10 mg/ml, 100 mg/ml, 150 mg/ml, or greater than 200 mg/ml. The claims also recite aqueous formulations wherein the protein is a therapeutic protein such as an antibody or antigen­ binding fragment thereof, including chimeric, human, humanized, or domain antibodies, and specifically, anti-IL-12 antibodies such as Humira (adalimumab). Also claimed are formulations comprising a non­ ionizable excipient, including polyols such as mannitol, and formulations which are stable in liquid form for at least about 3 months or at least about 12 months, and a device or article of manufacture comprising said formulation. Konstantinov discloses a method for concentration of proteins, wherein the resulting formulation has a conductivity of less than 2.5 mS/cm (paragraph 0013; claims 4 and 5). Specifically Konstantinov shows that a method of concentrating proteins using ultrafiltration followed by diafiltration against water can be used to lower the conductivity of the solution to less than 6 mS/cm, and preferably, from 0.5 to 5 mS/cm. Konstantinov is silent regarding formulation of a protein having a molecular weight of at least 47 kDa, or any specific concentration of protein. However, Matheus teaches formulations of antibodies and water (paragraph 0034), wherein the antibody concentration is at least 1 mg/ml, and up to 250 mg/ml (paragraph 0061). Matheus also suggests formulation of the anti-IL-12 antibody Humira (paragraph 0062), formulations comprising mannitol

IPR Page 1721 of 2482 Application/Control Number: 12/325,049 Page 6 Art Unit: 1646

(paragraph 0051), and formulations intended for in vivo administration via routs of administration such as intravenous and subcutaneous (paragraph 0064). It would have been obvious to a person of ordinary skill in the art, at the time the instant invention was conceived, to create formulations comprising a protein and water and having a conductivity of less than about 2.5 mS/cm by following the combined teachings of Konstantinov and Matheus. The motivation to do so comes from the teaching of Konstantinov, which teaches an ultrafiltration/diafiltration method of concentrating protein formulations, wherein the conductivity is less than 2.5 mS/cm. Further motivation comes from Matheus, which also teaches ultrafiltration-based methods of protein concentration, wherein antibodies can be formulated at high concentrations. Therefore, it would be obvious to a person of ordinary skill in the art that to formulate the concentrated antibodies of Matheus so that the conductivity of the formulation is less than 2.5 mS/cm, as suggested by Konstantinov. Furthermore, although Matheus exemplifies formulations comprising anti-EGFR antibodies, Matheus suggests that Humira can also be formulated at higher concentrations (paragraph 0062), providing the motivation to formulate anti-IL-12 antibodies/Humira in such a way as to provide a formulation with a higher antibody concentration and wherein the conductivity is less than 2.5 mS/cm. Also, Matheus provides the motivation to also create antibody formulations comprising excipients such as mannitol, and suggests in vivo administration by a variety of routes of administration. Similarly, although neither Konstantinov nor Matheus specifically teach a device or an article of manufacture comprising a protein formulation, it is noted that because Matheus suggests in vivo administration of said antibody formulations to a patient, it would be obvious to a skilled artisan to incorporate a formulation meeting the limitations of the instant claims into a device or article of manufacture for ease-of-use and/or convenience. It is also noted that while neither Konstantinov nor Matheus specifically teach that such formulations would be stable for at least 3 months or at least 12 months, the antibody formulations of Matheus are taught to be stable for extended periods of time (paragraphs 0013-0014, 0065). In absence of evidence to the contrary a formulation comprising an antibody and water and having a conductivity of less than 2.5 mS/cm, as suggested by the combination of Konstantinov and Matheus, would therefore be expected to be stable for at least about 3 months or at least about 12 months. Because the USPTO does not have the facilities for testing the formulation suggested by the prior art, the burden is on the Applicants to show a novel and unobvious difference between the claimed formulation and those which are suggested by the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and Ex parte Gray, 10 USPQ 2d 1922 1923 (PTO Bd. Pat. App. & Int.).

IPR Page 1722 of 2482 Application/Control Number: 12/325,049 Page 7 Art Unit: 1646

2. Claim 44 is rejected under 35 U.S.C. 103(a) as being unpatentable over Konstantinov et al ("Konstantinov" - US 20060149042) in view of Matheus et al ("Matheus" - US 20070172475), and further in view of Schuschnig (US 20070202051). The subject matter of the instant application is discussed above. Claim 44 is further drawn to the claimed formulation, wherein said formulation further comprises a non-ionic surfactant, including polysorbates 20, 40, 60, or 80. Konstantinov and Matheus teach as described above; both are silent regarding a formulation comprising a protein and water and further comprising a non-ionic surfactant. However, Schuschnig teaches formulation of various therapeutic proteins, including antibodies (paragraphs 0118, 0124), wherein said formulations can comprise non-ionic surfactants, including polysorbate 80 (paragraph 0140). Therefore, a person of ordinary skill in the art, at the time the instant invention was conceived, would have found it obvious to create an aqueous formulation comprising water and a therapeutic protein such as anti-IL-12 antibodies/Humira, wherein said formulation has a conductivity of less than 2. 5 mS/cm, and wherein the formulation also comprises polysorbate 80. The motivation to do so comes from Konstantinov and Matheus, which as discussed above provide the motivation to create an aqueous formulation comprising an antibody such as Humira, wherein the formulation has a conductivity of less than 2.5 mS/cm. Further motivation is provided by Schuschnig, which suggests formulation of agents such as antibodies compositions comprising a non-ionic surfactant such as polysorbate 80. Thus, a skilled artisan would find it obvious to further incorporate polysorbate 80, as suggested by Schuschnig, into an aqueous formulation comprising anti-IL-12 antibodies and water, wherein the conductivity of the formulation is less than 2.5 mS/cm, as suggested by Konstantinov and Matheus.

3. Claim 36 is rejected under 35 U.S.C. 103(a) as being unpatentable over Konstantinov et al ("Konstantinov" - US 20060149042) in view of Matheus et al ("Matheus" - US 20070172475), and further in view of Giles-Komar et al ("Giles-Komar" - US 20030143603). The subject matter of the instant application is discussed above. Claim 36 is further drawn to the claimed formulation, wherein the protein in said formulation is a therapeutic protein, and specifically can be Enbrel (Etanercept). Konstantinov and Matheus teach as described above; both are silent regarding an aqueous formulation comprising water and a protein, wherein the protein is Enbrel/Etanercept. However, Giles­ Komar teaches that aqueous formulations comprising inhibitors ofTNF-a, such as anti-TNF-a antibodies

IPR Page 1723 of 2482 Application/Control Number: 12/325,049 Page 8 Art Unit: 1646 and Etanercept, as useful for treatment of TNF-responsive disorders or conditions (paragraphs 0130, 0160, 0197) Therefore, a person of ordinary skill in the art, at the time the instant invention was conceived, would have found it obvious to create an aqueous formulation comprising water and a therapeutic protein wherein said formulation has a conductivity of less than 2.5 mS/cm, and wherein the therapeutic protein is an anti-TNF-a antibody or Enbrel/Etanercept The motivation to do so comes from Konstantinov and Matheus, which as discussed above provide the motivation to create an aqueous formulation comprising an antibody, wherein the formulation has a conductivity of less than 2.5 mS/cm. Further motivation is provided by Giles-Komar, which teaches that aqueous formulations of TNF-a inhibitors, such as Etanercept, are useful for treating various disorders. Therefore, it would have been obvious to a person of ordinary skill in the art to substitute an anti-TNF-a antibody or Enbrel/Etanercept as the antibody in the formulation which is obvious in view of Konstantinov and Matheus.

Conclusion No claim is allowable.

Any inquiry concerning this communication or earlier communications from the examiner should be directed to Bruce D. Hissong, Ph.D., whose telephone number is (571)272-3324. The examiner can normally be reached M-F from 8:30 am - 5:00 pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Gary Nickol, Ph.D., can be reached at (571) 272-0835. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair­ direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.

Bruce D. Hissong

IPR Page 1724 of 2482 Application/Control Number: 12/325,049 Page 9 Art Unit: 1646

Art Unit 1646

/Robert Landsman/ Primary Examiner, Art Unit 1647

IPR Page 1725 of 2482 Application/Control No. Applicant(s)/Patent Under Reexamination 12/325,049 FRAUNHOFER ET AL. Notice of References Cited Examiner Art Unit Page 1 of 1 Bruce D. Hissong, Ph.D. 1646

U.S. PATENT DOCUMENTS Document Number Date * Country Code-Number-Kind Code MM-YYYY Name Classification

* A US-2006/0149042 07-2006 Konstantinov et al. 530/412 * B US-2007/0172475 07-2007 Matheus et al. 424/143.1 * C US-2007/0202051 08-2007 Schuschnig, Uwe 424/045 * D US-2003/0143603 07-2003 Giles-Komar et al. 435/6 E US-

F US-

G US-

H US-

I US-

J US-

K US-

L US-

M US- FOREIGN PATENT DOCUMENTS Document Number Date * Country Code-Number-Kind Code MM-YYYY Country Name Classification

N

0 p

Q

R s

T NON-PATENT DOCUMENTS * Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)

u

V

w

X

*A copy of this reference 1s not being furnished with this Office action. (See MPEP § 707.05(a).) Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.

U.S. Patent and Trademark Office PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No. 20110225

IPR Page 1726 of 2482 Application/Control No. Applicant(s)/Patent Under Reexamination Search Notes 12325049 FRAUNHOFER ET AL.

Examiner Art Unit *1232504 Bruce D Hissong, Ph.D. 1646 9*

SEARCHED

Class I Subclass I Date I Examiner None I None I 2/26/2011 I BDH

SEARCH NOTES

Search Notes Date Examiner WEST, STN: protein, aqueous formulation, composition, conductivity, 2/26/2011 BDH osmolality, hydrodynamic diameter (search strateqy in case) PALM: inventor search 2/26/2011 BDH

INTERFERENCE SEARCH

Class I Subclass I Date I Examiner I I I

U.S. Patent and Trademark Office Part of Paper No. : 2011 0225

IPR Page 1727 of 2482 WEST Search History for Application 12325049

Creation Date: 2011022616:22

Prior Art Searches

Query DB Op. Plur. Thes. Date

((protein or polypeptide) with (water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition)) USOC, EPAB, JPAB, DWPI

( ( (protein or polypeptide) with ( water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, and conductivity JPAB,DWPI

(((protein or polypeptide) with (water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, same conductivity JPAB,DWPI

( ( (protein or polypeptide) with ( water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, same osmol$5 JPAB,DWPI

(((protein or polypeptide) with (water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, and (low adj osmol$5) JPAB,DWPI

( ( (protein or polypeptide) with ( water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, and (30 near2 (mosm or mosmol)) JPAB,DWPI

(((protein or polypeptide) with (water or PGPB, USPT, OR YES 02-26-2011 aqueous) with (formulat$3 or composition))) USOC, EPAB, and (hydrodynamic adj diameter) JPAB, DWPI

WEST Search History for Application 12325049 1

IPR Page 1728 of 2482 FILE 'MEDLINE, BIOSIS, EMBASE, SCISEARCH, CAPLUS, USPATFULL, PCTFULL' ENTERED AT 16:35:00 ON 26 FEB 2011 11 70903 S ((AQUEOUS OR WATER) (S) (PROTEIN OR POLYPEPTIDE) (S) (FORMULAT? 0 12 4690 S 11 AND (CONDUCTIVITY OR MOSM OR MOSMOL) 13 947 S Ll(P) (CONDUCTIVITY OR MOSM OR MOSMOL) 14 843 S Ll(S) (CONDUCTIVITY OR MOSM OR MOSMOL) 15 424 S Ll(S) (LOW(S) (CONDUCTIVITY OR MOSM OR MOSMOL)) 16 392 S 15 AND PY<2008 17 30429 S ((AQUEOUS OR WATER) (S) (ANTIBOD?) (S) (FORMULAT? OR COMPOSITION) 18 138 S L7(S) (LOW(S) (CONDUCTIVITY OR MOSM OR MOSMOL)) 19 138 S 18 AND PY<2008 110 159 S 11 AND (HYDRODYNAMIC(W)DIAMETER) 111 81 S 110 AND PY<2008 112 81 DUP REM 111 (0 DUPLICATES REMOVED)

=>

IPR Page 1729 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: October 19, 2010 Electronic Signature for Cristin Howley Cowles, Ph.D.: /Deborah L. Nagle/

ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned

Sheet I 1 I of I 4 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Pages, Columns, Lines, Examiner Document Number Cite Publication Date Name of Patentee or Where Relevant Passages or Initials* 2 No. 1 Number-Kind Code ( if MM-DD-YYYY Applicant of Cited Relevant known) Document Fiqures Appear A1 6,090,382 07-18-2000 Salfeld et al. A2 6,258,562 07-10-2001 Salfeld et al. A3 6,509,015 01-21-2003 Salfeld et al. A4 7,223,394 05-29-2007 Salfeld et al. A5 7,541,031 06-02-2009 Salfeld et al. A6 7,588,761 09-15-2009 Salfeld et al. A7 US 2003-0219438 A1 11-27-2003 Salfeld et al. A8 US 2010-0016557 A 1 01-21-2010 Salfeld et al. A9 US 2009-0155205 A 1 06-18-2009 Salfeld et al. A10 US 2003-0235585 A 1 12-25-2003 Fischkoff et al. A11 US 2004-0033228 A 1 02-19-2004 Krause et al. A12 US 2006-0153846 A 1 07-13-2006 Krause et al. A13 US 2003-0206898 A 1 11-06-2003 Fischkoff et al. A14 US 2004-0009172 A 1 01-15-2004 Fischkoff et al. A15 US 2004-0166111 A 1 08-26-2004 Kaymakcalan et al. A16 US 2006-0009385 A 1 01-12-2006 Hoffman et al. A17 US 2009-0304682 A 1 12-10-2009 Hoffman et al. A18 US 2006-00837 41 A 1 04-20-2006 Hoffman et al. A19 US 2007-0041905 A 1 02-22-2007 Hoffman et al. A20 US 2007-0081996 A 1 04-12-2007 Hoffman et al. A21 US 2007-0071747 A1 03-29-2007 Hoffman et al. A22 US 2007-0172897 A 1 07-26-2007 Maksymowych et al. A23 US 2007-0292442 A 1 12-20-2007 Wan et al. A24 US 2009-011 0679 A 1 04-30-2009 Li et al. A25 US 2009-0291062 A1 11-26-2009 Fraunhofer et al. A26 US 2008-0166348 A 1 07-10-2008 Kupper et al. A27 US 2009-0028794 A 1 01-29-2009 Medich et al. A28 US 2009-031 7399 A 1 12-24-2009 Pollack et al. A29 US 2008-0118496 A 1 05-22-2008 Medich et al. A30 US 2009-0123378 A 1 05-14-2009 Wong et al. A31 US 2008-0131374 A1 06-05-2008 Medich et al.

Examiner IDate Signature /Bruce Hissonq/ Considered 02/26/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 10293866v.1 IPR Page 1730 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned Sheet I 2 I 01 I 4 Attorney Docket Number 117813-26902

A32 US 2008-0311 043 A 1 12-18-2008 Hoffman et al. A33 US 2010-0021451 A1 01-28-2010 Wong et al. A34 US 2009-0017472 A1 01-15-2009 Stuhlmuller et al. A35 US 2010-0003243 A1 01-07-2010 Okun et al. A36 US 2009-0258018 A1 10-15-2009 Medich et al. A37 US 2008-0227136 A 1 09-18-2008 Pia et al. A38 US 2009-0239259 A 1 09-24-2009 Hsieh et al. A39 US 2009-0226530 A 1 09-10-2009 Lassner et al. A40 US 2009-02711 64 A 1 10-29-2009 Peng et al. A41 US 2010-0040630 A1 02-18-2010 Elden et al. A42 US 2004-0126372 A 1 07-01-2004 Banerjee et al. A43 US 2007-02021 04 A 1 08-30-2007 Banerjee et al. A44 US 2004-0131614 A1 07-08-2004 Banerjee et al. A45 US 2004-0126373 A 1 07-01-2004 Banerjee et al. A46 US 2004-0151722 A 1 08-05-2004 Banerjee et al. A47 US 2004-0136991 A 1 07-08-2004 Banerjee et al. A48 US 2008-0193466 A 1 08-14-2008 Banerjee et al. A49 US 2004-0136990 A 1 07-15-2004 Banerjee et al. A50 US 2004-0219142 A 1 11-04-2004 Banerjee et al. A51 US 2004-0136989 A 1 07-15-2004 Banerjee et al. A52 US 2003-0012786 A1 01-16-2003 Teoh et al. A53 US 2003-0161828 A 1 08-28-2003 Abdelghany et al. A54 US 2009-0280065 A 1 11-12-2009 Willian et al. A55 US 2010-0040604 A1 02-18-2010 Salfeld et al. A56 US 2010-034823 A1 02-11-2010 Borhani et al. A57 US 2009-0148513 A 06-11-2009 Fraunhofer et al. A58 US 2010-0160894 A 1 06-24-2010 Julian et al.

Examiner Date Signature iBruce Hissongi Considered 02/26i2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 10293866v.1 IPR Page 1731 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned Sheet I 3 I 01 I 4 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS I Foreign Patent Pages, Columns, Documet Publication Name of Patentee or Lines, 3 Date Applicant of Cited Document Where Relevant Country Code - Te Examiner Cite Number4-Kind Code5 MM-DD- Passages Or 1 yyyy Initials* No. (if known) Relevant Figures Appear

B1 EP 419251B 1-9-1993 Mitsui Toatsu Chemicals, Inc. and Machida Pharmaceutical Co., Ltd D B2 WO 97/45140 12-4-1997 Glaxo Group Limited D B3 WO 97/04801 12-13-1997 Genentech, Inc. D B4 WO 98/42376 10-1-1998 Common Services Agency D B5 WO 2000/67789 11-16-2000 CSL Limited D B6 WO 2002/013860 2-21-2002 Chugai Seiyaku Kabushiki Kaisha D B7 WO 2002/30463 4-18-2002 Genentech, Inc. D B8 WO 2002/43695 6-6-2002 Batelle Memorial Institute D B9 WO 2004/43750 6-6-2002 Batelle Memorial Institute D B10 WO 2002/051979 7-4-2002 Alpha Therapeutic Corporation D B11 WO 2002/096457 12-5-2002 Novartis AG, Novartis -Erfindungen Verwaltungsgesellschaft M.B.H. ad Genentech, Inc. D B12 WO 2003/053471 7-3-2003 Ortho-McNeil Pharmaceutical, Inc. D B13 WO 2004/001007 12-31-2003 IDEC Pharmaceuticals Corporation. D B14 WO 2004/016286 2-26-2004 Abbott Laboratories (Bermuda) Ltd. D B15 WO 2004/024 752 3-25-2004 Chugai Seiyaku Kabushiki Kaisha D B16 WO 2004/050059 6-17-2004 Elan Pharma International Ltd. D B17 WO 2004/055164 7-1-2004 Abgenix, Inc. D B18 WO 2004/066957 8-12-2004 Medimmune, Inc. D Examiner Date Signature /Bruce Hissong/ Considered 02/26/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 10293866v.1 IPR Page 1732 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned Sheet I 4 I 01 I 4 Attorney Docket Number 117813-26902

B19 WO 2004/1 02184 11-25-2004 Arizeke Pharmaceuticals, Inc. D B20 WO 2006/031560 3-23-2006 Genentech, Inc. and Novartis, AG D B21 WO 2007/003936 1-11-2007 lnsense Limited D B22 WO 2007/074880 7-5-2007 Chugai Seiyaku Kabushiki Kaisha D B23 WO 2007/095337 8-23-2007 lmclone Systems, Inc. D B24 WO 2008/015419 2-7-2008 Univ. Court of the Univesity of Dundee D B25 WO 95/03826 9-2-1995 Pasteur Merieux Serums Et Vaccins D B26 WO 98/044948 10-15-1998 Cangene Corporation D B27 WO 98/56418 12-17-1998 Genentech, Inc. D B28 WO 2006/138181 12-28-2006 Amgen Inc. D NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Examiner Cite 1 the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T' Initials No. number(s), publisher, city and/or country where published. C1 Zhao et al." Recent U.S. Patents on Protein Drug Formulation: 2000-2007" Protein Drug Formulation 2008

C2 Daugherty et al. "Formulation and Delivery Issues For Monoclonal Antibody Therapeutics," Advanced Drug Delivery Reviews 58:686-706 2006

Examiner Date Signature /Bruce Hissong/ Considered 02/26/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 10293866v.1 IPR Page 1733 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Dated: June 2, 2011 FIAdrnnir: ~inn.::::it1 lrA fnr nAhnr.:=ih I N:::inlA Ph n ' /n,:::,,hnr.:=ih I N:=inl,=,,/

Docket No.: 117813-26902 (PATENT) IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Application of: Wolfgang Fraunhofer

Attorney Docket No.: 117813-26902

Application No.: 12/325,049 Group Art Unit: 1646

Filed: November 28, 2008 Examiner: Hissong, Bruce D.

For: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

AMENDMENT AND RESPONSE TO NON-FINAL OFFICE ACTION

Dear Sir:

This communication is responsive to the Office Action dated March 2, 2011. Responsive to the Office Action, please amend the above-identified U.S. patent application as follows.

Amendments to the Specification begin on page 2 of this paper.

Amendments to the Claims begin on page 5 of this paper.

Remarks/Arguments begin on page 17 of this paper.

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AMENDMENTS TO THE SPECIFICATION

Please replace the paragraphs at page 23, line 28, through page 25, line 7 of the specification with the following amended paragraphs:

In one embodiment, the protein that is in solution is a therapeutic protein, including, but not limited to, fusion proteins and enzymes. Examples of therapeutic proteins include, but are not limited to, PULMOZYME® (Dornase alfa), REGRANEX® (Becaplermin), ACTIV ASE® (Alteplase), ALDURAZYME® (Laronidase), AMEVIVE® (Alefacept), ARANESP® (Darbepoetin alfa), Becaplermin Concentrate, BETASERON® (Interferon beta-I b ), BOTOX® (Botulinum Toxin Type A), ELITEK® (Rasburicase), ELSPAR® (Asparaginase), EPOGEN® (Epoetin alfa), ENBREL® (Etanercept), FABRAZYME® (Agalsidase beta), INFERGEN® (Interferon alfacon-1), INTRON® A (Interferon alfa-2a), KINERET® (Anakinra), MYOBLOC® (Botulinum Toxin Type B), NEULASTA® (Pegfilgrastim), NEUMEGA® (Oprelvekin), NEUPOGEN® (Filgrastim), ONTAK® (Denileukin diftitox), PEGASYS® (Peginterferon alfa-2a), PROLEUKIN® (Aldesleukin), PULMOZYME® (Dornase alfa), REBIF® (Interferon beta-la), REGRANEX® (Becaplermin), RETA VASE® (Reteplase), ROFERON®-A (Interferon alfa-2), TNKase® (Tenecteplase), and XIGRIS® (Drotrecogin alfa), ARCALYS® (Rilonacept), NPlate® (Romiplostim), MIRCERA® (methoxypolyethylene glycol-epoetin beta), CINRYZE® (Cl esterase inhibitor), ELAPRASE® (idursulfase), MYOZYME® (alglucosidase alfa), ORENCIA® (abatacept), NAGLAZYME® (galsulfase), KEPIVANCE® (palifermin) and ACTIMMUNE® (interferon gamma- I b ). The protein used in the invention may also be an antibody, or antigen-binding fragment thereof. Examples of antibodies that may be used in the invention include chimeric antibodies, non­ human antibodies, human antibodies, humanized antibodies, and domain antibodies (dAbs). In one embodiment, the antibody, or antigen-binding fragment thereof, is an anti-TNFa and/or an anti-IL- 12 antibody (e.g., it may be a dual variable domain (DVD) antibody). Other examples of antibodies, or antigen-binding fragments thereof, which may be used in the methods and compositions of the invention include, but are not limited to, 1D4.7 (anti-IL-12/IL-23 antibody; Abbott Laboratories), 2.5(E)mgl (anti-IL-18; Abbott Laboratories), 13C5.5 (anti-IL-13 antibody; Abbott Laboratories), J695 (anti-IL-12; Abbott Laboratories), Afelimomab (Fab 2 anti-TNF; Abbott

2 MEI 11765239v.1

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Laboratories), HUMIRA® (adalimumab) Abbott Laboratories), CAMPATH® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), ERBITUX® (Cetuximab), HERCEPTIN® (Trastuzumab), MYOSCINT®(Imciromab Pentetate), ProstaScint® (Capromab Pendetide), REMICADE® (lnfliximab), ReoPro® (Abciximab), RITUXAN® (Rituximab), SIMULECT® (Basiliximab), SYNAGIS® (Palivizumab), VERLUMA® (Nofetumomab), XOLAIR® (Omalizumab), ZENAPAX® (Daclizumab), ZEVALIN® (lbritumomab Tiuxetan), ORTHOCLONE OKT®3 (Muromonab-CD3), PANOREX® (Edrecolomab), MYLOTARG® (Gemtuzumab ozogamicin), golimumab (Centocor), CIMZIA® (Certolizumab pegol), SOURIS® (Eculizumab), CNTO 1275 (ustekinumab), VECTIBIX®(panitumumab), BEXXAR® (tositumomab and 1131 tositumomab), an anti-IL-17 antibody Antibody 7 as described in International Application WO 2007 /149032 (Cambridge Antibody Technology), the entire contents of which are incorporated by reference herein, the anti-IL-13 antibody CAT-354 (Cambridge Antibody Technology), the anti-human CD4 antibody CE9y4PE (IDEC-151, clenoliximab) (Biogen IDEC/GlaxoSmithKline), the anti-human CD4 antibody IDEC CE9.1/SB-210396 (keliximab) (Biogen IDEC), the anti-human CD80 antibody IDEC-114 (galiximab) (Biogen IDEC), the anti-Rabies Virus Protein antibody CR4098 (foravirumab), and the anti-human TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-2) antibody HGS-ETR2 (lexatumumab) (Human Genome Sciences, Inc.), and AV ASTIN® (bevacizumab ).

Please replace the paragraphs at page 45, line 25, through page 46, line 27 of the specification with the following amended paragraphs:

Examples of proteins that may be included in the aqueous formulation include antibodies, or antigen-binding fragments thereof. Examples of different types of antibodies, or antigen-binding fragments thereof, that may be used in the invention include, but are not limited to, a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb). In one embodiment, the antibody used in the methods and compositions of the invention is an anti-TNFa antibody, or antigen-binding portion thereof, or an anti-IL-12 antibody, or antigen binding portion thereof. Additional examples of an antibody, or antigen-binding fragment thereof, that may be used 3 MEI 11765239v.1

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in the invention includes, but is not limited to, 1D4.7 (anti-IL-12 / anti-IL-23; Abbott Laboratories), 2.5(E)mgl (anti-IL-18; Abbott Laboratories), 13C5.5 (anti-11-13; Abbott Laboratories), J695 (anti­ IL-12; Abbott Laboratories), Afelimomab (Fab 2 anti-TNF; Abbott Laboratories), HUMIRA® (adalimumab (D2E7); Abbott Laboratories), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint (Imciromab Pentetate), ProstaScint® (Capromab Pendetide), REMICADE® (lnfliximab), ReoPro® (Abciximab ), Rituxan® (Rituximab ), Simulect® (Basiliximab ), Synagis® (Palivizumab ), Verluma (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab-CD3), Panorex® (Edrecolomab), and Mylotarg® (Gemtuzumab ozogamicin) golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab) and Avastin® (bevacizumab). In one alternative, the protein is a therapeutic protein, including, but not limited to, Pulmozyme® (Dornase alfa), REGRANEX® (Becaplermin), Activase® (Alteplase), Aldurazyme® (Laronidase), Amevive® (Alefacept), Aranesp® (Darbepoetin alfa), Becaplermin Concentrate, BETASERON® (Interferon beta- I b ), BOTOX® (Botulinum Toxin Type A), Elitek® (Rasburicase), Elspar® (Asparaginase), EPOGEN® (Epoetin alfa), ENBREL® (Etanercept), Fabrazyme® (Agalsidase beta), INFERGEN® (Interferon alfacon-1), INTRON A® (Interferon alfa-2a), Kineret® (Anakinra), MYOBLOC® (Botulinum Toxin Type B), Neulasta® (Pegfilgrastim), NEUMEGA® (Oprelvekin), NEUPOGEN® (Filgrastim), Ontak® (Denileukin diftitox), PEGASYS® (Peginterferon alfa-2a), Proleukin® (Aldesleukin), Pulmozyme® (Dornase alfa), Rebif® (Interferon beta-la), REGRANEX® (Becaplermin), Retavase® (Reteplase), Roferon­ A® (Interferon alfa-2), TNKase® (Tenecteplase), and Xigris® (Drotrecogin alfa), ARCALYST® (Rilonacept), NPlate® (Romiplostim), MIRCERA® (methoxypolyethylene glycol-epoetin beta), Cinryze® (Cl esterase inhibitor), Elaprase® (idursulfase), Myozyme® (alglucosidase alfa), ORENCIA® (abatacept), Naglazyme® (galsulfase), Kepivance® (palifermin) and ACTIMMUNE® (interferon gamma-I b ).

4 MEI 11765239v.1

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AMENDMENTS TO THE CLAIMS

This listing of the claims will replace all prior versions, and listings, of claims in this application.

1. (Currently Amended) An aqueous formulation comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL a protein and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm and the antibody, or antigen-binding fragment thereof, protein has a molecular weight (Mw) greater than about 47 kDa.

2. (Currently Amended) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, protein has a Mw greater than about 57 kDa.

3. (Currently Amended) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, protein has a Mw greater than about 100 kDa.

4. (Currently Amended) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, protein has a Mw greater than about 150 kDa.

5. (Canceled)

6. (Currently Amended) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, protein has a Mw greater than about 250 kDa.

7. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 2 mS/cm.

8. (Canceled)

9. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 1 mS/cm. 5 MEI 11765239v.1

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10. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

11. (Currently Amended) An aqueous formulation comprising an antibody, or antigen-binding fragment thereof, a protein at a concentration of at least about 20 mg/mL 10 µg/mL and water, wherein the formulation has a conductivity of less than about 2 mS/cm.

12-14. (Canceled)

15. (Currently Amended) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, protein is at least about 100 mg/mL.

16. (Currently Amended) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, protein is at least about 150 mg/mL.

17. (Currently Amended) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, protein is at least about 200 mg/mL.

18. (Currently Amended) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, protein is greater than about 200 mg/mL.

19. (Canceled)

20. (Original) The formulation of claim 11 , wherein the formulation has a conductivity of less than about 0.5 mS/cm. 6 MEI 11765239v.1

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21. (Currently Amended) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, a protein at a concentration of at least about 50 mg/mL.,_ and water.,_ wherein the formulation has an osmolality of no more than about 30 mOsmol/kg.

22. (Original) The formulation of claim 21, wherein the osmolality of the formulation is no more than about 15 mOsmol/kg.

23. (Canceled)

24. (Currently Amended) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is at least about 150 mg/mL.

25. (Canceled)

26. (Currently Amended) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is greater than about 200 mg/mL.

27. (Currently Amended) An aqueous formulation comprising water and a given concentration of an antibody, or an antigen-binding fragment thereof, a protein, wherein the antibody, or an antigen-binding fragment thereof, protein has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or an antigen-binding fragment thereof, protein in a buffered solution at the same giveE: concentration.

28. ( Currently Amended) The formulation of claim 27, wherein the Dh of the protein is at least about 50% less than the Dh of the protein in phosphate buffered saline (PBS) at the same -given-concentration.

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29. (Currently Amended) The formulation of claim 28, wherein the Dh of the protein is at least about 60% less than the Dh of the protein in PBS at the same gi¥8H-concentration.

30. (Currently Amended) The formulation of claim 28, wherein the Dh of the protein is at least about 70% less than the Dh of the protein in PBS at the same givefl-concentration.

31. (Canceled)

32. (Currently Amended) The formulation of any one of claims 1, 11, 21, and 27 claim 31, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

33. (Currently Amended) The formulation of claim 1, 11, 21 or 27 M, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

34. (Currently Amended) The formulation of claim 33 M, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® (adalimumab), Campath (Alemtuzumab), CEA Scan Arcitumomab (fab fragment), ErbituJc (Cetuximab), Herceptin (Trastuzumab), Myoscint (Imciromab Pentetate), Prosta8cint (Capromab Pendetide), Remicade® (lnfliximab ), ReoPro (Abciximab), Rituxan (Rituximab), 8imulect (Basifocimab), 8ynagis (Palivizumab), Verluma (Nofetumomab), Xolair (Omalizumab), ZenapaJc (Daclizumab), Zevalin (Ibritumomab Timrntan), Orthoclone OKT3 (Muromonab CD3), PanornJc (Edrecolomab), Mylotarg (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), 8oliris (Eculizumab), and CNTO 1275 (ustekinumab), Vectibix (panitumumab), BeJocar (tositumomab and F tositumomab), and Avastin (bevacizumab).

35. (Canceled)

36. (Canceled) 8 MEI 11765239v.1

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37. (Original) An aqueous formulation comprising an antibody, or an antigen- binding fragment, at a concentration of at least about 10 mg/mL and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 5 µm.

38. (Original) The formulation of claim 37, wherein the antibody has a Dh of less than about 3 µm.

39. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

40. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

41. (Currently Amended) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® (adalimumab ), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (lmciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliximab), ReoPro® (Abciximab), Rituxan® (Rituximab), Simulect® (Basiliximab), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab­ CD3), Panorex® (Edrecolomab), and Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab) and Avastin® (bevacizumab ).

42. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, further comprising a non-ionizable excipient.

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43. (Previously Presented) The formulation of claim 42, wherein the non- ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

44. (Original) The formulation of claim 43, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

45. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

46. (Canceled)

47. (Canceled)

48. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

49. (Canceled)

50. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is suitable for in vitro or in vivo use.

51. (Canceled)

52. (Previously Presented) The formulation of claim 50, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group

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consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

53. (Withdrawn) Use of the formulation of claim 50 in the treatment of a disorder in a subject.

54. (Previously Presented) A device comprising the formulation of any one claims 1, 11, 21, 27, and 37.

55. (Canceled)

56. (Previously Presented) An article of manufacture comprising the formulation of any one of claims 1, 11, 21, 27, and 37.

57. (Currently Amended; Withdrawn) A method of preparing an aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, a protein and water, the method compnsmg: a) providing the antibody, or antigen-binding fragment thereof, protein in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved to thereby prepare the aqueous formulation.

58. (Currently Amended; Withdrawn) A method of preparing an aqueous formulation of an antibody, or an antigen-binding fragment thereof, a protein, the method comprising: a) providing the antibody, or an antigen-binding fragment thereof, protein in a first solution; b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five-fold volume exchange with the water has been achieved to thereby prepare a diafiltered antibody, or antigen-binding fragment thereof, protein solution; and

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c) concentrating the diafiltered antibody, or an antigen-binding fragment thereof, protein solution to thereby prepare the aqueous formulation of the antibody, or antigen-binding fragment thereof, protein.

59. (Currently Amended; Withdrawn) The method of claim 58, wherein the concentration of the diafiltered antibody, or antigen-binding fragment thereof, protein solution is achieved via centrifugation.

60. (Withdrawn) The method of claim 57 or 58, wherein the diafiltration medium consists of water.

61. (Withdrawn) The method of claim 57 or 58, wherein the first solution is subjected to diafiltration with water until at least about a six-fold volume exchange or at least about a seven­ fold volume exchange is achieved.

62. (Canceled)

63. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation has a final concentration of excipients which is at least about 95% less or 99% less than the first solution.

64. (Canceled)

65. (Currently Amended; Withdrawn) The method of claim 57 or 58, wherein the first antibody, or antigen-binding fragment thereof, protein solution is obtained from a mammalian cell expression system and has been purified to remove host cell proteins (HCPs).

66. (Currently Amended; Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen-binding fragment thereof, protein has a Mw greater than about 1 kDa.

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67. (Currently Amended; Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen-binding fragment thereof, protein has a molecular weight (Mw) selected from the group consisting of greater than about 10 kDa, greater than about 47 kDa, reater than about 57 kDa, greater than about 100 kDa, greater than about 150 kDa, greater than about 200 kDa, and greater than about 250 kDa.

68.-73. (Canceled)

74. (Withdrawn) The method of claim 57 or 58, further comprising adding an excipient to the aqueous formulation.

75. (Original) The method of claim 7 4, wherein the aqueous formulation is a pharmaceutical formulation.

76. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation is a pharmaceutical formulation.

77. (Original) The method of claim 76, further comprising loading the aqueous formulation into a device suitable for administering the aqueous formulation to a subject.

78. (Canceled)

79. (Canceled)

80. (Currently Amended) The method of claim 57 or 58+9, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

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81. (Currently Amended) The method of claim 57 or 58+9, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

82. (Currently Amended) The method of claim 57 or 58 +9, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira.@ (adalimumab), Campath.® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux.@ (Cetuximab), Herceptin® (Trastuzumab), Myoscint.@ (lmciromab Pentetate), ProstaScint.@ (Capromab Pendetide), Remicade.@ (lnfliximab), ReoPro.@ (Abciximab), Rituxan.@ (Rituximab), Simulect® (Basiliximab), Synagis.@ (Palivizumab), Verluma.@ (Nofetumomab), Xolair.@ (Omalizumab), Zenapax.@ (Daclizumab), Zevalin.@ (lbritumomab Tiuxetan), Orthoclone OKT3.® (Muromonab-CD3), Panorex.@ (Edrecolomab), Mylotarg.@ (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia.@ (Certolizumab pegol), Soliris.@ (Eculizumab), CNTO 1275 (ustekinumab), Vectibix.@ (panitumumab), Bexxar.@ (tositumomab and 1131 tositumomab), and Avastin.@ (bevacizumab ).

83.-85. (Canceled)

86. (Currently Amended) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, the antibody, or antigen-binding fragment thereof, prepared hy according to the methods of claim 58 or 59 a) providing the antibody, or antigen-binding fragment thereof, in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved.

87.-101. (Canceled)

102. (Previously Presented) The formulation of claim 43, wherein the polyol is mannitol or sorbitol.

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103. (New) An aqueous formulation comprising water and a given concentration of an antibody, or an antigen-binding fragment thereof, wherein the antibody, or antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50%-80% less than the Dh of the antibody, or an antigen-binding fragment thereof, in a buffered solution at the same concentration.

104. (New) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 150 mg/mL.

105. (New) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is greater than about 200 mg/mL.

106. (New) The formulation of claim 103, further comprising a non-ionizable excipient.

107. (New) The formulation of claim 106, wherein the non-ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

108. (New) The formulation of claim 107, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

109. (New) The formulation of claim 103, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

110. (New) The formulation of claim 103, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

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111. (New) The formulation of claim 103, wherein the formulation is suitable for in vitro or in vivo use.

112. (New) The formulation of claim 111, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

113. (New) Use of the formulation of claim 111 in the treatment of a disorder in a subject.

114. (New) A device comprising the formulation of claim 103.

115. (New) An article of manufacture comprising the formulation of claim 103.

116. (New) The formulation of claim 21 or 27, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® ( adalimumab ), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (lmciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliximab ), ReoPro® (Abciximab ), Rituxan® (Rituximab ), Simulect® (Basiliximab ), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab-CD3), Panorex® (Edrecolomab ), Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab), and Avastin® (bevacizumab).

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REMARKS Claims 1-4, 6, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52-54, 56, 61, 63, 65-67, 74-77, 79-84, 86, and 102 were pending in the application. Claims 13, 31, 35, 36, 79, 83, and 84 have been canceled without prejudice. Claims 1-4, 6, 11, 13, 15-18, 21, 24, 26-30, 32-34, 41, 57-59, 65-67, 80-82, and 86 have been amended and new claims 103-116 have been added. Claims 53, 57-61, 63, 65-67, 74-77, and 79-84 have been withdrawn by the Examiner as being directed to non-elected subject matter. The Examiner has also withdrawn claim 6 in view of the species election of Humira and Enbrel (see below). Accordingly, following entry of this amendment, claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52-54, 56-61, 63, 65-67, 74-77, 79-82, 85, and 102- 116 will be pending in the application.

Support for the amendments to the claims and the new claims may be found throughout the specification and claims as originally filed. Specifically, support for the amendments to claims 1, 11, 21, 27, 37, 57, 58, and 86 may be found in, for example, originally filed claims 31 and 33; support for new claim 103 may be found in, for example, originally filed claim 27 and at, for example, page 15, [0177] of the U.S. Publication, U.S. 2009/0291062; support for new claims 104- 115 may be found in, for example, originally filed claims 24, 26, 42-45, 48, 50, 52-54, and 56; and support for new claim 116 may be found in, for example, originally filed claim 34. No new matter has been added.

Any amendments to and/or cancellation of the claims are not to be construed as an acquiescence to any of the rejections set forth in the instant Office Action, and were done solely to expedite prosecution of the application. Applicants hereby reserve the right to pursue the subject matter of the claims as originally filed in this or a separate application(s).

Restriction Requirement

In the Response to Restriction Requirement dated December 16, 2010, Applicants elected, without traverse, the invention of Group I (claims 1-4, 6-7, 9-11, 13, 15-18, 20-22, 24, and 26-45, 48, 50, 52, 54, 56, and 86), drawn to aqueous solutions comprising a protein and water. Applicants

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further elected, without traverse and for search purposes only, the species of (1) Humira (adalimumab); (2) Enbrel (Etanercept); (3) Mannitol; and (4) Polysorbate 80.

Group I and Group II

The Examiner indicated that the claims of Group I and Group II are related as product and process claims. Accordingly, it is Applicants understanding that where Applicants elect claims directed to a product, and the product claims are subsequently found allowable, withdrawn process claims that depend from or otherwise require all the limitations of the allowable product claims will be considered for rejoinder. In the event of rejoinder, the requirement for restriction between the product claims and the rejoined process claims will be withdrawn, and the rejoined process claims will be fully examined for patentability in accordance with 37 C.F.R. § 1.104. See M.P.E.P. § 804.01.

As discussed below, the amendments to the claims presented herein have obviated all of the Examiner's objections and rejections. Accordingly, the claims are in condition for allowance. Therefore, since the withdrawn process claims of Group II (i.e., claims 57-61, 65-67, and 7 4-77) depend from or otherwise require all the limitations of the allowable product claims, Applicants respectfully request that the Examiner rejoin and examine claims 57-61, 65-67, and 74-77.

Group I and Group III

The Examiner also indicated, and it is Applicants' understanding that, the claims of Group I and Group III are related as product and process of use claims. Accordingly, it is Applicants understanding that where Applicants elect claims directed to a product, and the product claims are subsequently found allowable, withdrawn process of use claims that depend from or otherwise require all the limitations of the allowable product claims will be considered for rejoinder. In the event of rejoinder, the requirement for restriction between the product claims and the rejoined process of use claims will be withdrawn, and the rejoined process of use claims will be fully examined for patentability in accordance with 37 C.F.R. § 1.104. See M.P.E.P. § 804.01.

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As indicated above, the amendments to the claims presented herein have obviated all of the Examiner's objections and rejections. Accordingly, the claims are in condition for allowance. Therefore, since the withdrawn process of use claims of Group III (i.e., claim 53) depend from or otherwise require all the limitations of the allowable product claims, Applicants respectfully request that the Examiner rejoin and examine claim 53.

Species Election

It is Applicants' further understanding that the search will be extended to the remaining species upon a finding of allowability of a generic linking claim. As described in 37 C.F. R. § 1.144, it is Applicant's understanding that, upon the allowance of a generic linking claim, Applicant will be entitled to consideration of claims to additional species, i.e., non-elected species of claims 34, 36, 41, 43, 44, 82, and 84, which depend from or otherwise require all of the limitations of the allowed generic linking claim.

As indicated above, the amendments to the claims presented herein have obviated all of the Examiner's objections and rejections. Accordingly, the generic linking claims 21, 27, 37, 57, and 58 are in condition for allowance and Applicants respectfully request that the Examiner search the remaining species of claims 34, 36, 41, 43, 44, 82, and 84.

Claim Obiections

Claims 27-30

With respect to the Examiner's objection to claims 27-30, Applicants respectfully submit that the amendments to claims 27-30 based on the Examiner's suggestion have rendered this objection moot. Specifically, claims 27-30 have been amended to be directed to aqueous solutions comprising water and a given concentration of a protein, wherein the protein has a hydrodynamic diameter which is at least about 50% less (or 60% or 70% less) than the hydrodynamic diameter of the protein "in a buffered solution at the same concentration." Accordingly, Applicants respectfully request reconsideration and withdrawal of the foregoing objection. 19 MEI 11765239v.1

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Claim 86

The Examiner has objected to claim 86 "for depending from a non-elected invention" and suggests that claim 86 should be amended to include the limitations of withdrawn claims 58 and 59.

Applicants respectfully submit that the amendments to claim 86 to incorporate the limitations of claim 57 and the presentation of new claim 103 which is based on claim 86 and includes the limitations of claim 58 have rendered this objection moot. Accordingly, Applicants respectfully request reconsideration and withdrawal of the foregoing objection.

Rejection of Claims 1, 7-17, 19-22, 26-30, 91-93, and 98-100 Under 35 U.S.C. §112,

First Paragraph

The Examiner has rejected claims 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-45, 48, 50, 52, 54, 56, 86, and 102 under 35 U.S.C. § 112, first paragraph, as allegedly "containing subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention." In particular, the Examiner is of the opinion that

[a]s written, the claims encompass formulations comprising any protein. The specification has described formulations in which antibodies, such as anti-IL-12 ( adalimumab ), exhibit a conductivity of less than about 2.5 mS/cm and/or an osmolality of no more than about 30 mOSmol/kg. Additionally, the specification describes formulations comprising adalimumab wherein the antibody molecules have a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of adalimumab in PBS. However, the claims read on formulations comprising any protein, and the specification has not described any other formulations, or any other proteins, which can be formulated in such a way as to produce a formulation with a conductivity of less than about 2.5 mS/cm and/or an osmolality of no more than about 30 mOSmol/kg. The specification has also not described any proteins, other than antibodies, which would be expected to have a hydrodynamic diameter that is at least about 50% less than the hydrodynamic diameter of the protein in PBS at the given concentration when formulated in a manner which is commensurate with the instant invention. Furthermore, the claims do not 20 MEI 11765239v.1

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require the formulated proteins of the instant invention to have any biological activity, nor any particular structure. Thus, the claims are drawn to a genus of proteins which have not been described in the specification in such a way as to convey to a person of ordinary skill in the art that the inventors, at the time of invention, had possession of the full genus. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. In this case, the only factor present in the claims is a requirement that a formulation exhibiting various physical characteristics (conductivity and/or osmolality) comprise any protein. Accordingly, in the absence of sufficient distinguishing characteristics, the specification does not provide adequate written description of the claimed genus.

Applicants respectfully traverse this rejection and submit that based on the teachings in Applicants' specification and the extensive knowledge available to one of ordinary skill in the art at the time of the invention, one of ordinary skill in the art would understand that Applicants had possession of the claimed genus of proteins at the time of the invention.

For example, Applicants' specification teaches that the function of proteins suitable for use in the claimed compositions and methods is for therapeutic purposes (see, e.g., page 9, [0118] of the U.S. Publication, U.S. 2009/0291062). Applicants' specification teaches that such therapeutic proteins include fusion proteins, enzymes, and antibodies, or antigen-binding fragments thereof, such as chimeric, non-human, human, humanized, and domain antibodies. Applicants specification further provides a plethora of representative fusion proteins, enzymes, and antibodies, or antigen­ binding fragments thereof, for use in the claimed compositions and methods (see, e.g., [0119]­ [0147] and [0190] of the U.S. Publication, U.S. 2009/0291062). In addition, Applicants submit that the structure of suitable proteins for use in the claimed compositions and methods were known in the art at the time of the invention and remind the Examiner that "[a] patent need not teach, and preferably omits, what is well known in the art." See M.P.E.P. §2164.01.

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Furthermore, Applicants' specification also teaches in detail how to prepare the claimed compositions generally comprising a protein and water [see, e.g., [Ol 15]-[0116] and [0148]-[0186] of the U.S. Publication, U.S. 2009/0291062).

Applicants also respectfully point out to the Examiner that, without acquiescing to the Examiner's objection and solely in the interest of expediting prosecution of the application, claims 35 and 36 have been canceled, claims 1-4, 7, 9-11,13, 15-18, 20, 21, 22, 24, 26-34, and 86 have been amended to be directed to an antibody, or antigen-binding fragment thereof. Accordingly, Applicants submit that based on the foregoing, one of ordinary skill in the art would recognize that Applicant was in possession of the claimed antibodies and binding portions, such as anti-TNFa antibodies, or antigen-binding fragments there, based on the teachings contained within Applicant's specification and the level of knowledge available in the art.

In particular, the structural features of antibodies, including antibody-binding portions, were well known in the art at the time of Applicant's invention. Specifically, the word "antibody" denotes, for example, the basic structural unit of an antibody molecule includes two heavy chains and two light chains, wherein each heavy and light chain further includes constant and variable regions. The variable regions consist of the N-terminal regions of the heavy and light chains and come together to form the antigen-binding site of the antibody. Each light and heavy chain variable domain consists of three complement determining regions (CDRs), i.e., CDRl, CDR2, and CDR3. Thus, the term "antibody" inherently has a specific structure based on the commonly accepted definition of the word. In addition, the inherent function of such antibodies, or antigen-binding fragments thereof, is to bind to the specific target and, as discussed above, Applicants' specification teaches that such antibodies, or antigen-binding fragments thereof, are suitable for therapeutic purposes.

Moreover, in order to satisfy the Written Description requirement, the court expressly rejected the contention that the claims of the patent must be construed as being limited to a particular embodiment, even where a patent describes only a single embodiment. In Liebel­ Flarsheim Co. v. Medrad Inc. 358 F.3d 898, 906, 69 USPQ2d 1801, 1807 (Fed. Cir. 2004). Therefore, to satisfy the written description requirement, the claims, read in light of the specification, need only reasonably apprise those skilled in the art both of the utilization and scope 22 MEI 11765239v.1

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of the invention. Purdue Pharma L.P. v. Faulding Inc., 230 F.3d 1320 (Fed. Cir. 2000); Chiron Corp. v. Genentech, Inc., 363 F.3d 1247, 1253 (Fed. Cir. 2004). Even in an unpredictable art, patent applicants are not required to disclose every species encompassed by their claims. In re Vaeck, 947 F.2d 488,496 (Fed. Cir. 1991); In re Wallach, 378 F.3d 1330, 1334 (Fed. Cir. 2004). (Emphasis added). Therefore, Applicants submit that the data presented in the Examples section of the application fully supports the claimed compositions and methods.

Thus, based on the knowledge available to one of ordinary skill in the art at the time of the invention and the extensive teachings in Applicants' specification regarding the structural and functional properties of suitable antibodies, or antigen-binding fragments thereof, the requirement of 35 U.S.C. § 112, First Paragraph for Written Description has been satisfied since Applicants have provided structural and functional characteristics of the claimed antibodies, or antigen-binding fragments thereof, and enumerated numerous species falling within the claimed genus of antibodies, or antigen-binding fragments thereof.

Accordingly, Applicants respectfully request reconsideration and withdrawal of the foregoing rejection.

Rejections of Claims 34, 36, and 41 Under 35 U.S.C. §112, Second Paragraph

The Examiner has rejected claims 34, 36, and 41 under 35 U .S .C. § 112, second paragraph "as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention." In particular, the Examiner is of the opinion that "[c]laims 34, 36, and 41 recite numerous antibodies identified only by trademark/trade name." Since, according to the Examiner, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name", the identification/description is indefinite."

Applicants respectfully submit that, without acquiescing to the validity of the Examiner's rejection and solely in the interest of expediting prosecution, Applicants have canceled claim 36 and amended claims 34 and 41, thereby rendering this rejection moot. Accordingly, Applicants request that the rejection of claims 34, 36, and 41 under 35 U.S.C. § 112, second paragraph, be reconsidered and withdrawn. 23 MEI 11765239v.1

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Rejection of Claims 1-4, 7, 9-11, 13, 15-18, 20, 31-35, 42, 43, 45, 48, 50, 52, 54, 56, 86, and 102 Under 35 U.S.C. § 103(a) Konstantinov, et al. ( (US 2006/0149042) In View Of Matheus, et al.(US 2007/0172475)

The Examiner has rejected claims 1-4, 7, 9-11, 13, 15-18, 20, 31-35, 42, 43, 45, 48, 50, 52, 54, 56, 86, and 102 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475). Specifically, the Examiner is of the opinion that

Konstantinov discloses a method for concentration of proteins, wherein the resulting formulation has a conductivity of less than 2.5 mS/cm (paragraph 0013; claims 4 and 5). Specifically Konstantinov shows that a method of concentrating proteins using ultrafiltration followed by diafiltration against water can be used to lower the conductivity of the solution to less than 6 mS/cm, and preferably, from 0.5 to 5 mS/cm. Konstantinov is silent regarding formulation of a protein having a molecular weight of at least 47 kDa, or any specific concentration of protein. However, Matheus teaches formulations of antibodies and water (paragraph 0034), wherein the antibody concentration is at least 1 mg/ml, and up to 250 mg/ml (paragraph 0061). Matheus also suggests formulation of the anti-IL-12 antibody Humira (paragraph 0062), formulations comprising mannitol (paragraph 0051), and formulations intended for in vivo administration via routs of administration such as intravenous and subcutaneous (paragraph 0064). It would have been obvious to a person of ordinary skill in the art, at the time the instant invention was conceived, to create formulations comprising a protein and water and having a conductivity of less than about 2.5 mS/cm by following the combined teachings of Konstantinov and Matheus. The motivation to do so comes from the teaching of Konstantinov, which teaches an ultrafiltration/diafiltration method of concentrating protein formulations, wherein the conductivity is less than 2.5 mS/cm. Further motivation comes from Matheus, which also teaches ultrafiltration-based methods of protein concentration, wherein antibodies can be formulated at high concentrations. Therefore, it would be obvious to a person of ordinary skill in the art that to formulate the concentrated antibodies of Matheus so that the conductivity of the formulation is less than 2.5 mS/cm, as suggested by Konstantinov.

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Furthermore, although Matheus exemplifies formulations comprising anti-EGFR antibodies, Matheus suggests that Humira can also be formulated at higher concentrations (paragraph 0062), providing the motivation to formulate anti-IL-12 antibodies/Humira in such a way as to provide a formulation with a higher antibody concentration and wherein the conductivity is less than 2.5 mS/cm.

Applicants respectfully traverse this rejection for the reasons below. As set forth in M.P.E.P. §2143, "[t]o establish a primafacie case of obviousness, three basic criteria must be met. First, there must be some suggestion or motivation, either in the references themselves or in the knowledge generally available to one of ordinary skill in the art, to modify the reference or to combine the reference teachings. Second, there must be a reasonable expectation of success. Finally, the prior art reference (or references when combined) must teach or suggest all the claim limitations." Applicants respectfully submit that the Examiner has failed to establish a prima facie case of obviousness based on the combination of Konstantinov, et al. and Matheus, et al.

Claims 1 and 11, and Claims Dependent Therefrom As amended, claims 1 and 11, and claims dependent therefrom, are generally directed to formulations comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm and wherein the the antibody, or antigen binding portion thereof, has a molecular weight of greater than 47 kDa (claim 1, and claims dependent therefrom) or wherein the formulation has a conductivity of less than about 2 mS/cm and an antibody, or antigen binding portion thereof, at a concentration of at least about 20 mg/mL (claim 11, and claims dependent therefrom). Konstantinov et al. teach methods for concentrating a macromolecule, such as IL2SA (which is less than 18 kDa in weight; see US Patent Publication No. US2009/0104664, para. 0079, and US Patent No. 6,348,192; known molecular weight of IL-2 precursor is less than 18 kDa), from a solution comprising the macromolecule and an organic polymer, such as Pluronic F-68. Although Konstantinov, et al. provide a general teaching that the methods disclosed therein may be applied to macromolecules other than IL-2SA disclosed in the working examples therein (see, e.g., paragraph

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[0041] of Konstantinov, et al.), Konstantinov, et al. fail to teach or suggest antibody concentrations of at least about 20 mg/ml. While Konstantinov et al. teach that that methods disclosed therein may be used to concentrate the macromolecule by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov et al.), the working examples of Konstantinov et al. teach, for example, that the methods disclosed therein concentrate the macromolecule to achieve a concentration of macromolecule of at most 12 mg/mL (see, e.g., Example 3 at page 5 and Figure 6 of Konstantinov et al.). Accordingly, Konstantinov et al. fail to teach or suggest compositions comprising antibodies, or antigen-binding fragments thereof, at a concentration of at least 20 mg/mL. Accordingly, Konstantinov, et al. fail to teach or suggest all of the elements of claim 1 and 11, and claims dependent therefrom.

Furthermore, Applicants respectfully note that claim 10 describes an formulation having a conductivity of less than 0.5 mS/cm. In contrast, Konstantinov, et al. teach a conductivity of 0.5 to 5 mS/cm (see para. 0013).

The teachings of the secondary reference, Matheus, et al. relied on by the Examiner, fail to make up for the deficient teachings of Konstantinov, et al. In particular, Matheus et al. teach methods for the preparation of concentrated liquid formulations of an anti-EGFR antibody using tangential flow filtration and ultrafiltration (see, e.g., [0130]-[0134] of Matheus, et al.). However, Applicants submit, that one of ordinary skill would not be motivated to combine the teachings of Konstantinov et al. with Matheus et al., as one of ordinary skill would not look to the methods of Konstantinov et al. for achieving an aqueous formulation comprising a protein at a concentration of at least about 20 mg/mL, as described by the formulation of claims 1 and 11. As discussed above, Konstantinov et al. teach that that methods disclosed therein may be used to concentrate a macromolecule by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov et al.), and provide working examples which describe, for example, concentration of a macromolecule by about 25 fold to achieve a concentration of macromolecule of, at most, 12 mg/mL (see, e.g., Example 3 at page 5 and Figure 6 of Konstantinov et al.). Matheus et al. teach that it was known in the art that high protein concentration formulations were difficult to achieve, stating, for example, at paragraph [007] that "it is clear that the preparation of liquid highly concentrated antibody formulations which

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are stable for a sufficiently long time is proving to be extremely difficult for the person skilled in the art." Given the state of the art regarding high protein formulations described in Matheus et al., one of ordinary skill in the art would not look to the teachings of Konstantinov et al. for guidance, as the Konstantinov et al. reference does not teach or suggest formulations having the claimed concentration.

As such, Matheus et al. alone or in combination with Konstantinov et al. fail to teach all of the elements of the amended claims. Accordingly, Applicants respectfully request that the foregoing rejection of the claims be reconsidered and withdrawn.

Claim 86, and Claims Dependent Therefrom Claim 86, is directed to aqueous formulations comprising an antibody, or an antigen­ binding fragment thereof, the antibody, or antigen-binding fragment thereof, prepared by providing the antibody, or antigen-binding fragment thereof, in a first solution; and subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved. Konstantinov et al. are directed to methods for the concentration of a macromolecule isolated from cell culture, and, in particular, avoiding "Plurionic-induced protein precipitation" using diafiltration. Konstantinov, et al. teach that that methods disclosed therein may be used to concentrate a macromolecule by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov, et al.). However, Konstantibov, et al. fail to teach or suggest that the first solution is diafiltered using water as a diafiltration medium until at least a five-fold volume exchange with the water has been achieved, as required by claim 86. Furthermore, although Konstantibov, et al. teaches that the methods disclosed therein may be applied to macromolecules other than IL-2SA disclosed in the working examples therein (see, e.g., paragraph [0041] of Konstantinov, et al.), Konstantibov, et al. fails to teach or suggest that a suitable macromolecule is an antibody, or an antigen-binding fragment thereof, and wherein the antibody, or an antigen-binding fragment thereof Accordingly, Konstantinov, et al. fail to teach or suggest all of the elements of claim 86.

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The teachings of the secondary reference, Matheus, et al. relied on by the Examiner, fail to make up for the deficient teachings of Konstantinov, et al. In particular, Matheus et al. teach methods for the preparation of concentrated liquid formulations of an anti-EGFR antibody using tangential flow filtration and ultrafiltration (see, e.g., [0130]-[0134] of Matheus, et al.) However, Matheus, et al. fail to teach or suggest compositions prepared by diafiltering a first solution using water as a diafiltration medium until at least a five-fold volume exchange with the water has been achieved, as required by claim 86. As such, Konstantinov et al. and Matheus et al., alone or in combination, fail to teach all of the elements of the amended claims. Accordingly, Applicants respectfully request that the foregoing rejection of the claims be reconsidered and withdrawn.

Rejection of Claim 44 Under 35 U.S.C. § 103(a)

The Examiner has rejected claim 44 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475), and further in view of Schusching (U.S. Patent Application No. U.S. 2007 /0202051). Specifically, the Examiner is of the opinion that claim 44 is

further drawn to the claimed formulation, wherein said formulation further comprises a non-ionic surfactant, including polysorbates 20, 40, 60, or 80. Konstantinov and Matheus teach as described above; both are silent regarding a formulation comprising a protein and water and further comprising a non-ionic surfactant. However, Schuschnig teaches formulation of various therapeutic proteins, including antibodies (paragraphs 0118, 0124), wherein said formulations can comprise non­ ionic surfactants, including polysorbate 80 (paragraph 0140). Therefore, a person of ordinary skill in the art, at the time the instant invention was conceived, would have found it obvious to create an aqueous formulation comprising water and a therapeutic protein such as anti-IL-12 antibodieslHumira, wherein said formulation has a conductivity of less than 2.5 mS/cm, and wherein the formulation also comprises polysorbate 80. The motivation to do so comes from Konstantinov and Matheus, which as discussed above provide the motivation to create an aqueous formulation comprising an antibody such 28 MEI 11765239v.1

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as Humira, wherein the formulation has a conductivity of less than 2.5 mS/cm. Further motivation is provided by Schuschnig, which suggests formulation of agents such as antibodies compositions comprising a non­ ionic surfactant such as polysorbate 80. Thus, a skilled artisan would find it obvious to further incorporate polysorbate 80, as suggested by Schuschnig, into an aqueous formulation comprising anti-IL-12 antibodies and water, wherein the conductivity of the formulation is less than 2.5 mS/cm, as suggested by Konstantinov and Matheus.

Applicants respectfully traverse this rejection and submit that the Examiner has failed to establish a prima facie case of obviousness based on the combination of Konstantinov, et al.. Matheus, et al., and Schusching, alone or in combination. Claim 44, which depends from claims 1 and 11, is directed to formulations comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm (claim 1, and claims dependent therefrom) or wherein the formulation has a conductivity of less than about 2 mS/cm (claim 11, and claims dependent therefrom), further comprising a surfactant. As discussed above, Konstantinov et al. fail to teach or suggest all of the elements of the claimed compositions in that Konstantibov, et al. fail to teach or suggest that a suitable macromolecule for use in the methods disclosed therein is an antibody, or antigen-binding fragments thereof, let alone that such compositions have a concentration of at least 20 mg/mL. The teachings of the secondary reference, Matheus, et al. relied on by the Examiner, fail to make up for the deficient teachings of Konstantinov, et al. In particular, Applicants submit, that one of ordinary skill would not be motivated to combine the teachings of Konstantinov et al. with Matheus et al., as one of ordinary skill would not look to the methods of Konstantinov et al. for achieving an aqueous formulation comprising a protein at a concentration of at least about 20 mg/mL, as described by the formulation of claim 44. As discussed above, Konstantinov et al. teach that that methods disclosed therein may be used to concentrate a macromolecule by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov et al.), and provide working examples which describe, for example, concentration of a macromolecule by about 25 fold to achieve a concentration of macromolecule of, at most, 12 mg/mL (see, e.g., Example 3 at page 5 and Figure 6 of Konstantinov et al.). Matheus et al. teach that it was known in the art that high protein 29 MEI 11765239v.1

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concentration formulations were difficult to achieve, stating, for example, at paragraph [007] that "it is clear that the preparation of liquid highly concentrated antibody formulations which are stable for a sufficiently long time is proving to be extremely difficult for the person skilled in the art." Given the state of the art regarding high protein formulations described in Matheus et al., one of ordinary skill in the art would not look to the teachings of Konstantinov et al. for guidance, as the Konstantinov et al. reference does not teach or suggest formulations having the claimed concentration.

As such, Matheus et al. alone or in combination with Konstantinov et al. fail to teach all of the elements of the amended claims. Accordingly, Applicants respectfully request that the foregoing rejection of the claims be reconsidered and withdrawn.

Rejection of Claim 36 Under 35 U.S.C. § 103(a)

The Examiner has rejected claim 36 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475), and further in view of Giles-Komar, et al. (U.S. Patent Application No. U.S. 2003/0143603). Specifically, the Examiner is of the opinion that claim 36 is

drawn to the claimed formulation, wherein the protein in said formulation is a therapeutic protein, and specifically can be Enbrel (Etanercept). Konstantinov and Matheus teach as described above; both are silent regarding an aqueous formulation comprising water and a protein, wherein the protein is Enbrel/Etanercept. However, GilesKomar teaches that aqueous formulations comprising inhibitors oITNF-a., such as anti-TNF-a. antibodies and Etanercept, as useful for treatment of TNF-responsive disorders or conditions (paragraphs 0130, 0160,0197) Therefore, a person of ordinary skill in the art, at the time the instant invention was conceived, would have found it obvious to create an aqueous formulation comprising water and a therapeutic protein wherein said formulation has a conductivity of less than 2.5 mS/cm, and wherein the therapeutic protein is an anti-TNF-a antibody or Enbrel/Etanercept. The motivation to do so comes from Konstantinov and Matheus, which as discussed above provide the motivation to create an aqueous formulation comprising an antibody, wherein the formulation has a conductivity of less than 2.5 mS/cm. Further motivation is provided by Giles-Komar, which 30 MEI 11765239v.1

IPR Page 1763 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

teaches that aqueous formulations of TNF-a inhibitors, such as Etanercept, are useful for treating various disorders. Therefore, it would have been obvious to a person of ordinary skill in the art to substitute an anti-TNF-a antibody or Enbrel/Etanercept as the antibody in the formulation which is obvious in view of Konstantinov and Matheus.

Applicants respectfully traverse this rejection on the grounds that Konstantinov, et al., Matheus, et al., and Giles-Komar, et al., either alone or in combination, fail to teach or suggest the claimed compositions.

Nonetheless, without acquiescing to the Examiner's objection and solely in the interest of expediting prosecution of the application, claim 36 has been canceled, thereby rendering the Examiner's rejection moot. Accordingly, Applicants respectfully request reconsideration and withdrawal of the foregoing rejection.

31 MEI 11765239v.1

IPR Page 1764 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

SUMMARY

If a telephone conversation with Applicant's Attorney would expedite the prosecution of the above-identified application, the Examiner is urged to call the undersigned at (617) 449-6500.

The Commissioner is hereby authorized to charge any fees associated with the filing of this communication or any subsequent filing to our Deposit Account No. 50-4876, under Order No. 117813-26902 from which the undersigned is authorized to draw.

Dated: June 2, 2011 Respectfully submitted,

Electronic signature: / DLN /

Deborah L. Nagle, Ph.D. Registration No.: 59,978 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6500 (617) 607-9200 Attorney/Agent For Applicant

32 MEI 11765239v.1

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Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

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Customer Number: 87501

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New International A~~lication Filed with the USPTO as a Receiving Office If a new international application is being filed and the international application includes the necessary components for an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number and of the International Filing Date (Form PCT/R0/1 OS) will be issued in due course, subject to prescriptions concerning national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of the application.

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Dated: June 2, 2011 Electronic Signature for Cristin Howley Cowles, Ph.D.: /Deborah L. Nagle/

ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned

Sheet I 1 I of I 2 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Pages, Columns, Lines, Examiner Document Number Cite Publication Date Name of Patentee or Where Relevant Passages or Initials* 2 No. 1 Number-Kind Code ( if MM-DD-YYYY Applicant of Cited Relevant known) Document Fiqures Appear A1 us 20100160894 06-24-2010 Julian et al. A2 us 6,693,173 02-17-2004 Mamidi et al. A3 us 6,171,586 01-09-2001 Lam et al. A4 us 5,608,038 03-04-1997 Eibl et al. A5 us 2004-0170623 09-02-2004 Arvinte et al. A6 us 2006-0115472 06-01-2006 Li et al. A7 us 20090148406 06-11-2009 Jezek et al. A8 us 20040038878 02-26-2004 Tanikawa et al. A9 us 20050214278 09-25-2005 Kakuta et al. A10 us 200401 05889 06-03-2004 Ryde et al. A11 us 20060115472 06-01-2006 Li et al. A12 us 20060210557 09-21-2006 Luisi et al. A13 us 20080311078 12-18-2008 Gokarn et al. A14 us 20080139792 06-12-2008 Sek et al. A15 us 20080200655 08-21-2008 Sek et al. A16 us 20110054414 03-03-2011 Shanget al. A17 us 20100278822 11-04-2010 Fraunhofer et al.

IExaminer IDate ~ignature Considered

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11784332v.1 IPR Page 1769 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned Sheet I 2 I 01 I 2 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS Foreign Patent Pages, Columns, Documet Publication Name of Patentee or Lines, 3 Applicant of Cited Document Country Code - Date Where Relevant Te Examiner Cite Number4-Kind Code5 MM-DD- Passages Or 1 yyyy Initials* No. (if known) Relevant Figures Appear

B1 WO 95/03826 02-09-1995 Pasteur Merieux Serums ET Vaccins D B2 WO 98/044948 10-19-1998 Cangene Corporation D B3 WO 00/67789 11-16-2000 CSL Limited D NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Exami~er Cite 1 the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T' Initials No. number(s), publisher, city and/or country where published. C1 Li et al., "Resurrecting Abandoned Proteins with Pure Water: CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water," biophysj 91 :4201-4209 (2006)

C2 Manning et al., "Stability of Protein Pharmaceuticals," Pharm res 6:903-918 (1989)

C3 Shire et al., "Challenges in the Development of High Protein Concentration Formulations," J Pharm Sci 93:1390-1402 (2004)

C4 Tian et al., "Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations," Intl J Pharm 355:20- 31 (2007)

IExaminer IDate ~ignature Considered

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11784332v.1 IPR Page 1770 of 2482 WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 7 : (11) International Publication Number: WO 00/677891 A61K 39/395, A61P 7/00 Al (43) International Publication Date: 16 November 2000 (16.11.00) I

(21) International Application Number: PCT/AU00/00433 (81) Designated States: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG,BR,BY,CA,CH, CN,CR,CU,CZ, DE,DK,DM, (22) International Filing Date: 10 May 2000 (10.05.00) DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, L V, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, (30) Priority Data: RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, IT, TZ, PQ 0267 10 May 1999 (10.05.99) AU UA, UG, US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (71) Applicant (for all designated States except US): CSL LIMITED patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, [AU/AU]; 45 Poplar Road, Parkville, VIC 3052 (AU). IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and (75) Inventors/Applicants (for US only): COPPOLA, Germano [IT/AU]; 104 Hotham Street, Collingwood, VIC 3066 (AU). Published BERTOLINI, Joseph [AU/AU]; 4 Lae Court, Ashburton, With international search report. VIC 3147 (AU).

(74) Agent: F BRICE & CO; P.O. Box 668, Carlton South, 139 Rathdowne Street, Carlton, VIC 3053 (AU).

(54) Title: HIGH CONCENTRATION IMMUNOGLOBULIN PREPARATION AND METHOD FOR ITS PRODUCTION

(57) Abstract

The present invention provides intravenously tolerable immunoglobulin preparations which are stable over prolonged storage periods. The present invention also provides a method for the productionof a clinically acceptable immunoglobulin preparation. The method comprises the following steps: (1) obtaining an immunoglobulin solution; (2) subjecting the immunoglobulin solution to conditions which enhance the formation of immunoglobulin aggregates to form an immunoglobulin aggregate containing solution; and (3) removing the immunoglobulin aggregates from the immunoglobulin aggregate containing solution to obtain an immunoglobulin preparation substantially free of immunoglobulin aggregates.

IPR Page 1771 of 2482 FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AL Albania ES Spain LS Lesotho SI Slovenia AM Armenia FI Finland LT Lithuania SK Slovakia AT Austria FR France LU Luxembourg SN Senegal AU Australia GA Gabon LV Latvia sz Swaziland AZ Azerbaijan GB United Kingdom MC Monaco TD Chad BA Bosnia and Herzegovina GE Georgia MD Republic of Moldova TG Togo BB Barbados GH Ghana MG Madagascar TJ Tajikistan BE Belgium GN Guinea MK The former Yugoslav TM Turkmenistan BF Burkina Faso GR Greece Republic of Macedonia TR Turkey BG Bulgaria HU Hungary ML Mali TT Trinidad and Tobago BJ Benin IE Ireland MN Mongolia UA Ukraine BR Brazil IL Israel MR Mauritania VG Uganda BY Belarus IS Iceland MW Malawi us United States of America CA Canada IT Italy MX Mexico uz Uzbekistan CF Central African Republic JP Japan NE Niger VN Viet Nam CG Congo KE Kenya NL Netherlands YU Yugoslavia CH Switzerland KG Kyrgyzstan NO Norway zw Zimbabwe CI Cilte d'Ivoire KP Democratic People's NZ New Zealand CM Cameroon Republic of Korea PL Poland CN China KR Republic of Korea PT Portugal cu Cuba KZ Kazakstan RO Romania CZ Czech Republic LC Saint Lucia RU Russian Federation DE Germany LI Liechtenstein SD Sudan DK Denmark LK Sri Lanka SE Sweden EE Estonia LR Liberia SG Singapore

IPR Page 1772 of 2482 W000/67789 PCT/AU00/00433

1

High Concentration Immunoglobulin Preparation and Method for its Production

FIEW OF INVENTION 5 The present invention relates to a stable, high concentration immunoglobulin preparation and to a method of producing such an immunoglobulin preparation.

BACKGROUND OF THE INVENTION 10 Human plasma has long been a source of therapeutic proteins for use as replacement therapy for individuals suffering from various haematological conditions. For example, a deficiency in immunoglobulins results in a condition known as agammaglobulinaemia which results in a predisposition by the sufferer to infections. This condition may be treated by the 15 administration of IgG purified from the pooled plasma of healthy individuals. A shortcoming of this approach to therapy is that the irnmunoglobulin preparation must be relatively free of polymeric immunoglobulin species such as aggregates which following administration may induce severe adverse reactions. 20 It has been observed that irnmunoglobulin aggregates result from the use of destabilizing conditions during the manufacture of the plasma dedved IgG product. These protein destabilizing conditions include the use of ethanol to isolate the product and heat to virally inactivate the isolated IgG. Once unfolded a subpopulation of the IgG molecules may refold to form 25 various polymeric species including trimers, tetramers and aggregates. The formation of aggregated species appears to be cumulative with time. Thus it is imperative that IgG species which have aggregated or are prone to aggregate are removed prior to dispensing. This removal of aggregates typically occurs just prior to formulation so that aggregates formed 30 throughout the process are removed. The present inventors have now surprisingly found that if an immunoglobulin solution is exposed to conditions to deliberately enhance the formation of aggregates, and the aggregates are then removed, an irnmunoglobulin preparation is obtained which is stable at high 35 concentrations for prolonged periods. The present inventors have also made the observation that this clarification of the immunoglobulin solution to

IPR Page 1773 of 2482 W000/67789 PCT/AU00/00433

2

remove aggregates increases the stability and thus the clinical acceptability of the product. In fact the immunoglobulin solution prepared by this method is sufficiently stable that it may be formulated into a product which is room temperature stable and capable of being administered intramuscularly, 5 intravenously or subcutaneously.

SUMMARY OF THE INVENTION Accordingly, in a first aspect the present invention consists in a clinically acceptable immunoglobulin preparation, the preparation 10 comprising about 5% to about 25% (w/v) immunoglobulin and an osmolality agent and /or a stabilizer and having a pH of about 5.0 to about 8.0, wherein the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 2 7°C for 3 months is less than 2% (w/v). 15 In a second aspect the present invention consists in a clinically acceptable immunoglobulin preparation, the preparation comprising about 5% to about 25% (w/v) immunoglobulin and an osmolality agent and /or a stabilizer and having a pH of about 4.0 to about 8.0, wherein the concentration of immunoglobulin aggregates present in the immunoglobulin 20 preparation after storage at 4°C for 3 months is less than 1% (w/v). In a preferred embodiment the concentration of immunoglobulin is about 10% to about 25% (w/v), preferably about 20% (w/v) or about 25% (w/v). It is preferred that the pH of the immunoglobulin preparation is about 25 5.0 to about 6.0, more preferably about 5.5. This is particularly so when the immunoglobulin preparation is to be stored for prolonged periods at room temperature, eg 27°C. The preparation of the present invention will include an osmolality agent such that the osmolality of preparation is suitable for administration. 30 This osmolality agent may also be a compound which stabilizes the immunoglobulin. Stabilizers are well known in the art and include saccharides, such as sucrose, maltose and glucose, sugar alcohols, such as sorbitol and mannitol, and amino acids. In a preferred embodiment of the present invention the preparation 35 further comprises glycine.

IPR Page 1774 of 2482 W000/67789 PCTI AU00/00433

3

In other preferred embodiments the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 27°C for 6 months is less than 2% (w/v), and more preferably the concentration of immunoglobulin aggregates present in the immunoglobulin 5 preparation after storage at 27°C for 9 months is less than 2% (w/v). It is also preferred that the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 27°C for 6 months is less than 1% (w/v). In other preferred embodiments the concentration of 10 immunoglobulin aggregates present in the immunoglobulin preparation after storage at 4°C for 6 months is less than 1% (w/v), and preferably the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 4°C for 9 months is less than 1% (w/v). In a third aspect the present invention consists in a method for the 15 production of a clinically acceptable immunoglobulin preparation, the method comprising the following steps;- (1) obtaining an immunoglobulin solution; (2) subjecting the immunoglobulin solution to conditions which enhance the formation of immunoglobulin aggregates to form an 20 immunoglobulin aggregate containing solution; and (3) removing the immunoglobulin aggregates from the immunoglobulin aggregate containing solution to obtain an immunoglobulin preparation substantially free of immunoglobulin aggregates. 25 By "enhance the formation of immunoglobulin aggregates" we mean the inclusion or modification of a step in the production of an immunoglobulin preparation to deliberately cause or promote the formation of immunoglobulin aggregates. In a preferred embodiment step (2) comprises incubating the 30 immunoglobulin solution at a pH of about 5.8 to about 8.0 at temperature of about 4° to about 27°C for at least about 6 hours. It is preferred that the immunoglobulin solution is incubated at a pH of about 6.8 for about 12 hours. In a further preferred embodiment the aggregates are removed from 35 the aggregate containing solution by filtration, precipitation or size exclusion chromatography.

IPR Page 1775 of 2482 W000/67789 PCT/AU00/00433

4

In one preferred embodiment the aggregates are removed from the aggregate containing solution by precipitation using a precipitation enhancing agent. A preferred precipitation enhancing agent is polyethylene glycol. Preferably the precipitation is performed at a protein concentration of 5 about 2 to about 10% (w/v) and a polyethylene glycol concentration of about 6 to about 12% w/w and at a pH of about 6 to about 8. As will be understood by those skilled in this field numerous conditions can be used to promote aggregation. These include manipulation of pH together with incubation for various periods of time at various 10 temperatures. In addition incubation at elevated temperatures, eg 37°C to 42°C, and/or increasing ionic strength can be used to cause aggregate formation. The aggregates formed may be removed using any of a number of well known techniques, however it is presently preferred that the aggregates 15 are removed by filtration or precipitation, optionally using a precipitation enhancing agent such as PEG. For example the aggregates may be removed by membrane filtration using a filter of exclusion limits greater than 300,000 kDa. Alternatively the aggregates may be removed using size exclusion chromatography eg using a 20 resin of exclusion limits of 10,000 - 1,5000,000. It may be advantageous to remove the aggregates by membrane filtration in which the membrane filtration serves to remove viruses. It is believed that the pres£:nt invention will also have applicability for a range of protein preparations other than immunoglobulins, where the 25 removal of protein aggregates is desirable. As will be understood by those working in this field the preparation of the present invention may be administered intravenously, intramuscularly or subcutaneously. Throughout this specification the word "comprise", or variations such 30 as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

IPR Page 1776 of 2482 WO 00/67789 PCT/AU00/00433

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DETAILED DESCRIPTION In order that the nature of the present invention may be more readily understood preferred forms thereof will now be described with reference to the following non-limiting examples. 5 Example 1 Stabilitvof10% IgG preparations following aggregate removal bv membrane filtra lion

Starting material for this experiment was an IgG preparation 10 produced by a combination of Cohn fractionation and chromatographic methods. The preparation had been pasteurised in the presence of sucrose, followed by diafiltration against water, the final protein concentration being approximately 2% (w/v). The solution was then adjusted to pH 6.8 and maintained at this pH for 12hrs. Prior to membrane passage the solution was 15 adjusted to pH 4.2. The membrane was a Millipore PLCXK virus filter 2 membrane (surface area= 0.1 m ). Five hundred and fifty millilitres of the IgG preparation was processed through the filter under the following conditions: transmembrane pressure = 4.5 psi, cross flow rate = 500 mL/min. Water was added to the retentate pool at a rate of 40 mL/min to 20 maintain a constant protein concentration level in the permeate. At the conclusion of the run water was added to the retentate in 500 mL volumes to increase IgG recovery across the membrane. Samples of the starting material, final permeate pool, and final retentate pool were analysed by high performance liquid chromatography 25 (HPLC) for determination of aggregate content (Table 1.)

Table 1. Determination of monomer, dimer, and aggregate content of IgG before and after ultrafiltration across a PLCXK 1,000 kDa membrane.

Sample aggregate Dimer monomer

(%) (%) (%)

Starting material 0.6 4.9 93.3

Final rntentale 0.8 4.4 94.4

Pernieale pool 0.0 2.0 97.4 30 The process has thus resulted in complete removal of aggregate IgG.

IPR Page 1777 of 2482 WO 00/67789 PCT/AU00/00433

6

The filtered material was then diafiltered against PFW and concentrated to 10% w/v using a Amicon 30kDa membrane. Following concentration the solution was formulated to 10% w/v IgG containing 0.2 M glycine pH 5.5. Formulated samples were then incubated at various 5 temperatures and stability was assessed over time by comparing protein composition with that of non-filtered solutions (Table 2).

Table 2 Stability of 10% IgG solutions formulated to pH 5.5 following filtration to remove aggregates 10

Protein Time Time Time Time Time

Cmnposition o Month 4°C 1 Month4°C 9 Months 4°C 1 Month 27°C 9 Months 27°C

F* C** F C F C F C F C

Aggregate 0.0 0.3 0.0 0.3 0.1 1.1 0.2 1.76 1.2 4.8

Dimer 3.8 7.2 4.8 7.8 6.4 9.4 4.5 7.7 5.5 6.2

Monome1· 93.6 90.9 93.8 90.9 90.9 88.1 93.4 89.3 90.7 87.4

Fragments 2.6 1.6 1.7 0.9 2.5 1.4 1.0 1.2 2.6 1.7

F* Filtered material

C** Non-Filtered material

From Table 2 it can be seen that the filtered solution when compared 15 to the non-h·eated solution remains stable even at higher temperatures.

Example 2 Stability of16% IgG prepara lions following aggregate removal bv 111embrane .iltra tion

20 A similar procedure as described in Example 1 was used to prepare an aggregate depleted IgG solution. The solution was then concentrated and formulated to 16% w/v containing 0.25 Glycine pH 4.25 or 5.5. Samples of the final product were placed on stability trial and aggregate content determined (Tables 3a -3d.). 25

IPR Page 1778 of 2482 WO 00/67789 PCT/AU00/00433

7

Table 3a: 16% IgG, pH 4.25, 4 °C

Stora ~e Time (Months) 0 1 3 4 5 6 8 Protein Aggregate 0.1 0.1 0.1 0.1 0.2 0.1 0.1 Composition Dimer 3.3 2.7 2.6 2.0 4.0 2.6 2.9 tv!onomer 95.0 95.6 96.0 94.8 94.4 96.0 95.5 Fragments 1.8 1.6 1.5 3.2 1.5 1.4 1.6

Table 3b: 16% IgG, pH 4.25, 27 °C 5 Storage Time (Months) 0 1 3 4 5 6 8 Protein Aggregate 0.1 1.6 2.2 3.1 4.9 5.1 9.1 Composition Dimer 3.3 3.1 3.2 3.3 4.3 4.1 4.8 :tvionomer 95.0 93.4 91.3 88.9 86.9 86.9 81.3 Fragments 1.8 1.9 3.4 4.7 3.8 3.9 4.8

Table 3c: 16% IgG, pH 5.5, 4 °C

Storai e Time (Months)

0 1 3 4 5 6 8 Protein Aggregate 0.1 0.2 0.3 0.2 0.5 0.3 0.5 Composition Dimer 6.2 7.1 7.5 6.4 9.3 6.1 7.8 lvlonomer 90.6 91.1 90.7 90.0 88.8 92.2 90.3 Fragments 3.1 1.5 1.4 3.3 1.3 1.4 1.4

10 Table 3d: 16% IgG, pH 5.5, 27 °C

Storai e Time (Months) 0 1 3 4 5 6 8 Protein Aggregate 0.1 0.3 0.4 0.5 0.5 0.6 0.6 Composition Dimer 6.2 5.4 5.3 4.6 5.6 4.8 4.9 Monomer 90.5 92.5 91.9 91.5 91.B 92. 91.8 Fragments 3.1 1.7 2.3 3.3 2.0 2.2 2.6

IPR Page 1779 of 2482 WO 00/67789 PCT/A U00/00433

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Example 3 Stabilitvof16 % IgG preparations folloudngaggregate removal bv PEG precipita lion

Starting material for this experiment was an IgG preparation 5 produced by a combination of Cohn fractionation and chromatographic methods. The preparation had been pasteurised in the presence of sucrose and formulated for low pH incubation by concentrating to 5% w/v, adjusting the pH to 4.2 and incubating at 27°C for 14 days. Following incubation the material was adjusted to pH 6.8 and maintained at this pH for 12hrs. PEG 10 1000 was then added until a final concentration of 10% w/w PEG was achieved. The material was then left to settle for 1 hour and centrifuged. The supernatant was removed and analysed for aggregate content (Table 4).

Table 4. Determination of monomer, dimer, and aggregate contentoflgG 15 before and after PEG treatment.

Sample Aggregate dimer Monomer (%) (%) (%) Starting material pH 6.8 1.1 6.6 91.9 PEG precipitated material 0.0 6.3 92.0

The supernatant was then diafiltered against 8 exchanges of PFW to remove residual PEG. Following diafiltration the IgG solution was 20 concentrated and formulated to 16% w/v containing 0.25M glycine, pH 4.25, 5.5 or 6.8. The material was then dispensed and placed on stability trials where it was analysed for aggregate content (Table 5a and 5f).

IPR Page 1780 of 2482 C C C e ~ "t1 e = ("} ...:a ~ ~ \C> QO ...:a 0 ..0 following following 4.25 4.25 Months Months pH pH 3.65 19.37 60.22 8 Control Control 8 4.43 0.61 w/v, w/v, Months Montl1s 16% 16% 2.16 Control 4 2.03 0.15 97.69 93.85 4 10.22 86.25 Control IgG IgG Month Montl1 1 Control 4.28 0.42 95.30 4.50 1 88.79 Control 6.71 treated treated O PEG PEG 2.52 Control 97.33 2.52 0.14 TimeO 97.33 0.14 Control Time of of content content Months Months 3.44 95.22 8 PEG Treated 0.1 14.89 4.01 64.03 8 PEG aggregate aggregate Months Months 2.09 4 0.0 PEG Treated 2.10 86.99 4 9.65 PEG and and Month Month 1 3.01 PEG 0.12 96.87 97.91 Treated 1 3.84 89.71 6.44 PEG dimer, dimer, 0 1.63 98.37 0.01 Time 1.63 98.37 0.01 TimeO monomer, monomer, of of Protein Dimer Monomer Composition A12:cregate Dimer Composition Protein Monomer Am~regate Determination Determination 4°C. 27°C. at at 5a. 5b. storage Table storage Table

IPR Page 1781 of 2482 Q ~ tH Q ti c ~ > '"d n -.J -.J QO "° ~ ~ Q 0 ..... 0 following following 5.5 5.5 Months Months pH pH 7.05 Control 1.93 7.21 85.01 8 8 Control 5.94 89.95 w/v, w/v, Months Months 16% 16% 4 0.94 6.71 4 Control 5.87 89.77 4.36 lg(; IgG Month Month 7.32 1 1.46 Control Control 91.21 92.35 1 Control 89.71 4.50 6.71 treated treated 0 PEG PEG 3.56 TimeO Control 96.18 3.56 96.18 Conll'ol Time 0.26 of of content content Months Months 8 0.15 0.26 Treated 92.26 6.58 8.11 88.94 8 0.99 aggregate aggregate Months Months 4 0.12 PEG PEG Treated 94.04 5.83 PEG PEG 4 94.08 5.57 0.35 and and Month Month 1 0.21 94.32 5.47 PEG Treated 93.84 5.82 PEG 0.34 dimer, dimer, 0 1 0 2.01 97.93 2.01 Time 0.06 0.06 Time 97.93 monomer, monomer, of of Protein Composition Monomer Dimer Aggregate Protein Composition Agg-regate Dimer Monomer Determination Determination 27°C. 4°C. at at 5d. storage Table 5c. storage Table

IPR Page 1782 of 2482 !,;,I ~ ~ <:) = ~ "Cl e ~ ....:a ~ ~ <:) ...... following following 6.8 6.8 Months Months pH 7.22 75.07 3.84 8 0.15 Control Control 10.18 84.97 8 pH w/v, w/v, Months 16% 16% 4Months 4.06 Control 10.56 85.37 4 0.82 91.24 Control 6.58 IgG lg(,. Month Month 1 4.12 Control 86.18 9.69 7.35 1 Control 6.33 86.22 treated treated PEG PEG 1.58 TimeO 91.84 Control 6.58 1.58 Conh·ol 91.84 TimeO 6.58 of of content Months content Months 8 0.21 PEG 89.86 8.89 71.77 0.39 8 PEG 6.73 aggregate aggregate Months Months 4 90.55 9.25 PEG 4 0.50 n.23 ' I and and Month Month 1 0.26 0.20 91.32 PEG Treated Treated Treated 8.42 7.84 1 0.66 91.12 90.79 PEG PEG dimer, dimer, TimeO 0.0 0.0 94.78 5.22 TimcO 94.78 5.22 monomer, monomer, of of Protein Composition Aggregate Protein Monomer Dimer Composition Ae:irregate Dimer Monomer Determination 4°C. 27°C. Determination at at 5e. 5f. storage Table storage Table

IPR Page 1783 of 2482 WO 00/67789 PCT/AU00/00433

12

Example 4 Stabi1itvof10% IgG preparations follouingaggregate removal bv PEG precipita lion

A similar procedure as described in example 3 was used to prepare the 5 PEG treated IgG solution. Following aggregate removal the IgG solution was concentrated and formulated to 10% protein w/v containing 0.2M glycine, pH 4.25 or 5.5. The material was then dispensed and placed on stability trials where it was analysed for aggregate content. The results obtained are set out in Table 6a & b. 10 Table 6a Determination of mono:rrBr, dimer, and aggregate content of PEG treated IgG 10% w/v, pH 4.25 following storage at 4° and 27°C.

Storage Conditions ~gregate% Dimer% Monomer% 1 Month PEG Treated 4°C 0.18 3.33 96.49 4 Months PEG Treated 4°C 0.38 4.33 95.30 8 Months PEG Treated 4°C 0.17 5.29 94.54 12 Months PEG Treated 4°C 0.17 5.29 93.70 1 Month PEG Treated 27°C 1.01 4.01 94.98 4 Months PEG Treated 27°C 9.55 4.11 83.16 8 Months PEG Treated 27°C 13.74 4.66 63.92 12 Months PEG Treated 27°C 17.62 5.18 54.55

15 Table 6b Determination of monomer, dimer, and aggregate content of PEG treated IgG 10% w/v, pH 5.5 following storage at 4° and 27°C.

Storage Conditions A!!!!regate % Dimer% Monomer% 1 Month PEG Treated 4°C 0.21 8.73 91.07 4 Months PEG Treated 4°C 0.35 9.36 90.26 8 Months PEG Treated 4°C 0.29 12.21 87.49 12 Months PEG Treated 4°C 0.53 11.81 86.99 1 Month PEG Treated 27°C 0.41 9.54 90.06 4 Months PEG Treated 27°C 0.63 10.14 89.23 8 Months PEG Treated 27°C 0.88 10.43 87.34 12 Months PEG Treated 27°C 1.09 11.20 85.79

IPR Page 1784 of 2482 W000/67789 PCT/A U00/00433

13

Example 5 Stabilitvof20 and 25% IgG preparations following aggregate removal bvPEG predpita tion

5 A similar procedure as described in example 3 was used to prepare the PEG treated IgG solution. Following aggregate removal the IgG solution was concentrated and formulated to 20 and 25% protein w/v containing 0.3M glycine, pH 5.5. The material was then dispensed and placed on stability trials where it was analysed for aggregate content (Table 7a & 7b). 10 From Tables 5a - 7b it can be seen that the PEG treated material when compared to the non-treated solutions have significantly lower aggregate contents even after storage at higher temperatures.

15 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 20

IPR Page 1785 of 2482 ~ ~ ri "'O = i ~ ...... ~ ~ \C 00 .pa .... 37°c 4.03 Control 3.88 37°C 6.0 89.2 88.95 5.89 following following 5.5 5.5 32°c 3.32 6.30 :n°C 2.99 Control Control Control 6.44 89.74 89.50 pH pH w/v, w/v, 25% 20% 27°C 27°C 3.12 2.87 Control Control 90.24 6.65 89.43 6.17 IgG IgG 7.33 7.60 1.99 4°c Control 90.14 1.65 4°C Control 90.10 treated treated 0 O PEG PEG 1.36 Control 92.23 92.21 1.18 6.43 Time Control 6.59 Time of of content content 37°C 7.57 37°C 1.20 8.97 88.53 PEG Treated 0.78 90.5 Treated 32°c 32°c 8.40 PEG PEG 0.07 90.55 8.13 88.84 Treated 0.40 Treated aggregate aggregate and and 27°C 27°C 0.43 9.44 PEG Treated 0.33 90.4 8.33 89.19 PEG PEG Treated dimer, dimer, month. month. 9.67 4°C 4°c 0.30 90.24 8.74 89.16 PEG Treated 0.24 PEG Treated 1 1 for for monomer, monomer, of of PEG PEG O O 37°C 37°C & & 7.98 7.13 0.19 91.84 Time Treated 0.17 92.7 Time Treated 32° 32° 27°, 27°, Determination Determination lion 4°, 4°, at at 7a. 7b. Dimer Composition Aggregate Protein Monomer Composi Protein Dimer Monomer A1.m.regate storage storage Table Table

IPR Page 1786 of 2482 W000/67789 PCT/AU00/00433

15

CLAIMS:- 1. A clinically acceptable immunoglobulin preparation, the preparation comprising about 5% to about 25% (w/v) immunoglobulin and an osmolality agent and /or a stabilizer and having a pH of about 5.0 to about 8.0, wherein 5 the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 27°C for 3 months is less than 2% (w/v). 2. An immunoglobulin preparation as claimed in claim 1 in which the concentration of immunoglobulin is about 10% to about 25% (w/v). 10 3. An immunoglobulin preparation as claimed in claim 1 in which the concentration of immunoglobulin is about 20% (w/v). 4. An immunoglobulin preparation as claimed in claim 1 in which the concentration of immunoglobulin is about 25% (w/v). 5. An immunoglobulin preparation as claimed in any one of claims 1 to 15 4 in which the preparation has a pH of about 5.0 to about 6.0. 6. An immunoglobulin preparation as claimed in claim 5 in which the preparation has a pH of about 5.5. 7. An immunoglobulin preparation as claimed in claim 5 or claim 6 in which the concentration of immunoglobulin aggregates present in the 20 immunoglobulin preparation after storage at 27°C for 6 months is less than 2% (w/v). 8. An immunoglobulin preparation as claimed in anv one of claims 5 to 7 in which the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 27°C for 9 months is less than 25 2% (w/v). 9. An immunoglobulin preparation as claimed in any one of claims 5 to 6 in which the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 2 7°C for 6 months is less than 1% (w/v). 30 10. An immunoglobulin preparation as claimed in any one of claims 1 to 9 in which the preparation further comprises glycine. 11. A clinically acceptable immunoglobulin preparation, the preparation comprising about 5% to about 25% (w/v) immunoglobulin and an osmolality agent and /or a stabilizer and having a pH of about 4.0 to about 8.0, wherein 35 the concentration of immunoglobulin aggregates present in the

IPR Page 1787 of 2482 W000/67789 PCT/AU00/00433

16

immunoglobulin preparation after storage at 4°C for 3 months is less than 1 % (w/v). 12. An immunoglobulin preparation as claimed in claim 11 in which the concentration of immunoglobulin is about 10% to about 25% (w/v). 5 13. An immunoglobulin preparation as claimed in claim 11 in which the concentration of immunoglobulin is about 20% (w/v). 14. An immunoglobulin preparation as claimed in claim 11 in which the concentration of immunoglobulin is about 25% (w/v). 15. An immunoglobulin preparation as claimed in any one of claims 11 10 to 14 in which the preparation has a pH of about 5.0 to about 6.0. 16. An immunoglobulin preparation as claimed in claim 15 in which the preparation has a pH of about 5.5. 17. An immunoglobulin preparation as claimed in any one of claims 11 to 16 in which the preparation further comprises glycine. 15 18. An immunoglobulin preparation as claimed in any one of claims 11 to 17 in which the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 4°C for 6 months is less than 1% (w/v). 19. An immunoglobulin preparation as claimed in any one of claims 11 20 to 18 in which the concentration of immunoglobulin aggregates present in the immunoglobulin preparation after storage at 4°C for 9 months is less than 1% (w/v). 20. A method for the production of a clinically acceptable immunoglobulin preparation, the method comprising the following steps;- 25 (1) obtaining an immunoglobulin solution; (2) subjecting the immunoglobulin solution to conditions which enhance the formation of immunoglobulin aggregates to form an immunoglobulin aggregate containing solution; and (3) removing the immunoglobulin aggregates from the 30 immunoglobulin aggregate containing solution to obtain an immunoglobulin preparation substantially free of immunoglobulin aggregates. 21. A method as claimed in claim 20 in which step (2) comprises incubating the immunoglobulin solution at a pH of about 5.8 to about 8.0 at 35 temperature of about 4° to about 27°C for at least about 6 hours.

Substitute Sheet (Rule 26) RO/AU IPR Page 1788 of 2482 W000/67789 PCT/AU00/00433

17

22. A method as claimed in claim 20 in which the immunoglobulin solution is incubated at a pH of about 6.8 for about 12 hours. 23. A method as claimed in any one of claims 20 to 22 in which the aggregates are removed from the aggregate containing solution by filtration, 5 precipitation or size exclusion chromatography. 24. A method as claimed in claim 23 in which the aggregates are removed from the aggregate containing solution by precipitation using a precipitation enhancing agent. 25. A method as claimed in claim 24 in which the precipitation 10 enhancing agent is polyethylene glycol. 26. A method as claimed in claim 25 in which the precipitation is performed at a protein concentration of about 2 to about 10% (w/v) and a polyethylene glycol concentration of about 6 to about 12% w/w and at a pH of about 6 to about 8.

Substitute Sheet (Rule 26) RO/AU IPR Page 1789 of 2482 INTERNATIONAL SEARCH REPORT International application No. PCT/AU00/00433 A CLASSIFICATION OF SUBJECT MATTER

7 Int. Cl. : A61K 039/395; A61P 007/00

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED

Minimum documentation searched ( classification system followed by classification symbols) IPC: A6IK, SEARCH TERMS AS BELOW

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched AU: IPC AS ABOVE

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) WPAT, CA ONLINE (STN): immunoglobulin AND (stab: OR concentrat: OR storage OR pH) AND (haemato: OR blood OR plasma) AND immunoglobulin()aggregat:

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

AU-A-34297/97 (CSL LIMITED) 29 January 1998 X See whole document 1, 5-9, 11, 15-16, 18- 19

US 4,845,199A (HIRAO ET AL.) 4 July 1989 X See whole document 1-9, 11-16, 18-19

US 4,597,966A (ZOLTON ET AL.) 1 July 1986 X See whole document 1, 5, 7-9, 11, 15, 18- 19

[Kl Further documents are listed in the continuation of Box C [K] See patent family annex

* Special categories of cited documents: "T" later document published after the international filing date or nA'' document defining the general state of the art which is priority date and not in conflict with the application but cited to not considered to be of particular relevance understand the principle or theory underlying the invention "E" earlier application or patent but published on or after "X" document of particular relevance; the claimed invention cannot the international filing date be considered novel or cannot be considered to involve an "L., document which may throw doubts on priority claim( s) inventive step when the docmnent is taken alone or which is cited to establish the publication date of nyu document of particular relevance; the claimed invention cannot another citation or other special reason (as specified) be considered to involve an inventive step when the docmnent is "O" docmnent referring to an oral disclosure, use, combined with one or more other such documents, such exhibition or other means combination being obvious to a person skilled in the art "P" docmnent published prior to the international filing "&" document member of the same patent family date but later than the nrioritv date claimed Date of the actual completion of the international search Date of mailing of the international search report 26 June 2000 0 7 JUL 2000 Name and mailing address of the ISA/AU AUSTRALIAN PATENT OFFICE PO BOX 200, WODEN ACT 2606, AUSTRALIA ~ E-mail address: [email protected] MICHAEL GRIEVE Facsimile No. (02) 6285 3929 Telephone No : (02) 6283 2267

Form PCT/ISA/210 (second sheet) (July 1998)

IPR Page 1790 of 2482 INTERNATIONAL SEARCH REPORT International application No. PCT/AU00/00433 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. US 4,880,9I3A (DOLESCHEL ET AL.) 14 November 1989 X See whole document 1, 20, 19-24

EP 0123029A (SCHWAB & CO. GES.M.B.H.) 31 October 1984 X See whole document 20, 19, 21-22

WO 99/64462A (STATENS SERUM INSTITUT) 16 December 1999 P,X See whole document 20, 19, 21-22

Form PCT/ISA/210 (continuation of Box C) (July 1998)

IPR Page 1791 of 2482 INTERNATIONAL SEARCH REPORT International application No. Infonnation on patent family members PCT/AU00/00433

This Annex lists the known "A" publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document Cited in Search Patent Family Member Report

AU 34297/97 WO 9803550 EP 954534 CA 2261291 us 4845199 CA 1310267 EP 253313 JP 63146832 us 4597966 AU 51892/86 CA 1285225 EP 187712 JP 61218528 us 4880913 DE 3641115 EP 270025 EP 123029 AT 2126/83 DE 3310150 JP 59176215 WO 9964462 AU 42572/99

END OF ANNEX

Form PCT/ISA/210 (citation family annex) (July 1998)

IPR Page 1792 of 2482 ORGANISATION MONDIALE DE LA PROPRIETE INTEILECTUELLE PCT Bureau international DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU 'IRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT')

(51) Classification internationale des brevets 6 : (11) Numero de publication internationale: WO 95/03826 A61K 39/395 Al (43) Date de publication intemationale: 9 fevrier 1995 (09.02.95)

(21) Numero de la demande internationale: PCT/FR94/00955 (81) Etats designes: AU, CA, JP, US, brevet europeen (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, (22) Date de depot international: 28 juillet 1994 (28.07.94) SE).

(30) Donnees relatives a la priorite: Pobliee 93/()()416 30 juillet 1993 (30.07.93) FR Avec rappo11 de recherche internationale. Avant l'expiration du delai prevu pour la modification des revendications, sera republiee side telles modifications sont (71) Depognt (pour tousles Etats designes sauf US): PAS1EUR ref:ues. MERIEUX SERUMS ET VACCINS [FR/FR]; 58, avenue Leclerc, F-69007 Lyon (FR).

(72) Inventeurs; et (75) Inventeurs/Deposants (US seulement): GRANDGEORGE, Michel, Gaston, Joseph [FR/FR]; 12, rue du Recret, F-69670 Vaugneray (FR). GATI'EL, Paule, Annie [FR/FR]; 99, route de Strasbourg, F-69300 Caluire (FR). MAKULA, Marie-France, Marguerite, Andree [FR/FR]; 18D, rue de Tourvielle, F-69005 Lyon (FR).

(74) Mandataires: BERNASCONI, Jean etc.; Cabinet Bernasconi et Vigier, 13, boulevard des Batignolles, F-75008 Paris (FR).

(54) Title: STABILISED IMMUNOGLOBUUN PREPARATIONS AND METHOD FOR PREPARING SAME

(54) Titre: PREPARATIONS D'IMMUNOGLOBULINES STABIUSEES ET PROCEDE POUR LEUR PREPARATION

(57) Abstract

Substantially albumin-free human or animal and particularly polyclonal immunoglobulin preparations including a non-ionic surfactant in a concentration no higher than 0.1 g/1 as a liquid preservative stabilising agent.

(57) Abrege

Preparations d'immunoglobulines humaines ou animales, en particulier polyclonales, qui comprennent, a titre de stabilisant de conservation sous forme liquide, un tensioactif non ionique en concentration inferieure ou egale a 0,1 g/1 et qui sont essentiellement depourvues d'albumine.

...

IPR Page 1793 of 2482 UNIQUEMENT A TITRE D'INFORMATION

Codes utilises pour identifier les Etats parties au PCT, sur les pages de couverture des brochures publiant des demandes intemati.onales en vertu du PCT.

AT Autriche GB Royaume-Uni MR Mauritanie AU AUSlral.ie GE Georgie MW Malawi BB Barbade GN Guinee NE Niger BE Belgique GR ~ NL Pays-Bas BF Burldna Faso HU Hoogrie NO Norvege BG Bulgarie m Irlande NZ Nouvelle-:Ulande BJ Benin 1T Italic PL Pologoe BR Br6sil JP Japon PT Portugal BY B~larus KE Kenya RO Roumanie CA Canada KG KirgbizistaD RU Ft&ration de Rllssie CF R6publique centrafticaine KP R6publique populaire d&nocratique SD Soudan CG C.ongo de e.orce SE Sutde CB Suisse KR Rq,ublique de C.orce SI Slov6nie CI Olte d'Ivoire KZ Kauthstan SK Slovaquie CM Cameroon u Liechtenstein SN ~gal CN Oline LK Sri Lanka TD Tcbad cs Tcheco&lovaquie LU Luxembourg TG Togo CZ R6publique tcbeque LV Lettonie TJ Tadjikistan DE Allemagne MC Monaco TI Trini~-Tobago DK Dancmarlt MD R6publique de Moldova UA Ukraine ES Espagne MG Madagascar us Etats-Unis d'Amtrique FI Finlande ML Mali uz Ouzbeldstan FR France MN Mongolic VN Viet Nam GA Gabon

IPR Page 1794 of 2482 WO 95/03826 PCT/FR94/00955

Preparations d 1 immunoglobulines stabilisees et procede pour leur preparation

1 5 La presente invention a trait a des preparations' d'immunoglobulines (Ig) humaines ou animales, en particulier immunoglobulines polyclonales sanguines. La presente invention a egalement trait a un precede de stabilisation des immunoglobulines. 10 Les immunoglobulines sent largement utilisees dans la prophylaxie et la therapeutique. Leur mode d'administration par voie intraveineuse necessite de reduire au maximum leur activite anticomplementaire qui peut resulter d'une denaturation des Ig conduisant 15 notamment a la formation d'agregats et de polymeres. Ainsi, la norme etiropeenne publiee dans Pharmeuropa (3 (4), decembre 1991, 259-268) exige que les solutions d'immunoglobulines pour administration intraveineuse aient des taux d' agregats et de polymeres 20 inferieurs cu egaux a 3 % des proteines totales et une activite anticomplementaire inferieure ou egale a 1 unite CH 50 par mg d'Ig. La formation d'agregats n'intervient pas seulement au cours de la preparation des solutions d' immunoglobulines, mais aussi au cours de leur 25 conservation sous f.orme liquide, notamment lors de leurs manipulations. En effet, les immunoglobulines ont tendance a se denaturer aux interfaces liquide/gaz et liquide/solide, ce qui se traduit par une augmentation de l'activite anticomplementaire par formation d'agregats· 30 solubles ou insolubles. La demande de brevet franc;:ais FR-A-2 301 266 propose un precede de preparation de gamma-globulines inj ectables par voie intraveineuse. La mise sous f orme pharmaceutique comp rend la dissolution des gamma-

IPR Page 1795 of 2482 WO 95/03826 2 PCT/FR94/00955

globulines dans une solution aqueuse tamponnee et contenant du glycocolle et de l'albumine. En outre, pour empecher ou reduire toute denaturation de surface (aux interfaces liquide/air ou 5 liquide/solide), cette demande propose d'ajouter a la preparation pharmaceutique obtenue un agent tensio-actif non ionique. A titre d'agent tensio-actif, cette demande propose les Tweens ou le Pluronic 68. Dans le seul mode de realisation decrit, cette demande preconise 10 1 'utilisation de Tween 80 a une concentration de O, 1 % c'est-a-dire ~e 1 g/1. En consequence, la mise sous forme pharmaceutique des gamma-globulines passerait par ! 'utilisation combinee de glycocolle, d' albumine et de tensio-actif non-ionique a une concentration de 1 1 ordre 15 de- 1 g/1. La demande de brevet· europeen EP-A 448 075 decrit un precede de preparation d'Ig intraveineuse avec filtration sur membrane, dans lequel on cherche a empecher la denaturation des Ig lors de la filtration a 20 l 'aide d 'un stabilisant tensio-actif, qui peut etre un tensio-actif non ionique tel que le Pluronic. Le taux de stabilisant recommande est compris entre o,s et 50 g/1. Il en resul te que le produit final contient le tensio­ actif a un taux tres eleve. 25 H.L. Levine et al. (J. of Parenteral Science and Technology, volume 45 (3) mai-juin 1991, pages 160 a 165) rapportent une etude sur les effets protecteurs des tensio-actifs non ioniques centre la denaturation de surface de solutions d' anticorps monoclonaux faiblement 30 concentrees dans un test d'agitation. Les auteurs rapportent une concentration efficace de l'ordre de 0,1 %, c'est-a-dire correspondant a 1 g/1. Aucune protection n I est par centre obtenue avec une concentration de O, 1 g/1.

IPR Page 1796 of 2482 WO 95/03826 3 PCT /FR94/00955

or il est etabli qu'a ces concentrations elevees, ces agents tensio-actifs presentent des inconvenients serieux en cas d'injection intraveineuse. r Ainsi, le brevet US-A-4 439 421 recommande de ne 5 pas utiliser les tensio-actifs non ioniques tels que decrits notamment dans le brevet US-A-4 093 606 correspondant a la demande de brevet fran9ais ci-dessus, pour un probleme d'innocuite vis-a-vis des cellules sanguines. Cela confirme les observations de J.C. KRANTZ 10 et al. (J. Pharmacol Exp. Ther. 93, pages 188 a 195, 1948) sur le pouvoir hemolytique du Tween 20 a la concentration de 1 g/1. Le brevet US precite propose de stabiliser les solutions d'Ig en vue de leur lyophilisation en combinant plusieurs types de 15 stabilisants, macromolecules, proteines et polyols de bas poids moleculaire. Le polyethylene glycol est prefere en liaison avec de l'albumine humaine et du glucose. Il est en effet connu que 1 'albumine agi t comme un stabilisant efficace pour les immunoglobulines. 20 Mais dans le contexte medical actuel, on cherche a eviter autant que faire se peut !'utilisation de substances d'origine humaine ou animale, qui presentent un risque de contamination virale. La demanderesse a maintenant decouvert de f a9on 25 surprenante qu' il etait possible de preparer des solutions d'immunoglobulines stabilisees sous forme liquide a 1 'aide d 'un tensio-actif non ionique en tres faible concentration, tout en se passant des stabilisants habituels tels que l'albumine. 30 La presente invention a done pour objet des preparations d'immunoglobulines humaines ou animales, en particulier polyclonales, et notamment IgG polyclonales, qui comprennent, a titre de stabilisant de conservation sous forme liquide, un tensio-actif non ionique en 35 concentration inferieure ou egale a 0,1 g/1 et qui sent

IPR Page 1797 of 2482 WO 95/03826 4 PCT/FR94/00955

essentiellement depourvues d'albumine. Par essentiellement depourvues d'albumine, il faut comprendre qu•aucune trace d'albumine n'est detectee par la methode de reference Pharmeuropa en electrophorese sur acetate de 5 cellulose , ce qui correspond a une quantite d 'albumine· inferieure al% des IgG. De preference, la concentration en tensio-actif non ionique est comprise entre 0,02 et 0,05 g/1 environ et est de preference de l'ordre de 0,025 g/1. 10 De preference, les preparations selon !'invention comprennent entre 30 et 120 g/1 d'immunoglobulines. Le tensio-actif non-ionique est choisi de preference dans le groupe consistant en monooleate de sorbitanne polyoxyethylene (20), ether octylphenylique du 15 deoaethylene-glycol, monolaurate de sorbitanne polyoxyethylene (20), copolymere mixte polyoxyethylene/polyoxypropylene et polyoxyethylene et Laurate de polyethylene glycol 600. Comme cela est d 'usage, les preparations selon 20 l 'invention peuvent aussi comprendre un stabilisant de lyophilisation usuel tel que le saccharose, notamment en concentration de l'ordre de 50 a 100 g/1. Les preparations d'immunoglobulines selon !'invention ant egalement de preference les 25 caracteristiques preconisees par les normes . en vigueur, tel que pH compris entre 4,0 et 7,4, osmolalite superieure a 280 mosmol/kg par l 'addition de solutes osmotiquement actifs, tels que des sels mineraux (tel que NaCl) ou des sucres (tel que glucose, saccharose, 30 maltose) cu des sucres-alcools (tels que mannitol, sorbitol) cu des acides amines (tel que glycocolle). Les preparations obtenues repondent aux criteres de securite d'emploi particuliers aux solutions d'Ig pour administration intraveineuse, a savoir sterilite 35 bacterienne et fongique, apyrogenicite, taux reduit

IPR Page 1798 of 2482 WO 9S/03826 5 PCT/FR94/00955

d'agregats et de polymeres et activite anticomplementaire reduite. Les immunoglobulines presentes dans la preparation peuvent etre obtenues a partir de plasma, de 5 serum cu de placenta par les methodes classiques de fractionnement des proteines, completees le cas echeant par des traitements specifiques visant a reduire le taux d'agregats et de polymeres et/cu a reduire l'activite anticomplementaire de la preparation (par exemple 10 traitement modere a la pepsine ou a la plasmine, traitement dissociant a pH acide, modification chimique par reduction et/cu alkylation ou precipitation des agregats et polymeres par le PEG). Les preparations selon l 'invention peuvent etre 15 des solutions pretes a 1 1 emploi, done conservees sous forme liquide, ou etre des solutions obtenues extemporanement par dissolution d'un concentre lyophilise. La presente invention a egalement trait a un 20 precede de stabilisation des preparations d'immunoglobulines humairtes ou animales, polyclonales, dans lequel on ajoute a la preparation un stabilisant de conservation sous forme liquide qui est un tensio-actif non ionique de fa9on a obtenir une concentration 25 inferieure ou egale a 0,1 g/1 dans la preparation finale. De preference, le tensio-actif non ionique est ajoute en concentration comprise entre 0,02 et 0,05 g/1 environ et de preference de l'ordre de 0,025 g/1. Le tensio-actif non ionique est choisi parmi ceux indiques plus haut. 30 L'agent tensio-actif est avantageusement ajoute juste avant le conditionnement final en flacons, mais il peut egalement etre aj cute a un stade anterieur du precede d'extraction et de purification des Ig.

IPR Page 1799 of 2482 WO 95/03826 6 PCT /FR94/00955

L' invention va etre maintenant decrite plus en detail a l' aide d I essais realises avec des preparations d'inununoglobulines selon l'invention. on a simule des conditions severes de 5 manipulation de flacons d'Ig intraveineuse a l 1 aide d'un, test d' agitation. Des flacons de verre borosilicate de type I de 50 ml sent remplis aseptiquement avec 20 ml d I une solution sterile d' Ig a 50 g/1. Les flacons sent agites a temperature ambiante de 20°c pendant 1 ou 2 h 10 sur un agitateur de type oscillant/alternatif regle pour 80 oscillations horizontales par minute. L' activite anticomplementaire (AcA) de la solution est mesuree selon le test decrit dans "Pharmeuropa 11 (supra). 15 Essai 1: effet protecteur du Tween 80 Composition de la solution d'Ig intraveineuse: Ig = 50 g/1 Saccharose = 100 g/1 20 NaCl= 1 g/1 pH= 4,3 Concentration variable de Tween 80: de O a 100 mg/1

25 Tableau 1: Tween 80 (mg/1) 0 10 25 100 * AcA avant agitation 0,34 0,35 0,35 0,36 * AcA apres agitation 2h >1,19 1,19 0,75 0,38 30 * CH 50/mg Ig

Resultat: Une protection nette s'observe des 25 mg/1. Elle est quasiment totale avec 100 mg/1 de tensio- 35 actif.

IPR Page 1800 of 2482 WO 95/03826 7 PCT/FR94/00955

Essai 2 : effet protecteur de Triton X 100 Idem essai 1, mais avec du Triton X 100 au lieu du Tween 80 comme tensio-actif Tableau 2 : ( 5 Triton X 100 (mg/1) 0 25 50 100 * AcA avant agitation 0,37 0,36 0,36 0,31 * AcA apres agitation 2h 0,50 0,34 0,36 0,19 10 * CH 50/mg Ig

Resultat: Une protection totale est obtenue avec la plus faible concentration essayee, 25 mg/1. 15 Essai 3: effet protecteur du Tween so Idem essai 1, mais avec une agitation de 1 h au lieu de 2h. Tableau 3 20 Tween 80 (mg/1) 0 25 * AcA avant agitation 0,41 0,33 * AcA apres agitation 1 h 0,70 0,27 25 * CH 50/mg Ig

Resul tat : Dans cet essai mains severe, la concentration de 25 mg/1 de Tween suffit a proteger 30 totalement l'Ig.

Essai 4: effet protecteur de l 1 albumine Idem essai 1, mais utilisation d'albumine humaine au lieu de Tween 80 comme protecteur. 35

IPR Page 1801 of 2482 WO 95/03826 8 PCT /FR.94/00955

Tab1eau 4 Albumine (mg/1) 0 100 1000 10.000 * AcA avant agitation 0,34 0,36 0,26 0,29 ': 5 * AcA apres agitation 2h 1,24 0,64 0,27 0,28

* CH 50/mg Ig

Resultat : L'albumine exerce un effet protecteur 10 sur l'Ig a partir d'une concentration comprise entre 100 et 1000 mg/1.

Tensio-actifs non ioniques preferes : - Tween 80 (ester oleique du sorbitanne polyoxyethylene 15 fabrique par Atlas). - Triton X 100 (ether octyl-phenylique du polyoxyethylene fabrique par Rohm et Haas). - Tween 20 (ester laurique du sorbitanne polyoxyethylene) Pluronic F 68 ( copolymere de polyoxyethylene et de 20 polyoxypropylene fabrique par Ugine Kuhlmann). Laurate de polyethylene glycol 600 (fabrique par Gattefosse). Le Tween 80 est prefere du fait de son absence de toxicite et de son emploi en formulation pharmaceu~tique 25 ou alimentaire bien documente (Vair l'ouvrage "Non Ionic surfactants" M. Schick ed. Marcel Dekker NY, 1967, 28, 923-970).

30

35

IPR Page 1802 of 2482 WO 95/03826 PCT /FR94/00955

9

REVENDICATIONS 1. Preparations d'irnmunoglobulines humaines ou animales, en particulier polyclonales, qui comprennent, a titre de stabilisant de conservation sous forme liquide, UD 5 tensio-actif non ionique en concentration inferieure ou egale a 0,1 g/1 et qui sent essentiellement depourvues d'alburnine. 2. Preparations selon la revendication 1, caracterisees en ce que la concentration en tensio-actif 10 non-ionique est comprise entre 0,02 et 0,05 g/1 environ, de preference 0,025 g/1 environ. 3. Preparations selon la revendication 1 ou 2, caracterisees en ce qu'elles comprennent entre 30 et 120 g/1 d'irnrnunoglobulines. 15 4. Preparations selon l 'une quelconque des revendications 1 a 3, caracterisee en ce que le tensio-actif non ionique est choisi dans le groupe consistant en monooleate de sorbitanne polyoxyethylene (20), ether octylphenyl ique du decaethyl ene-gl ycol, rnonolaurate de 20 sorbitanne polyoxyethylene (20), copolymere mixte polyoxyethylene/polyoxypropylene et polyoxyethylene et Laurate de polyethylene glycol 600. 5. Preparations selon l 'une quelconque des revendications 1 a 4, caracterisees en ce qu'elles 25 comprennent en outre un stabilisant de lyophilisation. 6. Precede de stab i 1 i sat ion des prepara ti ens d'immunoglobulines polyclonales, dans lequel on ajoute a la preparation un stabilisant de conservation sous forme liquide qui est un tensio-actif non ionique de fagon a 30 obtenir une concentration inferieure ou egale a 0,1 g/1 dans la preparation finale.

IPR Page 1803 of 2482 INTERNATIONAL SEARCH REPORT lntan. .al Application No PCT/FR 94/00955 A. CLASSIFICATION OF SUWECT MATIER IPC 6 A61K39/395

According to International Patent Oassific:ation (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols} IPC 6 C07K

Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search tams used}

C. DOCUMENTS CONSIDERED TO BE RELEVANT Callegory" Qtation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X PATENT ABSTRACTS OF JAPAN 1-6 vol. 5, no. 41 (C-047) 18 March 1981 & JP,A,55 164 630 (GREEN CROSS CORP) 22 December 1980 see abstract --- X EP,A,O 448 075 (MITSUBISHI RAYON CO., LTD) 1-6 25 September 1991 cited in the application see column 5, line 45 - column 6, line 16 see column 8, line 33 ---- line 40 -!--

[]] Further documents arc listed in the continuation of box C. OCJ Patent family members arc liswi in annex.

• Special categories of cited documents : "T' law document published after the intcmational filing date or priority date and not in conflict with the application but • A" document defining the general state of the art which is not cited to understand the principle or theory underlying the coosidcrcd to be of particular relevance invention ·E· earlier document but published on or after the international ·x· document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considcrcd 1D ·L• document which may throw doubts on priority daim(s) or involve an inventive step whm the document is taken alone which is cited to establish the publication date of another •y• document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the ·o· document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu· other means ments, such combination being obvious to a person skilled •p- document published prior to the international filing date but in the art. later than the priority date claimed • &" document member of the same patent family 1 Date of the actual completion of the international search Date of mailing of the international search report 9 November 1994 29 -11- 199\ Name and mailing address of the ISA Authorized officer European Patent Office, P.B. 5818 Patcntlaan 2 NL • 2280 HV Rijswijk Tel. ( + 31 • 70) 340-2040, Tx. 31 6Sl epo nl, Fax: ( + 31-70) 340-3016 Sitch, w

Ferm PCT/ISA,r.uo (second sheet) (July 1'!12) page 1 of 2

IPR Page 1804 of 2482 INTERNATIONAL SEARCH REPORT Intem aal Application No PCT/FR 94/00955 C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category• Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X JOURNAL OF PARENTERAL SCIENCE AND 1-4,6 TECHNOLOGY, vol.45, no.3, 1991, PHILADELPHIA,PA,USA pages 160 - 165 LEVINE ET AL 1THE USE OF SURFACE TENSION MEASUREMENTS IN THE DESIGN OF ANTIBODY-BASED PRODUCT FORMULATIONS' cited in the application Y see the whole document 5 cited in the application y WO,A,89 11297 (CENTOCOR,INC.) 30 November 5 1989 see claims 1-21 y DE,A,32 08 523 (LABORATORIOS 5 LANDERLAN,S.A.) 5 May 1983 A see claims 1-10 1-4,6

1

Fann PCT/ISA(.110 (continuation of ncood lheet) (July 1992) page 2 of 2

IPR Page 1805 of 2482 INTERNATIONAL SEARCH REPORT Intern; ;ii Application No m1ormation on patent family members PCT/FR 94/00955

Patent document Publication Patent family Publication cited in search report I date I member(s) I date EP-A-0448075 25-09-91 JP-A­ 3271234 03-12-91 US-A- 5219999 15-06-93 WO-A-8911297 30-11-89 DE-T­ 68908175 03-03-94 EP-A,B 0417193 20-03-91 JP-T- 3504605 09-10-91 DE-A-3208523 05-05-83 JP-A- 57206625 18-12-82 •

Farm PCT/ISA/210 !patent famUy annex) (July 1992)

IPR Page 1806 of 2482 RAPPORT DE RECHERCHE INTERNATIONALE Demai. .nt.emalionale No PCT/FR 94/00955 A. CLASSEMENT DE L'OBJET DE LA DEMANDE CIB 6 A61K39/395

Selan la classification intemationale des brevets {CIB) ou 4 la fois scion la classification nationale et la CIB B. DOMAINES SUR LESQUELS LA RECHERCHE A PORTE Documentation minimale consulU:e {systane de classification suivi des symboles de classcmcnt) CIB 6 C07K

Documentation c:onsulU:e autre que la documentation minimale dans la mesure ol) ces documents rclevent des domaincs sur lesqucls a pone la rccherche "

Base de donnees tlectronique consulU:e au cours de la rechm:he intemationale {nom de la base de donn~ et si cela est realisable, tames de recberche utilisa)

C. DOCUMENTS CONSIDERES COMME PERTINENTS Categoric• Identification des documents cites, avcc, le cas ecMant, l'indication des passages pertinents no. des revendications visees

X PATENT ABSTRACTS OF JAPAN 1-6 vol. 5, no. 41 (C-047) 18 Mars 1981 & J~,A,55 164 630 (GREEN CROSS CORP) 22 Decembre 1980 voir abrege --- X EP,A,O 448 075 (MITSUBISHI RAYON CO., LTD) 1-6 25 Septembre 1991 cite dans la demande voir colonne 5, ligne 45 - colonne 6, ligne 16 voir colonne 8, ligne--- 33 - ligne 40 -/--

CT] Voir la suite du cadre C pour la fin de la listc des documents III Les documents de familles de brevets sont indiques en annexe • Categories spcciales de documents ciU:s: T document ulU:rieur publie apres la date de depOt intmlational ou la date de priorite et n'llppllrtfflenant pas 41' etat de la •A• document definissant I'etat gtneral de la technique, non tcchnite pertinent, mais cite pour comprendre le principe consideri comme particulicrcmcnt pertinent ou la eerie constituant la base de l'invention •e• document anterieur, mais publie 4 la date de depOt international documentcculicrcment pertinent; )'invention revendiquee ne ~ OU cette date ·x· a.pres etre consi me comme nouvelle ou comme impliquant une activite "L" document pouvant jeter un doutc sur une revcndication de inventive par rapport au document consideri isolcment priorite ou cite pour dttcrminer la date de publication d'une •y• document particulicrcment pertinent; )'invention revcndiquee aulre citation ou pour une raison speciale (telle qu'indiquee) ne peut etre consideree comme impliquant une activiU: inventive ·o· document se rererant 4 une divulgation orale, 4 un usage, 4 Jorsque le document est associe a un ou plusieurs autres une exposition ou tous autres moyens documents de meme nature, cette combinaison etant evidcnte •p• document publie avant la date de d:f Ot international, mais pour une personne du metier posterieurcrnent a la date de priori revendiquec • &.· document qui fait partie de la meme Camille de brevets Date 4 laquelle la recherche intemationale a ete effectivcmcnt acbevee Date d'expedition du present rapport de recherche intemationalc 9 Novembre 1994 29 -11- 1994

Nom et adresse postale de I' adminimation chargcc de la rechercbe intemationale Fonctionnaire autorise Office Europeen des Brevets, P.B. 5818 Patmtlaan 2 NL • 2280 HV Rijswijk Td. {+31-70) 340-2040, Tx. 31 651 epo nl, Fu: ( + 31·70) 340-3016 Sitch, w

Fmmlllaire PCTJlSA/210 (dewaeme.. feuille) (juiUet 1992) page 1 de 2

IPR Page 1807 of 2482 RAPPORT DE RECHERCHE INTERNATIONALE Dc:ma.. intcmationale No PCT/FR 94/00955 C(IUite) DOCUMENTS CONSIDERES COMME PERTINENTS

C.pe • Identification des documents citbs, avec, le cas echt:ant, l'indication des passages pertinents no. des rcvcndic:ationa viKCS

X JOURNAL OF PARENTERAL SCIENCE AND 1-4,6 TECHNOLOGY, vol.45, no.3, 1991, PHILADELPHIA,PA,USA pages 160 - 165 LEVINE ET AL 'THE USE OF SURFACE TENSION .. MEASUREMENTS IN THE DESIGN OF ANTIBODY-BASED PRODUCT FORMULATIONS' cite dans la demande • y voir le document en entier 5 cite dans la demande y WO,A,89 11297 (CENTOCOR,INC.) 30 Novembre 5 1989 voir revendications 1-21 y DE,A,32 08 523 (LABORATORIOS 5 LANDERLAN,S.A.) 5 Mai 1983 A voir revendications 1-10 1-4,6

1

Fmmulalre PCT/ISA,1.210 (suite de la dewcieme feullle) (julllet 1992) page 2 de 2

IPR Page 1808 of 2482 RAPPORT DE RECHERCHE INTERNATIONALE Dema. mtemationale No Rcnscignements rdatifs aux membres de families de brevets PCT/FR 94/00955

Document brevet cite Date de Membre(s) de la Date de au rapport de recherche I publication I famille de brevet(s) I publication EP-A-0448075 25-09-91 JP-A­ 3271234 03-12-91 US-A- 5219999 15-06-93 WO-A-8911297 30-11-89 DE-T­ 68908175 03-03-94 EP-A,B 0417193 20-03-91 JP-T_._ 3504605 09-10-91 DE-A-3208523 05-05-83 JP-A- 57206625 18-12-82 'f

Fonnulaire PCT/ISA/210 (annexe t.mllles de brevets} (jui:let 199l)

IPR Page 1809 of 2482 WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent CJassification 6 : (11) International Publication Number: WO 98/44948 A61K 39/395 A2 (43) International Publication Date: 15 October 1998 (15.10.98)

(21) International Application Number: PCT/CA98/00325 (81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, (22) Internationa] Filing Date: 7 April 1998 (07.04.98) GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, (30) Priority Data: TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO 60/041,921 7 April 1997 (07.04.97) us patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, (71) Applicant (for all designated States except US): CANGENE IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CORPORATION [CA/CA]; 104 Chancellor Metheson CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG). Road, Winnipeg, Manitoba R3T 2N2 (CA).

(72) Inventors; and Published (75) Inventors/Applicants (for US only): PRICE, Hugh, W. Without international search report and to be republished [CA/CA]; 350 Kingston Crescent, Winnipeg, Manitoba upon receipt of that report. R2M OT8 (CA). WOLOSKI, B., Michael, R. [CA/CA]; Cangene Corporation, 104 Chancellor Matheson, Winnipeg, Manitoba R3T 2N2 (CA).

(74) Agent: BERESKlN & PARR; 40th floor, 40 King Street West, Toronto, Ontario M5H 3Y2 (CA).

(54) Title: INTRAVENOUS IMMUNE GLOBULIN FORMULATION CONTAINING A NON-IONIC SURFACE ACTIVE AGENT WITH IMPROVED PHARMACOKINETIC PROPERTIES

(57) Abstract

Addition of a non-ionic surface active agent to an immune globulin formulation extends the serum half-life of relatively pure and non-aggregated immune globulin suitable for intravenous injectionor infusion. The non-ionic surface active agent may be a sorbitan ester or a polyoxyethylene sorbitan ester of a fatty acid. Formulations of the present invention is therapeutically advantageous over conventional formulations in that an extended serum half-life of the immune globulin improves its therapeutic effectiveness, reduces the frequency of drug administration and/or lowers the therapeutic effective dosage required and cost of treatment. 1 ---.---.--.--.---r--r----.---.-----.---.----1 0 20 4o 60 ao 100 lime after Injection (days)

IPR Page 1810 of 2482 FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AL Albania ES Spain LS Lesotho SI Slovenia AM Armenia FI Finland LT Lithuania SK Slovakia AT Austria FR France LU Luxembourg SN Senegal AU Australia GA Gabon LV Latvia sz Swaziland AZ Azerbaijan GB United Kingdom MC Monaco TD Chad BA Bosnia and Herzegovina GE Georgia MD Republic of Moldova TG Togo BB Barbados GH Ghana MG Madagascar TJ Tajikistan BE Belgium GN Guinea MK The former Yugoslav TM Turkmenistan BF Burkina Faso GR Greece Republic of Macedonia TR Turkey BG Bulgaria HU Hungary ML Mali TT Trinidad and Tobago BJ Benin IE Ireland MN Mongolia UA Ukraine BR Brazil IL Israel MR Mauritania UG Uganda BY Belarus IS Iceland MW Malawi us United States of America CA Canada IT Italy MX Mexico uz Uzbekistan CF Central African Republic JP Japan NE Niger VN Viet Nam CG Congo KE Kenya NL Netherlands YU Yugoslavia CH Switzerland KG Kyrgyzstan NO Norway zw Zimbabwe CI C6te d'Ivoire KP Democratic People's NZ New Zealand CM Cameroon Republic of Korea PL Poland CN China KR Republic of Korea PT Portugal cu Cuba KZ Kazakstan RO Romania CZ Czech Republic LC Saint Lucia RU Russian Federation DE Germany LI Liechtenstein SD Sudan DK Denmark LK Sri Lanka SE Sweden EE Estonia LR Liberia SG Singapore

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INTRAVENOUS IMMUNE GLOBULIN FORMULATION CONTAINING A NON-IONIC SURFACE ACTIVE AGENT WITH IMPROVED PHARMACOKINETIC PROPERTIES

FIELD OF THE INVENTION The present invention relates to an improved method and immune globulin formulation containing a non-ionic surface active agent to prolong the serum half-life and to alter the immunom:odulatory 10 effect of immune globulin. BACKGROUND OF THE INVENTION Immune globulins (also known as immunoglobulins or antibodies) are proteins produced by lymphoreticular tissues. There are 6 known classes of immune globulin: IgG, IgM, IgA, IgD, IgE and secretory 15 IgA. IgG (also known as gamma-globulin) is the most abundant and the most therapeutically relevant class of immune globulin. The primary function of immune globulins is to specifically recognize and bind antigens through reversible bonding thereby facilitating the immune systems ability to eliminate antigens. 20 IgG is a glycoprotein of approximately 150,000 Daltons consisting of 2 "heavy" (gamma) chains and 2 "light" (kappa or gamma) chains held together by disulphide as well as weak covalent bonds. Within the class of IgG, there are 4 subclasses of IgGl, IgG2, IgG3 and IgG4 comprising about 70%, 15%, 10% and 5% of total IgG in normal human 25 serum respectively. These subclasses possess minor antigenic differences among their "heavy" chains resulting in distinct biological actions. There are principally two types of immune globulins that are available as therapeutic agents: standard immune serum globulin preparations for general use, and immune globulin preparations that 30 recognize specific antigens for use in specific disorders. Commercial examples of products in the former category include numerous brands of intravenous immune globulin (Gamimune N® by Bayer; Sandoglobulin® by Sandoz; Gammar-IV® by Armour) as well as intramuscular immune globulin (Gamastan® by Cutter; Gammar® by Armour). Examples of

IPR Page 1812 of 2482 W098/44948 PCT/CA98/00325

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products in the latter category are hepatitis B immune globulin (H-BIG® by Abbott; Hep-B-Gammagee® by MSD; HyperHep® by Cutter), varicella zoster immune globulin (VZIG by Massachusetts Public Health Biologic Labs.), cytomegalovirus immune globulin (CytoGam® by Connaught) and 5 Rh immune globulin (WinRho® and WinRho SD® by Cangene; HypRho­ D® by Miles; Gamulin Rh® by Armour; RhoGAM® by Ortho Diagnostics). The primary therapeutic basis for immune globulins is passive immunity conferred to the recipient through the direct introduction of extraneous "ready-made" antibodies. The major clinical utilities of immune 10 globulins are prophylaxis and/ or treatment of antigen-associated disorders. Immune globulins may be prepared by isolation of natural immune globulins from mammalian serum. Immune globulins prepared using Cohn's cold ethanol fractionation method suffers from 15 relatively low product yield and IgG purity. The resultant product contains significant amounts of aggregated immune globulin which combines with complement (also termed anticomplementary activity) and produces adverse reactions in recipients if given by intravenous injection or infusion (see Huchet, J. et al., Rev. Fr. Transfus. 13:231, 1970; 20 Chown, B. et al., Can. Med. Assoc. J. 100:1021, 1969; Barandun, S. et al., Vox Sang. 7: 157-174, 1962). Correspondingly, these immune globulin preparations must be injected intramuscularly (therefore termed intramuscular immune globulin). Intramuscular injections are painful. Drug absorption and peak levels of immune globulin (hence onset of 25 therapeutic action) are slow, and approximately half of the injected dose is lost due to local proteolysis and incomplete absorption. Significant amounts of IgA and IgM are also present in Cohn-prepared intramuscular immune globulin preparations which can cause anaphylactic reactions in certain recipients. 30 Compared with other parenteral routes, such as those just discussed, intravenous injection or infusion of immune globulin is the preferred route of drug administration in the clinical setting due to instant bioavailability and rapid onset of therapeutic protection.

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Intravenous immune globulin products differ from intramuscular products in two fundamental ways. First, an intravenous preparation must contain a significantly smaller amount of aggregated immune globulin molecules (over about 94% monomeric immune globulin) S thereby causing fewer anticomplementary adverse reactions. Second, the IgG content and product purity of intravenous immune globulin products are significantly higher (over about 95% IgG content) than intramuscular products. The low level of contamination with IgA or IgM in intravenous immune globulin (less than about 40 ug/mL) is also 10 associated with lower incidences of adverse events such as anaphylactic reactions especially in agammaglobulinemic recipients. Improved methods involving further purification of the Cohn immune globulin fractions (see Jouvenceaux, A. et al., Rev. Fr. Trans/us. 12 (suppl.): 341, 1969) were developed to render immune 15 globulin produced from cold ethanol fractionation suitable for intravenous administration. Ultracentrifugation of the immune globulin-containing fraction, or treatment of immune globulin with pepsin, plasmin, a sulfitolytic agent or beta-propriolactone, reduces the anticomplementary activity of the final preparation (see U.S. Patent No. 20 4,160,763; Barandun, S. et al., Monogr. Allergy 9: 39-60, 1975; Stephan, Vox Sang. 28: 422-437, 1975; Wells, J.L.V. et al., Austr. Ann. Med. 18: 271, 1969; Baumgarten, W. et al., Vox Sang. 13: 84, 1967; Merler, E. et al., Vox Sang. 13: 102, 1967; Sgouris, J.T. et al., Vox Sang. 13: 71, 1967; Barandun, S. et al., Vox Sang. 7: 157-174, 1962; Nisonoff, A. et al., Science 132: 1770-1771, 1960). 25 U.S. Patent No. 3,903,262 describes the reduction of intermolecular disulphide bonds of immune globulin and alkylation of the resultant sulfhydryl groups. Schura of Germany also developed an immune globulin for intravenous injection by adsorption of immune globulin onto hydroxy-ethyl starch. However, these approaches are often 30 technically unfeasible for manufacturing or residual reactants in the final preparation have been shown to cause undesirable outcomes such as a reduction in serum half-life of the immune globulin and elicitation of immunogenic reactions in recipients.

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Further improved chromatographic techniques (e.g. using DEAE-Sephadex ion-exchange columns in combination with ultrafiltration) were also developed for the manufacture of immune globulins from human plasma to produce immune globulins suitable for 5 intravenous injection or infusion (see Canadian Patent number 1,168,152; Canadian Patent number 1,201,063; Cunningham, C.J. et al., Biochem. Soc. Trans. 8: 178, 1980; Hoppe, H.H. et al., Vox. Sang. 25: 308, 1973; Hoppe, H.H. et al., Miinch. Med. Wochenschr. 109: 1749, 1967; Baumstark, J.S. et al., Arch. Biochem. 108:514, 1964). The use of such chromatographic 10 manufacturing processes also substantially increases product yield to over about 90%. Immune globulin prepared by improved processes may be administered by intravenous injection or infusion as well as other parenteral routes. For instance, Cangene's WinRho® and WinRho SD® 15 are produced using a proprietary anion exchange chromatographic

process and are the only commercial anti-Rh0 D immune globulin preparations that can be administered safely by intravenous injection or infusion to humans. This is due to their relatively higher IgG purity and monomeric protein content as well as lower IgA/IgM contamination. 20 Monoclonal immune globulins can be produced using recombinant and hybridoma techniques (see Canadian Patent number 1,303,534; Canadian Patent number 1,303,533; European Patent Application 87302620.7 published as EP 239,400; European Patent Application 93102609.0 published as EP 557,897; Fletcher, A. and 25 Thompson, A., Transfus. Med. Rev. 9: 314-326, 1995; Alting-Mees, M. et al., Strat. Mol. Biol. 3: 1-9, 1990; Huse, W.D. et al., Science 246: 1275-1281, 1989; Sastry, L. et al., Proc. Natl. Acad. Sci. USA 86: 5728-5732, 1989). Similarly, binding partners or domains may also be constructed using recombinant DNA techniques to incorporate the variable regions of a 30 gene encoding a specific antibody (see PCT Patent Application PCT /GB93/00605 published as WO 93/19172; PCT Patent Application PCT /GB93/02492 published as WO 94/13804; PCT Patent Application

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PCT /EP90/01964 published as WO 91/07492; Bird et al., Science 242: 423- 426, 1988). Immune globulin preparations suitable for parenteral injection commonly consist of an immune globulin distributed in a 5 physiologically compatible medium. This medium may be sterile water for injection (WFI) with or without isotonic amounts of sodium chloride. For example, the recommended diluent for reconstituting commercial intravenous immune globulins such as Iveegam®, Gammagard®, or, Venoglobulin®, is sterile WFI. Sandoglobulin® is supplied with 0.9% 10 (w /v) sodium chloride solution as diluent (see Gahart, B.L. & Nazareno, A.R., Intravenous Medications: a handbook for nurses and allied health professionals, p. 516-521, Mosby, 1997). WinRho SD®, is reconstituted in 0.9% sodium chloride solution for intravenous injection. The immune globulin product by Schura (supra) is formulated as a solution of 165 15 mEq/L sodium ion and 120 mEq/L chloride ion with a final pH of 6.7. The Miles' intravenous immune globulin preparation, Gammimune®, when constituted, has an osmolality of 278 mOsm/L and a pH of 4.0-4.5. U.S. patent Nos. 4,396,608 and 4,499,073 also disclose a low pH (3.5-5.0) and low ionic strength ( <0.001) immune globulin formulation for 20 intravenous injection. The globulin protein concentration in the above preparations ranges from 0.5% to 20%. Carbohydrates and their derivatives such as glucose, maltose or mannitol may be included in immune globulin formulations to adjust the tonicity of the preparation. For example, maltose (10%) is 25 included in Miles' intravenous immune globulin preparation, Gammimune®, to achieve isotonicity. Sucrose (5%) is included in Sandoz's intravenous immune globulin preparation, Sandoglobulin® and in Armour's intravenous immune globulin preparation, Gammar­ IV®. The commercial intravenous immune globulin preparation, 30 Venoglobulin®, contains 50 mg/mL D-sorbitol. Likewise, amino acids such as glycine or histidine may be added to improve storage stability of the protein. For example, glycine (0.3M) is included in commercial immune globulin preparations such as

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American Red Cross' intravenous immune globulin preparations, Polygam® and Polygam SID®; the intramuscular varicella zoster immune globulin preparation by Massachusetts Public Health Biologic

Laboratories; and the intramuscular anti-Rh0 D immune globulin 5 preparations by Armour and Miles. U.S. patents Nos. 4,396,608 and 4,597,966 describe the use of glycine and histidine to stabilize immune globulin formulations. European Patent Application No. EP 392,717 describes the use of mannitol and glycine to stabilize and prevent aggregation of immune globulin in formulation. 10 The prolongation of storage shelf-life of immune globulin preparations may also be accomplished by the addition of preservatives including organic mercurial derivatives such as thimerosal. Surface active agents (also termed surfactants or detergents) are compounds that can lower the surface tension of water. AH surface 15 active agents are amphipathic possessing a hydrophobic end (e.g. one or more hydrocarbon chain(s)) as well as a hydrophilic moiety (which may or may not be ionic). A surface active agent may be classified as anionic, cationic, or non-ionic depending on the nature of its hydrophilic moiety. Soaps with carboxylate or sulphonate groups carry net negative charges 20 and are examples of anionic surface active agents. Benzalkonium, an N­ benzyl quaternary ammonium chloride and an antibacterial agent, carries a net positive charge and is an example of a cationic surface active agent. A non-ionic surface active agent contains a neutral group such as a carbohydrate which can hydrogen-bond with water. 25 Tween® and Span® are two types of non-ionic surface active agent. Agents such as Span® are partial esters of common fatty acids and sugar alcohol anhydrides derived from sorbitol. Agents such as Tween® are derivatives of Span® products with polyoxyethylene chains attached to non-esterified hydroxyl groups. Their hydrophilic property is 30 due to free hydroxyl and/ or oxyalkylene groups, and their hydrophobic property is due to their long chain fatty acids. Examples of commonly used Span® type surface active agents are sorbitan monolaurate {Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60),

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sorbitan tristearate (Span 65), sorbitan monooleate (Span 80), and sorbitan trioleate (Span 85). A commonly used member of Tween® type surface active agent, Tween 80, is also known as Polysorbate 80, sorbitan mono-9- octadecenoate poly(oxy-1,2-ethanediyl) derivative, polyoxyethylene 5 sorbitan monooleate, polyethylene oxide sorbitan monooleate, sorethytan monooleate, Sorlate, Monitan or Olothorb. Other examples of polyoxyethylene sorbitan surface active agents comprise polyoxyethylene sorbitan monolaurate (Tween 20 or 21), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate 10 (Tween 60 or 61), polyoxyethylene sorbitan tristearate (Tween 65), and polyoxyethylene sorbitan trioleate (Tween 85). The safety of non-ionic surface active agents in mammals has been studied extensively and established. Previous acute toxicity studies indicated that the LD50 values for Tween® and Span® type non- 15 ionic surface active agents in rats are relatively high at > 15 g/kg for oral ingestion and >1.4 g/kg for parenteral injection (J. Am. Coll. Toxicol. 3: 1- 82, 1984; Varma, R.K. et al., Arzneimittelforschung 35: 804-808, 1985; Farkas, W.R. et al., Pharmacol. Toxicol. 68:154-156, 1991). Subacute and chronic toxicity studies also showed minimal toxicity after administration 20 of relatively high oral doses to rats (100-200 mg per kg body weight) (Nityanand, S and Kapoor, N.K., Indian J. Med. Res. 69: 664-670, 1979). A number of reproductive toxicology studies also did not identify a hazard with the clinical use of non-ionic surface active agents (Kitchin, K.T. and Ebron, M.D., Toxicology 30: 45-47, 1984; Ema, M. et al., Drug Chem. 25 Toxicol. 11: 249-260, 1988; Gajdova, M. et al., Med. Toxicol. 3: 128-165 and 209-240, 1988). The inclusion of surface active agents in protein drug preparations has been practised extensively to improve product stability in storage and/ or to increase product solubility. A commercially available 30 preparation of granulocyte colony stimulating factor, Neupogen®, contains 0.004% Polysorbate 80 to improve storage stability. Turbersol® is a sterile isotonic solution of Tuberculin in phosphate buffered saline containing 0.0005% Polysorbate 80 as a stabilizer.

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With respect to immune globulin preparations, U.S. Patent No. 4,902,500 discloses immune globulin formulations with improved storage stability containing at least one polyoxypropylene-polyoxyethylene block polymer (Pluronic 68). PCT Patent Application FR93/00584 5 published as W094/16728 describes the inclusion of Polysorbate 80 in a parenteral formulation of an anti-LFA-1 monoclonal antibody for stabilization purposes. U.S. Patent No. 5,215,743 describes the use of Polysorbate 80 to stabilize parenteral formulations of tumour necrosis factor (TNF). 10 U.S. Patent No. 5,151,266 teaches a method of treating antibodies with an anionic detergents such as sodium dodecylsulfate (sodium lauryl sulfate), cetyl ammonium sulfate, or taurocholic acid, to increase the solubility of the antibody and to reduce its reticuloendothelial uptake. The method claimed in U.S. Patent No. 15 5,151,266 involves preincubation of an antibody with anionic detergent: any unreacted anionic detergent is removed before drug administration.

Current commercial anti-Rh0 D immune globulin preparations containing <0.01 % Polysorbate 80 are RhoGAM® and MICRhoGAM® both produced by Ortho Diagnostics Systems Inc. These 20 preparations must be administered only by intramuscular injection due to relatively high protein aggregation and low product purity (as discussed supra for such preparations). Similarly, Polygam® and Polygam SID® (Red Cross) contains <0.01% Polysorbate 80 to improve immune globulin solubility and storage stability. These products contain relatively 25 low IgG content at 90%. With respect to the use of surface active agents in the production of immune globulin, U.S. Patent Nos. 4,371,520 and 4,379,086 describes the use of alkylene oxide polymers such as polyethylene glycol in the fractionation process for isolating immune globulin-rich fractions 30 from plasma. Similarly, U.S. Patent Nos. 4,276,283 and 5,132,406 describe the use of alkylene oxide polymers such as polyethylene glycol in a precipitation step for isolating and purifying immune globulin-rich fractions.

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The ability of surface active agents to alter the pharmacologic properties of drugs has been examined to a limited extent, but their usefulness in immune globulin formulations in a clinical setting has not been established. Jekunen, A. et al. (Acta Oncol. 35: 267- 5 271, 1996) and Kairemo et al. (Anticancer Res. 16(6B);3542-3550, 1996) reported the in vivo modulation of antibody kinetics in mice by Polysorbate 80. Intra-tumour administration of the non-ionic surface active agent, Polysorbate 80, improved the targeting of a radiolabeled monoclonal antibody to the tumour and accelerated antibody clearance 10 from the blood. Ellis, A.G. et al. (Cancer Chemother. Pharmacol. 38: 81-87, 1996) describes the effects of two surface active agents, Cremophor EL and Tween 80, on the pharmacokinetics of a chemotherapeutic non-protein drug, etoposide, in an isolated perfused rat liver experimental model. Co- 15 administration of either surface active agent decreased the elimination half-life of etoposide. Masters, J.R. et al. (Cancer Chemother. Pharmacol. 25: 267-273, 1990) decreased the in vivo half-life of the chemotherapeutic drug, thioTEPA in human subjects. These decreases in plasma half-life correspondingly increase the need for more frequent drug administration 20 to maintain effective plasma drug concentrations which in turn increases the cost associated with such therapy in the clinical setting. Liu, F. and Liu, D. (Pharm. Res. 12: 1060-1064, 1995) demonstrated the ability of Polysorbate 80 to attenuate the clearance of parenterally administered oil-in-water emulsions. Such oil-in-water 25 emulsions are physicochemically and biochemically different from the immune globulin proteins of the present invention. The benefits and methods for covalent bonding of amphipathic polymer moieties to proteins are known in the art. For example, the chemical conjugation of polyethylene glycol (PEG) or 30 monomethoxy-polyethylene glycol (MPEG) to a variety of proteins by a variety of different methods has been described (see PCT Patent Application GB94/01844 published as WO 95/06058; U.S. Patent No. 5,349,052; Delgado, C. et al., Crit. Rev. Ther. Drug Carrier Sys. 9: 249-304,

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1992). One of the major observed advantages of PEG conjugation to a protein is to decrease its rate of clearance from the body and to increase plasma half-life. However, the polymer (MPEG) by itself without conjugation was shown to elicit no effect on the plasma half-life of 5 proteins. SUMMARY OF THE INVENTION Immune globulin preparations with novel clinical characteristics and benefits are presented. Immune globulin preparations of the present invention contain an immune globulin with relatively 10 high IgG and low aggregated protein contents and are suitable for intravenous injection or infusion. The immune globulin preparation contains one or more non-ionic surface active agents in a physiologically compatible buffered medium. Inclusion of one or more such non-ionic surface active agents in the preparation surprisingly prolongs the serum 15 half-life of the immune globulin in vivo and improves the safety profile of the product. An immune globulin preparation with an increased serum half-life is clearly advantageous and contrary to the teachings of the prior art. A preparation with an extended half-life means that the active therapeutic ingredient would have a longer survival time in the 20 bloodstream to exert its desired therapeutic effect. A longer serum survival time would also alow for a reduced frequency of drug administration. This results in a more convenient dosing schedule with fewer injections which thereby improves patient compliance. A consequence is to reduce indirect costs associated with parenteral 25 administration of the drug. Moreover, a longer serum survival time of a time may translate into the requirement of smaller maintenance doses to maintain an effective serum drug concentration thereby minimizing the direct cost of drug therapy. In one aspect, the present invention provides an immune 30 globulin preparation comprising an immune globulin and a non-ionic surface active agent, where the non-ionic surface active agent is in a concentration sufficient to increase the serum half-life of the immune globulin. The immune globulin preparation may have more than one

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non-ionic surface active agent, so long as the non-ionic surface active agents are in a concentration sufficient to increase the serum half-life of the immune globulin. According to one embodiment, the immune globulin is

5 anti-Rh0D immune globulin wherein the anti-Rh0 D immune globulin has an IgG purity of greater than 95% and a monomeric protein content of greater than 94%. In a preferred embodiment, the preparation is aqueous. According to another embodiment, the immune globulin is anti-c immune globulin which has an IgG purity of greater than 95% 10 and a monomeric protein content of greater than 94%. According to another embodiment, the preparation has a concentration of the immune globulin of about 2 weight percent to about 10 weight percent. The non-ionic surface active agent may be a sorbitan ester 15 of a fatty acid selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate and sorbitan trioleate. The non-ionic surface active agent may also be a polyoxyethylene sorbitan ester of a fatty acid selected from the group 20 consisting of polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (4) sorbitan monolaurate; polyoxyethylene (20) sorbitan monopalmitate; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene (4) sorbitan monostearate; polyoxyethylene (20) sorbitan tristearate; polyoxyethylene (20) sorbitan monooleate; polyoxyethylene (5) sorbitan monooleate; and 25 polyoxyethylene (20) sorbitan trioleate. According to one embodiment, the preparation will have a concentration of the non-ionic surface active agent of about 0.01 weight percent to about 0.5 weight percent. According to another embodiment there is provided an 30 aqueous immune globulin preparation wherein the immune globulin has an increased serum half-life comprising: about 3-8% human anti­ Rh0D immune globulin with an IgG purity of greater than 95% and a monomeric protein content of greater than 94%; sodium chloride at about

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0.25% (w /v); very low level buffer with essentially no ionic strength; Polysorbate 80 at about 0.01 % to about 0.5% (w /v); and L-glycine at about O.lM. In another aspect, the present invention provides a method 5 of increasing the serum half-life of an immune globulin comprising administering a preparation comprising an immune globulin and a non­ ionic surface active agent in a physiologically acceptable medium to an animal in need thereof. In yet another aspect, the present invention provides a 10 method of reducing the elevation of neutrophil counts in a recipient comprising administering a preparation comprising an immune globulin and a non-ionic surface active agent in a physiologically acceptable medium to an animal in need thereof. According to either method the immune globulin preparation may be administered intravenously. 15 In all cases, the preparation possesses the novel characteristic of an extended serum half-life in vivo and reduced immunogenicity in comparison with equivalent immune globulin preparations not containing the non-ionic surface active agent. A preparation according to the present invention may be in 20 the format of a liquid formulation or may be lyophilized to form a powder formulation. The liquid formulation may be administered directly, while the lyophilized powder format may be reconstituted in a physiologically compatible medium before drug administration. In an embodiment, the immune globulin in a preparation 25 of the present invention is a human immune globulin prepared by extraction from human plasma using conventional cold ethanol fractionation method followed by a method to render the preparation suitable for intravenous administration or by chromatographic procedures. The immune globulin or binding partner may also be a 30 monoclonal antibody or binding partner produced by recombinant DNA or hybridoma technology. In another embodiment, the immune globulin in a

preparation of the present invention is an anti-D (also known as anti-Rh0

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or anti-Rh0 D) immune globulin; an anti-C (also known as anti-rh') immune globulin; an anti-E (also known as anti-rh") immune globulin; an anti-c (also known as anti-hr') immune globulin or anti-e (also known as anti-hr") immune globulin. This immune globulin may be prepared 5 by conventional cold ethanol fraction followed by a method to render the preparation suitable for intravenous administration, by chromatographic techniques or by recombinant DNA or hybridoma technology. Another aspect of the invention provides a method of extending the serum half-life or altering the immunomodulatory effect of 10 and immune globulin comprising the addition of a sufficient amount of one or more non-ionic surface active agents to the immune globulin formulation. The immune globulin may a human immune globulin extracted from plasma using conventional ethanol fractionation followed by a method to render the preparation suitable for intravenous 15 administration, by chromatographic methods, or it may be a monoclonal antibody or binding partner produced by recombinant DNA or hybridoma technology. More specifically, the immune globulin may be an anti-D

(also known as anti-Rh0 or anti-Rh0 D) immune globulin; an anti-C (also known as anti-rh') immune globulin; an anti-E (also known as anti-rh") 20 immune globulin; an anti-c (also known as anti-hr') immune globulin or anti-e (also known as anti-hr") immune globulin. In a further aspect of the invention, the immune globulin formulation is administered to a mammal by parenteral injection or infusion to elevate circulating immune globulin levels. 25 Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only and are not intended to limit, in any 30 way, the scope of the present invention. As will become apparent to those skilled in the art, various changes and modifications may be made based on the following detailed description, however, all are within the spirit and scope of the prersent invention.

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BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which:

Figure 1 is a graph of mean anti-Rh0 D immune globulin 5 blood levels after an intramuscular injection of WinRho SD™. Mean

serum anti-Rh0 D immune globulin results are shown for formulations with and without 0.01 % (w /v) Polysorbate 80. The solid diamonds show the results from subjects injected with the conventional formulation of WinRho SD™ in 0.9% (w /v) sodium chloride solution. The shaded 10 boxes show the results in subjects injected with new formulation of WinRho SD™ with Polysorbate 80 in 0.9% (w /v) sodium chloride solution.

Figure 2 is a graph of mean anti-Rh0 D immune globulin blood levels for up to 82 days after an intramuscular injection of WinRho

15 SD™. Mean serum anti-Rh0 D immune globulin results are shown for formulations with and without 0.01 % (w /v) Polysorbate 80. The solid diamonds show the results from subjects injected with the conventional formulation of WinRho SD™ in 0.9% (w/v) sodium chloride solution. The open squares show the results from subjects injected with the 20 formulation of WinRho SD™ with Polysorbate 80 in 0.9% (w /v) sodium chloride solution. DETAILED DESCRIPTION OF THE INVENTION As mentioned above, the present inventors have found that the addition of a non-ionic surface active agent to a preparation of an 25 immune globulin favourably alters the pharmacokinetics of the immune globulin as a therapeutic agent. The inclusion of said non-ionic surface active agent in the formulation prolongs the survival time or serum half­ life of said immune globulin. Accordingly, broadly stated, the present invention provides 30 an immune globulin preparation comprising an immune globulin and a non-ionic surface active agent, where the non-ionic surface active agent is in a concentration sufficient to increase the serum half-life of the immune globulin. The immune globulin preparation may have more

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than one non-ionic surface active agent, so long as the non-ionic surface active agents are in a concentration sufficient to increase the serum half­ life of the immune globulin. The phrase "surface active agent" means an agent that 5 reduces surface tension when dissolved in a solution, such as in water or an aqueous solution. The term "surfactant" is synonomous. The phrase "sufficient to increase the serum half-life of the immune globulin" means that the serum half-life of the immune globulin with at least one surface active agent is increased as compared to the serum half-life of the 10 immune globulin when administered without a surface active agent. The immune globulin of the present invention can be any immune globulin including IgG (all subclasses), IgA, IgD, IgE, and IgM and includes fragments of the immune globulins such as Fab' and F(ab'h fragments. The immune globulin is preferably non-aggregated (over 15 about 94% monomeric immune globulin) and has a purity of greater than about 95%. More preferably, the immune globulin preparation is in a form suitable for intravenous injection or infusion. An example of immune globulin that can be used in the present invention is Rh immune globulin or Rh antibodies. Rh

20 antibodies include anti-D (also known as anti-Rh0 or anti-Rh0 D); anti-C (also known as anti-rh'); anti-E (also known as anti-rh"); anti-c (also known as anti-hr') and anti-e {also known as anti-hr"). The Rh antibodies of the present invention may be preparations from plasma enriched for Rh antibodies, polyclonal antibodies, monoclonal antibodies, 25 antibody fragments (e.g. Fab, and F(ab')z), and those produced by recombinant DNA technology. Other immune globulin preparations suitable for intravenous injection or infusion, such as varicella zoster immune globulin (Varitect® by Biotest Pharma) or cytomegalovirus immune globulin (Cytogam® by Connaught), can also benefit from the 30 present invention. The inventors have also found that the addition of a non­ ionic surface active agent to an immune globulin reduced the elevation

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of patient neutrophil counts observed with a conventional immune globulin without the non-ionic surface active agent. Immune Globulin Production Preparations with a high Rh antibody content suitable for 5 intravenous injection or infusion may be isolated as an immune globulin fraction from plasma, preferably human plasma, using conventional techniques. For example, they may be isolated using: (a) the Cohn cold ethanol fractionation method or modifications thereto (see Huchet, J. et al., Rev. Fr. Trans/us. 13:231, 1970; Chown, B. et al., Can. Med. Assoc. J. 10 100:1021, 1969; Jouvenceaux, A. et al., Rev. Fr. Transfus. 12 (suppl.): 341, 1969; Barandun, S. et al., Vox Sang. 7: 157-174, 1962); (b) ion-exchange chromatographic methods (e.g. using DEAE-Sephadex) and modifications thereto may be used to produce Rh antibodies of higher product yield and quality (Cunningham, C.J. et al., Biochem. Soc. Trans. 8: 178, 1980; Hoppe, 15 H.H. et al., Vox. Sang. 25: 308, 1973; Hoppe, H.H. et al., Munch. Med. Wochenschr. 109: 1749, 1967; Baumstark, J.S. et al., Arch. Biochem. 108:514, 1964); and/ or (c) anion-exchange chromatographic method as taught in Canadian Patent No. 1,201,063, and modifications thereto.

Commercially available anti-Rh0 D immune globulin preparations may

20 also be used in the methods. For example, anti-Rh0 D preparations such as WinRho® or WinRho SD® (Cangene Corporation) may be used in the present invention.

In an embodiment of the invention, an anti-Rh0 D immune globulin fraction is prepared by contacting an aqueous plasma 25 fraction containing IgG with one or more chromatographic separation columns to produce a purified IgG-rich fraction. The aqueous plasma fraction used in the process may be normal non-immunized plasma from an animal source, preferably a human source, or hyperimmune plasma such as plasma from Rh alloimmunized donors.

30 For example, Rh0 D antigen is used to immunize an animal through intramuscular, subcutaneous, intraperitoneal, or intraocular injection, with or without an adjuvant such as Freund's complete or incomplete adjuvant. With the option of booster

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immunizations, samples of serum are collected and tested for reactivity to the antigen in standard assays (described below). Particularly preferred is polyclonal antisera which will give a signal on one of the assays that is at least three times greater than background. Once the titre of the animal 5 has reached a plateau in terms of antigen reactivity, larger quantities of the antisera may be obtained readily either by periodic bleeding or by exsanguinating the animal.

Human anti-Rh0 D immune globulin may also be produced

in human volunteers. For example, an anti-Rh0 D immune globulin 10 preparation may be obtained from a subject who is naturally immunized (e.g. from an Rh incompatible pregnancy) or artificially immunized using

Rh-positive blood cells or Rh0 D antigen.

Anti-Rh0 D immune globulin-containing plasma collected from animal or human is modified to the ionic strength and pH of the 15 initial buffer used with a chromatographic separation column. According to one embodiment of the invention, an aqueous animal plasma fraction is contacted with one or more, preferably one to two, anionic exchangers to produce a purified IgG-rich fraction. By way of example, an aqueous animal or human plasma 20 fraction is applied to an anion exchange column which may contain an agarose cross-linked anionic exchange resin such as DEAE-Sepharose CL6B, TMAE Fractogel or DEAE Sephadex A-50. An IgG-rich fraction is obtained from the column by eluting with an equilibrating buffer. The IgG-rich fraction may be concentrated, for example, by ultrafiltration. 25 The purified IgG protein may optionally be treated with a solvent and detergent to inactivate lipid envelope viruses. Suitable solvents and detergents which may be used include Triton X-100 and tri(n-butyl) phosphate (Horowitz, B., Curr. Stud. Hematol. Blood Transfus. 56: 83-96, 1989). After this process, the solvents and detergents 30 may be removed using conventional methods such as reverse phase chromatography. Monoclonal immune globulins may also be readily produced using recombinant and hybridoma techniques (see Canadian

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Patent number 1,303,534; Canadian Patent number 1,303,533; European Patent Application 87302620.7 published as EP 239,400; European Patent Application 93102609.0 published as EP 557,897; Fletcher, A. and Thompson, A., Trans/us. Med. Rev. 9: 314-326, 1995; Alting-Mees, M. et 5 al., Strat. Mol. Biol. 3: 1-9, 1990; Huse, W.D. et al., Science 246: 1275-1281, 1989; Sastry, L. et al., Proc. Natl. Acad. Sci. USA 86: 5728-5732, 1989). Similarly, binding partners or domains may be constructed using recombinant DNA techniques to incorporate the variable regions of a gene encoding a specific antibody (see PCT Patent Application 10 PCT /GB93/00605 published as WO 93/19172; PCT Patent Application PCT /GB93/02492 published as WO 94/13804; PCT Patent Application PCT /EP90/01964 published as WO 91/07492; Bird et al., Science 242: 423- 426, 1988). It will be apparent to one skilled in the art that fractionation and recombinant approaches may be applied to diverse types of immune 15 globulins. For example, specific monoclonal immune globulins against different antigens may be generated by techniques based on the same principle of recombinant DNA technology. Non-Ionic Surface Active Agents Non-ionic surface active agents of the present invention 20 can be any agent that can prolong the serum half-life of an immune globulin. Preferably, the surface active agent is of the Tween® or Span® type surface active agents. Span® type agents are partial esters of common fatty acids and sugar alcohol anhydrides derived from sorbitol. The common fatty 25 acids derived from sorbitol are preferably lauric acid, palmitic acid, stearic acid or oleic acid derived from sorbitol. For example, Span 20 is sorbitan monolaurate, Span 40 is sorbitan monopalmitate, Span 60 is sorbitan monostearate, Span 65 is sorbitan tristearate, Span 80 is sorbitan monooleate, and Span 85 is sorbitan trioleate. 30 Tween® type agents are derivatives of Span® products with polyoxyethylene chains attached to non-esterified hydroxyl groups. The common fatty acids derived from sorbitol are preferably lauric acid, palmitic acid, stearic acid or oleic acid derived from sorbitol. For example,

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Tween 20 is polyoxyethylene (20) sorbitan monolaurate, Tween 21 is polyoxyethylene (4) sorbitan monolaurate, Tween 40 is polyoxyethylene (20) sorbitan monopalmitate, Tween 60 is polyoxyethylene (20) sorbitan monostearate, Tween 61 is polyoxyethylene (4) sorbitan monostearate, 5 Tween 65 is polyoxyethylene (20) sorbitan tristearate, Tween 80 is polyoxyethylene (20) sorbitan monooleate, Tween 81 is polyoxyethylene (5) sorbitan monooleate, and Tween 85 is polyoxyethylene (20) sorbitan trioleate. Non-ionic surface agents such as sorbitan esters or 10 polyoxyethylene sorbitan esters of fatty acids may be prepared by methods well known in the art. Such surface active agents may also be obtained commercially from J.T. Baker Inc. (Phillipsburg, New Jersey, USA), ICI Atkemix (Brantford, Ontario, Canada), Van Waters and Rogers Ltd. (Richmond, British Columbia, Canada), or Nikkol Co. Gapan). 15 Immune Globulin Preparation

Formulation of anti-Rh0 D immune globulin of the present invention involves the addition of an amount of a non-ionic surface active agent sufficient to extend the serum half-life or to alter the immunomodulatory effect of the immune globulin to the IgG-rich 20 concentrate obtained as described above. The immune globulin preferably is at least about 95% pure, more preferably about 99.5% pure, contains at least 94% monomeric IgG, and has not been subjected to chemical or enzymatic modification. A preferred non-ionic surface active agent is Polysorbate 80 which is added to a final concentration of about 25 0.01 % to about 0.5%. Sodium chloride may be added to the formulation to a final concentration of up to about 0.9%. An additional stabilizing agent such as L-glycine or L-histidine may be added to a final concentration of about 0.025M to 0.05M. A preferred preparation contains

the following: a pharmacologically effective amount of human anti-Rh0 D 30 immune globulin (about 3-8%); sodium chloride at about 0.25% (w /v); no or very low level buffer with essentially no ionic strength; Polysorbate 80 at a concentration of about 0.01 %-0.02% (w /v); and L-glycine at a concentration of about O.lM.

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The anti-Rh0 D immune globulin formulation is aseptically filtered again through a 0.22 micron filter and put into vials or ampoules. Filling operations are conducted under aseptic conditions and the fill volume per vial is calculated so that each vial contains a

5 pharmacologically effective amount of anti-Rh0 D immune globulin. This specific amount or volume can vary depending upon the intended route of administration and therapeutic use. The target filling volume is also calculated with sufficient excess to allow for variation in the potency assay and/ or possible loss of potency during storage. 10 The final aqueous formulation may be lyophilized using a Virtus 251 SRC-5 Sublimator (or equivalent). Lyophilization (also termed vacuum freeze-drying or sublimation) is commonly used in the manufacture of protein pharmaceuticals to improve the stability of the product and extend its shelf life. The lyophilization process is often 15 described as being divided into three stages: freezing; primary drying (also termed ice sublimation); and secondary drying (also termed water desorption). The starting aqueous solution containing the protein is frozen and the ice is subsequently sublimed thereby leaving a dry porous mass of protein which is stable and can be reconstituted rapidly in water. 20 The technical parameters of the lyophilization process including temperature (eutectic and collapse}, vacuum pressure and atmospheric gas composition, are fully automated by the Virtus 251 SRC-5 Sublimator. The basic theories and more practical aspects of protein lyophilization and formulation are described in detail in Skrabanja, A.T. et al. (J. Pharm. Sci. 25 Technol. 48: 311-317, 1994); Rey, L.R. (Dev. Biol. Stand. 74: 3-8, 1992); Pikal, M.J. (BioPharm October, 26-30, 1990); Pikal, M.J. (J. Parenter. Sci. Technol. 39: 115-138, 1985); Williams, N.A. and Polli, G.P. (J. Parenter. Sci. Technol. 38: 48-59, 1984; Nail, S.L. (J. Parenter. Drug Assoc. 34: 358-368, 1980); Ito, K. (Chem. Pharm. Bull. 19: 1095-1102, 1971). 30 In the case of a lyophilized powder formulation, the powder comprising the immune globulin and the non-ionic surface active agent is reconstituted in a physiologically compatible diluent such as sterile water for injection or saline before parenteral administration.

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For example, 120 ug (600 IU) or 300 ug (1,500 IU) of a commercial anti­ Rh0D immune globulin product, WinRho SD™, is reconstituted in 2.5 mL diluent. Therapeutic dosage of immune globulin preparation 5 Dosages of anti-Rh0D immune globulin in formulations of the present invention depend on individual needs, on the protein content/ concentration of the immune globulin preparation, on the desired effect in a particular therapeutic indication, and on the chosen route of drug administration. Daily dosages of an anti-Rh0D immune 10 globulin preparation (3% to 8% wt-solution) for humans by intramuscular or intravenous injection generally vary between about 50 IU (10 ug) to 2,000 IU (400 ug) per kg body weight. For intramuscular injection, the preferred dosage is about 100 IU (20 ug) to 2,000 IU (400 ug) per kg body weight. For intravenous injection, the preferred dosage is 15 about 50 IU (10 ug) to 1,000 IU (200 ug) per kg body weight, preferably 250 IU ( 50 ug) per kg body weight. The recommended dosage of Biotest Pharma's intravenous varicella zoster immune globulin preparation, Varitect®, is about 50 IU per kg body weight for shingles therapy and are lower (about 12 to 25 IU per kg body weight) for chickenpox prophylaxis. 20 In contrast, the recommended dosages of general intravenous immune globulin products (4.5-5.5 wt-% solution) such as Gamimune®, lveegam® or Sandoglobulin® are significantly higher at about 100 mg to 800 mg per kg body weight. EXAMPLES 25 EXAMPLE1

Pharmacokinetics of anti-Rh0 D immune globulin given as Polysorbate 80 formulation over a period of 30 days

Methods. The Pharmacokinetics of anti-Rh0 D immune globulin formulations with and without Polysorbate 80 were assessed in a single- 30 centre, randomized, parallel arm study. Twenty-four human subjects (normal, healthy male and female volunteers of age 18 to 55 years) were randomized into two groups to receive 600 µg (3,000 IU) of a commercial

brand of anti-Rh0 D immune globulin, WinRho SD®. Twelve subjects

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received the conventional WinRho SD® formulation without Polysorbate 80, and the other 12 subjects received the WinRho SD® formulation with 0.01 % (w /v) Polysorbate 80. Each formulation was given intramuscularly as two 1.25 mL injections with the test articles 5 being lyophilized human anti-Rh0D immune globulin in 0.9% (w /v) saline for intravenous injection with or without Polysorbate 80. Screening assessments were conducted within three weeks of drug administration. Baseline assessments were conducted on the morning of the day that WinRho SD™ was administered to the subjects 10 and before drug administration. These included haematology, blood chemistry and urinalysis. Demographics, vital signs and baseline laboratory tests were compared for study subjects randomized to the different arms of treatment. There was no statistically significant difference between the two treatment groups in any assessments prior to 15 drug administration. Subjects remained under observation for eight hours and blood samples for anti-Rh0D immune globulin analysis were drawn from the study subjects to provide 5 mL serum samples at the following times after study drug administration: 8 hours, 24 hours, 3 days, 7 days, 11 days, 20 14 days, 21 days, and 28 days. Subjects also underwent haematology, blood chemistry and urinalysis laboratory testing at 7 days and 28 days after WinRho SD™ injection. Anti-Rh0D immune globulin concentration in patient samples was analyzed by conventional techniques (see Auto­ Analyzer technique in Moore, B.P.L., Can. Med. Assoc. J. 100: 381-387, 25 1969). Pharmacokinetics. Regressions were performed on log transformed

corrected serum anti-Rh0 D immune globulin levels against time to determine lambda and subsequently, the estimated half-life of drug in the study subjects. Blood levels after the day 3 draw were used in these 30 regressions as peak serum levels of anti-Rh0D immune globulin were usually obtained by day 3 after injection of drug. Significant linear relationships (p<0.02) existed between the variables Log (corrected anti­ Rh0D immune globulin) and Time for all study subjects.

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The mean time to peak anti-Rh0 D immune globulin levels after intramuscular injection of 600 µg of WinRho SD™ was about 3 days

and peak serum anti-Rh0 D immune globulin levels that were achieved were about 70 ng/mL. There was no statistically significant difference 5 between the two formulations and the mean time to peak or peak levels

of drug. Similarly, the AUC28 day was about 1250 days•ng/mL and there was no difference between the two formulations in these values. The mean half-life was different for the two formulations of WinRho SD™. The formulation of drug without Polysorbate 80 had a 10 mean apparent serum half-life of 16.4 ± 3.8 days, while the mean apparent half-life in subjects whom received the formulation with Polysorbate 80 was 20.3 ± 3.4 days. This difference is statistically significant (p=0.012) in a Student t test of the difference between means. Key pharmacokinetic data are presented in Table 1. 15 TABLE1 Formulation without Formulation with Significance Polysorbate 80 Polysorbate 80 Time to Peak (dS)s) Mean± D 2.83 ± 0.14 3.65 ± 1.55 p=0.100 Median 3.00 2.99 Range 1.00 - 3.04 2.96 - 6.99 PeakAnti-D (ng/mL) Mean±SD 76.2 ± 12.2 68.8 ± 11.8 p=0.157 Median 79.7 68.8 Range 54.76 - 99.09 49.28 - 88.39 AUC (days•ng/mL) Mean±SD 1225 ± 195 1273 ± 268 p=0.673 Median 1278 1174 Range 938 - 1564 982 - 1803 Serum Half-Life (days) Mean±SD 16.4 ± 3.6 20.3± 3.4 P=0.012 Median 17.0 20.6 Range 9.04 - 21.91 15.07 - 26.74

Pharmacokinetics of anti-Rh0 D immune globulin given as Polysorbate 80 formulation over a period of 84 days. 20 5 mL serum samples were also collected from the same

subjects on 42, 56 and 84 days post injection and serum anti-Rh0 D immune globulin levels were determined for up to 84 days post administration of WinRho SD™.

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Individual subject pharmacokinetics are presented in Table 2. The slope from the regression of log (corrected anti-D) on actual time after injection of WinRho SD (up to day 84) data was included in the regression analysis.

TABLE2

Subject Time to Peak PeakAnti-D Corrected Peak A. t 1/2 AUC (Days) (ng/mL) Anti-D {ng/mL) (days•ng/mL) (/day) (days)

WinRho SD™ Formulation (without Polysorbate 80)

SS02 3 91.54 83.22 0.040118 17.278 1940.45

SS03 3 80.04 72.76 0.040598 17.073 1853.01

SS05 1 88.67 79.06 0.028732 24.125 2544.84

SS06 2.99 89.3 81.18 0.050862 13.628 1135.8

SS08 2.98 62 56.36 0.033751 20.537 1597.15

SSlO 3 55.42 54.76 0.032355 21.423 1435.6

SS14 2.98 86.74 78.85 0.035784 19.37 1979.34

SS16 2.96 66.71 70.19 0.035764 19.381 1489.86

SS18 2.99 95.05 99.09 0.036003 19.252 2263.9

SS19 3.03 85.31 84.67 0.029916 23.17 2034.25

SS20 3.04 80.59 81.04 0.040478 17.124 1866.07

SS22 3 74.15 71.41 0.042734 16.22 1600.78

WinRho SD™ Formulation (with Polysorbate 80)

SS01 3 82.8 75 0.031157 22.247 2131.76

SS04 3.08 66.56 60.29 0.027221 25.464 1769.77

SS07 6.97 88.32 80 0.018439 37.591 3043.64

SS09 2.96 77.23 69.95 0.031561 21.962 1759.42

SSll 2.96 82 74.28 0.033716 20.558 1986.99

SS12 6.99 97.58 88.39 0.02761 25.105 2963.95

SS13 3 91.59 82.96 0.035027 19.789 2300.9

S515 2.96 74.57 67.55 0.028825 24.047 1998.26

SS17 2.96 62.85 56.93 0.033925 20.432 1702.44

SS21 2.98 71.01 64.32 0.033193 20.882 1710.56

SS23 2.99 62.97 57.04 0.030317 22.863 1679.9

S524 2.98 54.41 49.28 0.024491 28.302 1737.46

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Mean peak values of anti-Rh0 D immune globulin levels were 79.6 + 12.6 ng/mL for the conventional formulation and 76.0 + 13.0 ng/mL for the new formulation which are not significantly different (t = -0.69603; p = 5 0.4937). Mean time-to-peak values were 3.65 + 2.56 days for the conventional formulation and 2.83 + 0.58 days for the new formulation which are not significantly different (t = -1.71648; p = 0.10012). Similarly,

mean area under the curve (AUC84 day) values were 1811.8 + 385.3 days for the conventional formulation and 2065.4 + 479.6 days.ng/mL for the new 10 formulation which are not significantly different (t = -1.4284; p = 0.16722). Conversely, the mean apparent half-life values for the two WinRho SD™ formulations were different. The formulation of drug without Polysorbate 80 had a mean apparent serum half life of 19.1 + 3.00 days, while the corresponding value for the drug formulation with Polysorbate 15 80 was 24.1 + 4.9 days (t = -3.03582; p = 0.006). Safety Of Administration Twenty-four subjects completed 28 days of participation in this study and no subject was withdrawn because of adverse experience. There were a total of 33 adverse events reported in this study (see Table 3). 20 The majority of the events occurred in the Body As A Whole (13 events) and the Respiratory System (9 events). For the most part, the events were evenly distributed between the two different arms of treatment. An exception was 3 adverse events that occurred in the Digestive System: two reports of dyspepsia and one report of vomiting occurred in subjects 25 receiving WinRho SD™ without polysorbate 80. However, since none of the events that occurred in the Digestive System were believed to be related to the WinRho SD™ injection, this observation is not considered significant.

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TABLE3 Preferred Term All Reports Formulation without Formulation with Polysorbate 80 Polysorbate 80 #Events #{%) #Events #(%) # #{%) Events BODY AS A WHOLE 13 13 (54%) 6 6 (50%) 7 7 (58%) CARDIOVASCULAR 1 1 (4%) 0 0 1 1 (8%) SYSTEM DIGESTIVE SYSTEM 3 3 (12%) 3 3 (25%) 0 0 NERVOUS SYSTEM 3 3 (12%) 2 2 {17%) 1 1 (8%) RESPIRATORY SYSTEM 9 6 {25%) 4 3 {25%) 5 3 (25%) SPECIAL SENSES 2 2 (8%) 0 0 2 1 (17%) UROGENITAL SYSTEM 2 1 (4%) 2 1 (8%) 0 0

On the morning of study drug administration and before drug injection, a baseline assessment was conducted which included Vital Signs. The Vital 5 Signs were then assessed in study subjects at 1 hour, 3 hour, 8 hour, 24 hour, 7 day and 28 days after drug administration. There were no statistically significant changes in group Vital Signs from baseline and all mean group changes in Vital Signs were within a standard deviation of zero. The largest changes in vital signs relative to the variance was in the 10 temperature of the subjects receiving WinRho SD™ without polysorbate 80 at early times after drug administration. Body temperature was elevated by 0.3±0.4 °C at 1 hour, 0.3±0.3 °C at 3 hours and 0.5±0.5 °C at 8 hours after WinRho SD™ injection compared to increases in this group of 0.2±0.4 oc at 24 hours, 0.1±0.6 °C at 7 days and 0.1±0.4 °C at 28 days after 15 drug administration. In contrast, the study subjects who received WinRho SD™ with polysorbate 80 had body temperature increases of 0.0±0.5 °C, 0.1±0.5 °C, 0.2±0.5 °C, 0.0±0.4 °C, 0.1±0.7 °C and 0.1±0.4 °C at the assessments after drug administration. Given the low statistical significance to these changes in body temperature, it is not clear if they are 20 related to WinRho SD™ administration. If they are, then the changes are subclinical and the pyrogenic effect is smaller with the new formulation of WinRho SD™, i.e., WinRho SD™ with polysorbate 80. On the morning of study drug administration and before drug injection, a baseline assessment was conducted that included

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laboratory testing. These laboratory tests were repeated in study subjects at 7 days and 28 days after injection of WinRho SD™. Day 7 laboratory data was not significantly different from baseline data and mean Li parameter values were within a standard deviation of zero. However, 5 the mean neutrophil count for the patients receiving the conventional, or old formulation, i.e., WinRho SD™ without polysorbate 80, was 4.67% ± 4.72% seven days after drug administration and this change was statistically different (p=0.040) from the mean increase of -0.75% ± 7.20% in neutrophils 7 days after administration of the new formulation of 10 WinRho SD™. This difference accounted, in part, for the differences between arms in the proportion of WBC that were neutrophils (61 % vs. 53%) and in the bands (4.44xl09 /L vs. 3.07xl09 /L) at 7 days after drug administration. There are no statistically significant differences in 15 treatment arms in the haematology laboratory data obtained 28 days after drug administration. There was an overall decrease of 1.3% in the haematocrit of the study subjects 28 days after WinRho SD™ that may have been related to the frequent phlebotomy associated with participation in the study. 20 There were differences in the 7 day LiALT and LiBilirubin with data from the subjects receiving new formulation being closer to baseline values than data from subjects receiving old formulation. However, the mean differences reflected subclinical changes and there were no differences in mean ALT and mean Bilirubin data for these 25 groups. As such, these differences were believed to be fortuitous and result from the frequent hypothesis testing in this analysis. There were no statistically significant differences between the treatment arms in the clinical chemistry data obtained 28 days after study drug administration. Conclusion. The formulation 6f WinRho SD™ that contains Polysorbate 30 80 had the same, or a better, safety profile as the currently licensed WinRho SD™. This is consistent with the relatively high LDSO values for non-ionic surface active agents in mammals (supra.). This new formulation also improved the appearance and stability of WinRho SD™

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when reconstituted. Surprisingly, the formulation with Polysorbate 80 has a longer half-life (20.3 days) than the formulation without Polysorbate 80 (16.4 days). This difference is beneficial in the therapeutic use of the drug. For example, the new WinRho SD™ formulation with Polysorbate

5 80 leads to higher passive anti-Rh0 D immune globulin levels for long times after drug administration in prophylaxis of Rh Immunization of Rh negative patients. Moreover, inclusion of Polysorbate 80 in the anti­ Rh0D immune globulin preparation significantly minimized drug­ induced elevations of neutrophil counts in the recipients and altered the 10 immunomodulatory effect of the immune globulin. While the present invention has been described with reference to what are presently considered to be preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various 15 modifications and equivalents included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and 20 individually indicated to be incorporated by reference in its entirety.

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We Claim:

1. An immune globulin preparation comprising an immune globulin and at least one non-ionic surface active agent, said one or more 5 non-ionic surface active agent{s) in a concentration sufficient to increase the serum half-life of the immune globulin.

2. The preparation according to claim 1 wherein the immune globulin is anti-Rh0D immune globulin.

3. The preparation according to claim 2 wherein the anti­ Rh0D immune globulin has an IgG purity of greater than about 95% and a monomeric protein content of greater than about 94%.

15 4. The preparation according to claim 3 which is aqueous.

5. The preparation according to claim 1 wherein the immune globulin is anti-c immune globulin.

20 6. The preparation according to claim 5 wherein the anti-c immune globulin has an IgG purity of greater than about 95% and a monomeric protein content of greater than about 94%.

7. The preparation according to claim 6 which is aqueous.

8. The preparation according to claim 1 wherein the concentration of the immune globulin is about 2 weight percent to about 10 weight percent.

30 9. The preparation according to claim 1 wherein the one or more non-ionic surface active agent(s) is(are) a sorbitan ester of a fatty acid.

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10. The preparation according to claim 9 wherein the non­ ionic surface active agent(s) is(are) selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate.

11. The preparation according to claim 1 wherein the one or more non-ionic surface active agent(s) is(are) a polyoxyethylene sorbitan ester of a fatty acid.

10 12. The preparation according to claim 11 wherein the non- ionic surface active agent(s) is(are) selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (4) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (4) sorbitan 15 monostearate, polyoxyethylene (20) sorbitan tristearate, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (5) sorbitan monooleate, and polyoxyethylene (20) sorbitan trioleate.

13. The preparation according to claim 1 wherein two or more 20 non-ionic surface active agents are selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (4) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate; polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, polyoxyethylene 25 (20) sorbitan monooleate, polyoxyethylene (5) sorbitan monooleate, and polyoxyethylene (20) sorbitan trioleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate.

30 14. The preparation according to claim 1 wherein the concentration of the one or more non-ionic surface active agent(s) is(are) about 0.01 weight percent to about 0.5 weight percent.

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15. The preparation according to claim 1 wherein the aqueous immune globulin preparation is lyophilized to form a dry powder preparation.

5 16. An aqueous immune globulin preparation wherein the immune globulin has an increased serum half-life comprising:

about 3-8% human anti-Rh0 D immune globulin with an IgG purity of greater than 95% and a monomeric protein content of greater than 94%; 10 sodium chloride at about 0.25% (w /v); very low level buffer with essentially no ionic strength; Polysorbate 80 at about 0.01 % to about 0.5% (w /v); and L-glycine at about 0.1M.

15 17. A use of an immune globulin preparation according to any one of claims 1 to 16 to increase the serum half-life of an immune globulin.

18. A use of an immune globulin preparation according to any 20 one of claims 1 to 16 to reduce the elevation of neutrophil counts.

19. A method of increasing the serum half-life of an immune globulin comprising administering an immune globulin preparation according to claims 1 to 16 to an animal in need thereof.

20. A method of reducing the elevation of neutrophil counts in a recipient of immune globulin comprising administering an immune globulin preparation according to claims 1 to 16 to an animal in need thereof.

21. A method according to claim 19 or 20 wherein said immune globulin preparation is administered intravenously.

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1/2 FIGURE 1

-~ 70 c, 60 C - 50 C ;; 40 C cu 30 ~ §.. 20 : 10

10 20 30 Time after Injection (days)

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2/2

FIGURE2

100 ~- 1--:-=1 i ~.. -0 10 1 ' ~~·~D i • 1 0 20 40 60 80 100 lime after Injection (days)

IPR Page 1844 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: June 2, 2011 Docket No. 117813-26902 Electronic Signature for Deborah L. Nagle, Ph.D.: /Deborah L. Nagle/

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Patent Application of: Wolfgang Fraunhofer

Application No.: 12/325,049 Confirmation No.: 1766

Filing Date: November 28, 2008 Art Unit: 1636

For: PROTEIN FORMULATIO NS AND METHODS Examiner: Not yet assigned OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

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Pursuant to 37 C.F.R. §§ 1.56, 1.97 and 1.98, the attention of the Patent and Trademark Office is hereby directed to the references listed on the attached PTO/SB/08. It is respectfully requested that the information be expressly considered during the prosecution of this application, and that the references be made of record therein and appear among the "References Cited" on any patent to issue therefrom.

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In accordance with 37 C.F.R. § 1.97, Applicants cite the commonly owned co-pending applications directed to related subject matter:

U.S. Application/ Patent Filing Date Publication No. Status No. 12/770,557 April 29, 2010 US 2011-0054414 Al !Pending 12/772,595 May 3, 2010 US-2010-0278822-Al Pending

ME1 11784587v.1

IPR Page 1845 of 2482 Application No.: 12/325,049 Docket No.: 117813-29602

Applicants note that although U.S. Application Numbers 12/770,557 and 12/772,595 were previously listed and cited in the Information Disclosure Statement dated October 19, 2010, these two applications have recently published. Applicants, therefore, have included them again to reflect the updated publication information as A16 and Al 7 on the SB-08 attached herewith. Applicant understands the Examiner can access papers from the prosecution of the cited cases electronically. However, if the Examiner has difficulty obtaining papers from that source he is invited to call the undersigned who will be happy to supply them.

In accordance with 37 CFR 1.97(g), the filing of this Information Disclosure Statement shall not be construed to mean that a search has been made or that no other material information as defined in 37 CFR 1.56(a) exists. In accordance with 37 CFR 1.97(h), the filing of this Information Disclosure Statement shall not be construed to be an admission that any patent, publication or other information referred to therein is "prior art" for this invention unless specifically designated as such.

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Please charge our Deposit Account No. 50-4876 in the amount of $180.00 covering the fee set forth in 37 CFR 1.17(p). The Director is hereby authorized to charge any deficiency in the fees filed, asserted to be filed or which should have been filed herewith (or with any paper hereafter filed in this application by this firm) to our Deposit Account No. 50-4876, under Order No. 117813-26902.

Dated: June 2, 2011 Respectfully submitted,

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2 MEI 11784587v.1

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D SEARCH FEE N/A N/A N/A N/A (37 CFR 1.16(k), (i), or (ml)

D EXAMINATION FEE N/A N/A N/A N/A (37 CFR 1.16(0), (p), or (q)) TOTAL CLAIMS X $ OR X $ (37 CFR 1.16(i)) minus 20 = * = = INDEPENDENT CLAIMS X $ X $ (37 CFR 1.16(h)) minus 3 = * = = If the specification and drawings exceed 100 sheets of paper, the application size fee due 0APPLICATION SIZE FEE is $250 ($125 for small entity) for each (37 CFR 1.16(s)) additional 50 sheets or fraction thereof. See 35 U.S.C. 41(a)(1)(G) and 37 CFR 1.16(s). D MULTIPLE DEPENDENT CLAIM PRESENT (37 CFR 1.16(j)) * If the difference in column 1 is less than zero, enter "O" in column 2. TOTAL TOTAL

APPLICATION AS AMENDED - PART II OTHER THAN (Column 1) (Column 2) (Column 3) SMALL ENTITY OR SMALL ENTITY CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) f-- 06/02/2011 AFTER PREVIOUSLY EXTRA FEE($) FEE($) z AMENDMENT PAID FOR w Total (37 CFR Minus X $ = OR X $52= ~ 1.16(i)) · 72 ** 839 = 0 0 0 Independent z * 9 Minus *** 7 = 2 X $ = OR X $220= 440 w (37 CFR 1 .16(h)) ~ D Application Size Fee (37 CFR 1.16(s)) <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L 440 FEE FEE

(Column 1) (Column 2) (Column 3) CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) AFTER PREVIOUSLY EXTRA FEE($) FEE($) AMENDMENT PAID FOR f-- Total (37 CFR z * Minus ** X $ = OR X $ = w 1.16(i)) = Independent ~ Minus X $ OR X $ 0 (37 CFR 1 .16(h)) * *** = = = z w D Application Size Fee (37 CFR 1.16(s)) ~ <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L FEE FEE * If the entry in column 1 is less than the entry in column 2, write "O" in column 3. Legal Instrument Examiner: ** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 20, enter "20". /TONYA MCBRIDE/ *** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 3, enter "3". The "Highest Number Previously Paid For" (Total or Independent) is the highest number found in the appropriate box in column 1. This collection of 1nformat1on 1s required by 37 CFR 1.16. The 1nformat1on 1s required to obtain or retain a benefit by the public which 1s to file (and by the US PTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1 .14. This collection is estimated to take 12 minutes to complete, including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and Trademark Office, U.S. Department of Commerce, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450. If you need assistance in completing the form, ca/11-800-PT0-9199 and select option 2.

IPR Page 1847 of 2482 PTO/SB/06 (07-06) Approved for use through 1/31/2007. 0MB 0651-0032 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid 0MB control number. PATENT APPLICATION FEE DETERMINATION RECORD Application or Docket Number Filing Date Substitute for Form PT0-875 12/325,049 11/28/2008 D To be Mailed

APPLICATION AS FILED - PART I OTHER THAN (Column 1) (Column 2) SMALL ENTITY D OR SMALL ENTITY FOR NUMBER FILED NUMBER EXTRA RATE($) FEE($) RATE($) FEE($)

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D EXAMINATION FEE N/A N/A N/A N/A (37 CFR 1.16(0), (p), or (q)) TOTAL CLAIMS X $ OR X $ (37 CFR 1.16(i)) minus 20 = * = = INDEPENDENT CLAIMS X $ X $ (37 CFR 1.16(h)) minus 3 = * = = If the specification and drawings exceed 100 sheets of paper, the application size fee due 0APPLICATION SIZE FEE is $250 ($125 for small entity) for each (37 CFR 1.16(s)) additional 50 sheets or fraction thereof. See 35 U.S.C. 41(a)(1)(G) and 37 CFR 1.16(s). D MULTIPLE DEPENDENT CLAIM PRESENT (37 CFR 1.16(j)) * If the difference in column 1 is less than zero, enter "O" in column 2. TOTAL TOTAL

APPLICATION AS AMENDED - PART II OTHER THAN (Column 1) (Column 2) (Column 3) SMALL ENTITY OR SMALL ENTITY CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) f-- 06/02/2011 AFTER PREVIOUSLY EXTRA FEE($) FEE($) z AMENDMENT PAID FOR w Total (37 CFR Minus X $ = OR X $52= ~ 1.16(i)) · 72 ** 839 = 0 0 0 Independent z * 9 Minus *** 7 = 2 X $ = OR X $220= 440 w (37 CFR 1 .16(h)) ~ D Application Size Fee (37 CFR 1.16(s)) <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L 440 FEE FEE

(Column 1) (Column 2) (Column 3) CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) AFTER PREVIOUSLY EXTRA FEE($) FEE($) AMENDMENT PAID FOR f-- Total (37 CFR z * Minus ** X $ = OR X $ = w 1.16(i)) = Independent ~ Minus X $ OR X $ 0 (37 CFR 1 .16(h)) * *** = = = z w D Application Size Fee (37 CFR 1.16(s)) ~ <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L FEE FEE * If the entry in column 1 is less than the entry in column 2, write "O" in column 3. Legal Instrument Examiner: ** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 20, enter "20". /TONYA MCBRIDE/ *** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 3, enter "3". The "Highest Number Previously Paid For" (Total or Independent) is the highest number found in the appropriate box in column 1. This collection of 1nformat1on 1s required by 37 CFR 1.16. The 1nformat1on 1s required to obtain or retain a benefit by the public which 1s to file (and by the US PTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1 .14. This collection is estimated to take 12 minutes to complete, including gathering, preparing, and submitting the completed application form to the USPTO. Time will vary depending upon the individual case. Any comments on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and Trademark Office, U.S. Department of Commerce, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450. If you need assistance in completing the form, ca/11-800-PT0-9199 and select option 2.

IPR Page 1848 of 2482 Document code: WFEE

United States Patent and Trademark Office Sales Receipt for Accounting Date: 06/21/2011

TMCBRIDE SALE #00000002 Mailroom Dt: 06/02/2011 504876 12325049 01 FC : 1201 440.00 DA

IPR Page 1849 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with § 1.6(a)(4). Dated: July 6, 2011 Electronic Signature for Deborah L. Nagle, Ph.D.: /Deborah L. Nagle/ PTO/SB/08b (11-08) Approved for use through 12/31/2008. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid 0MB control number.

Complete if Known Substitute for form 1449/PTO Application Number 12/325,049-Conf.# 1766 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1646 (Use as many sheets as necessary) Examiner Name Hissong, Bruce D. Sheet I 1 I 01 I 7 Attorney Docket Number 117813-26902

Pages, Columns, Document Number Publication Date Name of Patentee or Examiner Cite Lines, Where Relevant Initials' No. 1 Number-Kind Code2 ( if known) MM-DD-YYYY Applicant of Cited Document Passages or Relevant Fi~ u res Appear

A1* US-20070184045 08-09-2007 Doctor et al

A2* US-20070269463 11-22-2007 Donavan et al

A3* US-20060142549 06-29-2006 Takeda et al

A4* US-20070237762 10-11-2007 Winter

!Examiners;ga,rurn I ICorn;d,rndDate

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901 .04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11955346v. 1

IPR Page 1850 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Filer: Deborah L. Nagle

Attorney Docket Number: 117813-26902

Filed as Large Entity

Utility under 35 USC 111 (a) Filing Fees

Sub-Total in Description Fee Code Quantity Amount USO($)

Basic Filing:

Pages:

Claims:

Miscellaneous-Filing:

Petition:

Patent-Appeals-and-Interference:

Post-Allowance-and-Post-Issuance:

Extension-of-Time:

IPR Page 1851 of 2482 Sub-Total in Description Fee Code Quantity Amount USO($)

Miscellaneous:

Submission- Information Disclosure Stmt 1806 1 180 180

Total in USD ($) 180

IPR Page 1852 of 2482 Electronic Acknowledgement Receipt

EFSID: 10460485

Application Number: 12325049

International Application Number:

Confirmation Number: 1766

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Customer Number: 87501

Filer: Deborah L. Nagle

Filer Authorized By:

Attorney Docket Number: 117813-26902

Receipt Date: 06-JUL-2011

Filing Date: 28-NOV-2008

Time Stamp: 14:30:49

Application Type: Utility under 35 USC 111 (a)

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Payment was successfully received in RAM $180

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Deposit Account 504876

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The Director of the USPTO is hereby authorized to charge indicated fees and credit any overpayment as follows: Charge any Additional Fees required under 37 C.F.R. Section 1.16 (National application filing, search, and examination fees) Charge any Additional Fees required under 37 C.F.R. Section 1.17 (Patent application and reexamination processing fees)

IPR Page 1853 of 2482 Charge any Additional Fees required under 37 C.F.R. Section 1.19 (Document supply fees)

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File Listing:

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This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents, characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a Post Card, as described in MPEP 503.

New A~~lications Under 35 U.S.C. 111 If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR 1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this Acknowledgement Receipt will establish the filing date of the application.

National Stage of an International A~~lication under 35 U.S.C. 371 If a timely submission to enter the national stage of an international application is compliant with the conditions of 35 U.S.C. 371 and other applicable requirements a Form PCT/DO/E0/903 indicating acceptance of the application as a national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt, in due course.

New International A~~lication Filed with the USPTO as a Receiving Office If a new international application is being filed and the international application includes the necessary components for an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number and of the International Filing Date (Form PCT/R0/1 OS) will be issued in due course, subject to prescriptions concerning national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of the application.

IPR Page 1854 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Docket No.: 117813-29602

Dated: July 6, 2011 Electronic Signature for Deborah L. Nagle, Ph.D.: /Deborah L. Nagle/

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Patent Application of: Wolfgang Fraunhofer

Application No.: 12/325,049 Confirmation No.: 1766

Filing Date: November 28, 2008 Art Unit: 1636

For: PROTEIN FORMULATIO NS AND METHODS Examiner: Hissong, Bruce D. OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

SUPPLEMENTAL INFORMATION DISCLOSURE STATEMENT {IDS)

Dear Sir:

Pursuant to 37 C.F.R. §§ 1.56, 1.97 and 1.98, the attention of the Patent and Trademark Office is hereby directed to the references listed on the attached PTO/SB/08. It is respectfully requested that the information be expressly considered during the prosecution of this application, and that the references be made of record therein and appear among the "References Cited" on any patent to issue therefrom.

This Supplemental Information Disclosure Statement is filed under 37 C.F.R. § 1.97(c), after the mailing date of a non-Final Office Action, but before the mailing date of any of a final action under § 1.113, a notice of allowance under § 1.311, or an action that otherwise closes prosecution in the application, and it is accompanied by the fee set forth in§ 1.17(p).

In accordance with 37 C.F.R. § 1.98(a)(2)(ii), Applicants have not submitted copies of U.S. patents and U.S. patent applications. Applicants submit herewith copies of foreign patents and non­ patent literature in accordance with 37 C.F.R. § 1.98(a)(2).

In accordance with 37 CFR 1.97(g), the filing of this Information Disclosure Statement shall not be construed to mean that a search has been made or that no other material information as defined in 37 CFR 1.56(a) exists. In accordance with 37 CFR 1.97(h), the filing of this Information Disclosure Statement shall not be construed to be an admission that any patent, publication or other information referred to therein is "prior art" for this invention unless specifically designated as such.

1 MEI 11955674v.1

IPR Page 1855 of 2482 Application No.: 12/325,049 Docket No.: 117813-29602

It is submitted that the Information Disclosure Statement is in compliance with 37 CFR 1.98 and the Examiner is respectfully requested to consider the listed references.

Please charge our Deposit Account No. 50-4876 in the amount of $180.00 covering the fee set forth in 37 CFR 1.17(p). The Director is hereby authorized to charge any deficiency in the fees filed, asserted to be filed or which should have been filed herewith (or with any paper hereafter filed in this application by this firm) to our Deposit Account No. 50-4876, under Order No. 117813-26902.

Dated: July 6, 2011 Respectfully submitted,

By I Deborah L. Nagle/ Deborah L. Nagle, Ph.D. Registration No.: 59,978 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6512 (617) 607-9200 (Fax) Attorney/Agent For Applicants

2 MEI 11955674v.1

IPR Page 1856 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: July 9, 2011 Electronic Signature for Cristin Howley Cowles, Ph.D.: /Cristin Cowles/

ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name B. Hissong

Sheet I 1 I of I 2 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Pages, Columns, Lines, Examiner Document Number Cite Publication Date Name of Patentee or Where Relevant Passages or Initials* 2 No. 1 Number-Kind Code ( if MM-DD-YYYY Applicant of Cited Relevant known) Document Fiqures Appear A1 us 4877608 10-31-1989 Lee et al. A2 us 6436397 08-20-2002 Baker et al. A3 US20040197324 10-07-2004 Liu et al. A4 US200602694 79 11-30-2006 Colton et al. A5 US20070065440 03-22-2007 Tomlinson et al. A6 US20070122402 05-31-2007 Balli et al.

IExaminer IDate ~ignature Considered

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11968784v.1 IPR Page 1857 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name B. Hissong

Sheet I 2 I of I 2 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS

Foreign Patent Documet Pages, Columns, Publication Name of Patentee or Lines, Applicant of Cited Document Where Relevant 3 4 Date TB Examiner Cite Country Code -Number - Passages Or 1 MM-DD-YYYY Initials* No. Kind Code5 (if known) Relevant Figures Appear D NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Examiner Cite 1 the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T2 Initials No. number(s), publisher, citv and/or countrv where published. C1 LIU et al. "Reversible Self-Association Increases the viscosity of a Concentrated Monoclonal Anitbody in Aqueous Solution", J Pharma Sci ,Vol. 94, No. 9, pp. 1928-1940 (2005) C2 WANG et al. "Opalescence of an lgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients", BioPharm lnternatl, Vol. 22(4) (2009)

IExaminer IDate ~ignature Considered

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11968784v.1 IPR Page 1858 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Docket No.: 117813-29602

Dated: July 9, 2011 Electronic Signature for Cristin Cowles, Ph.D.: /Cristin Cowles/

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Patent Application of: Wolfgang Fraunhofer

Application No.: 12/325,049 Confirmation No.: 1766

Filing Date: November 28, 2008 Art Unit: 1636

For: PROTEIN FORMULATIO NS AND METHODS Examiner: Hissong, Bruce D. OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

SUPPLEMENTAL INFORMATION DISCLOSURE STATEMENT {IDS)

Dear Sir:

Pursuant to 37 C.F.R. §§ 1.56, 1.97 and 1.98, the attention of the Patent and Trademark Office is hereby directed to the references listed on the attached PTO/SB/08. It is respectfully requested that the information be expressly considered during the prosecution of this application, and that the references be made of record therein and appear among the "References Cited" on any patent to issue therefrom.

This Supplemental Information Disclosure Statement is filed under 37 C.F.R. § 1.97(c), after the mailing date of a non-Final Office Action, but before the mailing date of any of a final action under § 1.113, a notice of allowance under § 1.311, or an action that otherwise closes prosecution in the application, and it is accompanied by the fee set forth in§ 1.17(p).

In accordance with 37 C.F.R. § 1.98(a)(2)(ii), Applicants have not submitted copies of U.S. patents and U.S. patent applications. Applicants submit herewith copies of non-patent literature in accordance with 37 C.F.R. § 1.98(a)(2).

In accordance with 37 CFR 1.97(g), the filing of this Supplemental Information Disclosure Statement shall not be construed to mean that a search has been made or that no other material information as defined in 37 CFR 1.56(a) exists. In accordance with 37 CFR 1.97(h), the filing of this Supplemental Information Disclosure Statement shall not be construed to be an admission that any patent,

1 MEI 11968901 v.1

IPR Page 1859 of 2482 Application No.: 12/325,049 Docket No.: 117813-29602 publication or other information referred to therein is "prior art" for this invention unless specifically designated as such.

It is submitted that the Supplemental Information Disclosure Statement is in compliance with 37 CFR 1.98 and the Examiner is respectfully requested to consider the listed references.

Please charge our Deposit Account No. 50-4876 in the amount of $180.00 covering the fee set forth in 37 CFR 1.17(p). The Director is hereby authorized to charge any deficiency in the fees filed, asserted to be filed or which should have been filed herewith (or with any paper hereafter filed in this application by this firm) to our Deposit Account No. 50-4876, under Order No. 117813-26902.

Dated: July 9, 2011 Respectfully submitted,

By I Cristin Cowles/ Cristin Howley Cowles, Ph.D. Registration No.: 55,291 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6512 (617) 607-9200 (Fax) Attorney For Applicants

2 MEI 11968901 v.1

IPR Page 1860 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Filer: Cristin E. Howley

Attorney Docket Number: 117813-26902

Filed as Large Entity

Utility under 35 USC 111 (a) Filing Fees

Sub-Total in Description Fee Code Quantity Amount USO($)

Basic Filing:

Pages:

Claims:

Miscellaneous-Filing:

Petition:

Patent-Appeals-and-Interference:

Post-Allowance-and-Post-Issuance:

Extension-of-Time:

IPR Page 1861 of 2482 Sub-Total in Description Fee Code Quantity Amount USO($)

Miscellaneous:

Submission- Information Disclosure Stmt 1806 1 180 180

Total in USD ($) 180

IPR Page 1862 of 2482 Electronic Acknowledgement Receipt

EFSID: 10484511

Application Number: 12325049

International Application Number:

Confirmation Number: 1766

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Customer Number: 87501

Filer: Cristin E. Howley

Filer Authorized By:

Attorney Docket Number: 117813-26902

Receipt Date: 09-JUL-2011

Filing Date: 28-NOV-2008

Time Stamp: 16:05:09

Application Type: Utility under 35 USC 111 (a)

Payment information:

Submitted with Payment yes

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Payment was successfully received in RAM $180

RAM confirmation Number 16128

Deposit Account 504876

Authorized User

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This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents, characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a Post Card, as described in MPEP 503.

New A~~lications Under 35 U.S.C. 111 If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR 1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this Acknowledgement Receipt will establish the filing date of the application.

National Stage of an International A~~lication under 35 U.S.C. 371 If a timely submission to enter the national stage of an international application is compliant with the conditions of 35 U.S.C. 371 and other applicable requirements a Form PCT/DO/E0/903 indicating acceptance of the application as a national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt, in due course.

New International A~~lication Filed with the USPTO as a Receiving Office If a new international application is being filed and the international application includes the necessary components for an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number and of the International Filing Date (Form PCT/R0/1 OS) will be issued in due course, subject to prescriptions concerning national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of the application.

IPR Page 1864 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Dated: August 9, 2011 Electronic Signature for Cristin H. Cowles / Cristin Cowles/

Docket No.: 117813-26902 (PATENT) IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Application of: Wolfgang Fraunhofer

Attorney Docket No.: 117813-26902

Application No.: 12/325,049 Group Art Unit: 1646

Filed: November 28, 2008 Examiner: Hissong, Bruce D.

For: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

SUPPLEMENTAL AMENDMENT AND RESPONSE

TO NON-FINAL OFFICE ACTION

Dear Sir:

This communication is supplemental to the Amendment and Response to Non-Final Office Action dated June 2, 2011. In addition to the amendments described in the June 2, 2011 Amendment and Response, please amend the above-identified U.S. patent application as follows.

Amendments to the Specification begin on page 2 of this paper.

Amendments to the Claims begin on page 4 of this paper.

Remarks/Arguments begin on page 25 of this paper.

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AMENDMENTS TO THE SPECIFICATION

Please insert the following paragraphs at page 28, line 31 of the specification:

In one embodiment, the human neutralizing, antibody, or an antigen-binding portion thereof, having a high affinity for human TNFa is an isolated human antibody, or an antigen-binding portion thereof, with a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5 (i.e., the D2E7 VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). Even more preferably, the LCVR further has CDRl domain comprising the amino acid sequence of SEQ ID N0:7 (i.e., the D2E7 VL CDRl) and the HCVR has a CDRl domain comprising the amino acid sequence of SEQ ID N0:8 (i.e., the D2E7 VH CDRl).

In another embodiment, the human neutralizing, antibody, or an antigen-binding portion thereof, having a high affinity for human TNFa is an isolated human antibody, or an antigen­ binding portion thereof, with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:l (i.e., the D2E7 VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID N0:2 (i.e., the D2E7 VH). In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG 1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG 1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region.

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Please amend the application to include the sequence listing provided herewith containing SEQ ID NOS: 1-8.

Please insert the following on page 1, after the priority paragraph of the specification:

Sequence Listing The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 3, 2011, is named SeqList and is 3,883 bytes in size.

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AMENDMENTS TO THE CLAIMS

Please amend the claims as follows:

1. (Previously Presented) An aqueous formulation comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm and the antibody, or antigen­ binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa.

2. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 57 kDa.

3. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 100 kDa.

4. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 150 kDa.

5. (Canceled)

6. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 250 kDa.

7. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 2 mS/cm.

8. (Canceled)

9. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 1 mS/cm.

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10. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

11. (Currently Amended) An aqueous formulation comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL.,_ and water, wherein the formulation has a conductivity of less than about 2 mS/cm.

12-14. (Canceled)

15. (Previously Presented) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 100 mg/mL.

16. (Previously Presented) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 150 mg/mL.

17. (Previously Presented) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 200 mg/mL.

18. (Previously Presented) The formulation of claim 11, wherein the concentration of the antibody, or antigen-binding fragment thereof, is greater than about 200 mg/mL.

19. (Canceled)

20. (Original) The formulation of claim 11 , wherein the formulation has a conductivity of less than about 0.5 mS/cm.

21. (Previously Presented) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, at a concentration of at least about 50 mg/mL, and water, wherein the formulation has an osmolality of no more than about 30 mOsmol/kg. 5 MEI 12104094v.1

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22. (Original) The formulation of claim 21, wherein the osmolality of the formulation is no more than about 15 mOsmol/kg.

23. (Canceled)

24. (Previously Presented) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is at least about 150 mg/mL.

25. (Canceled)

26. (Previously Presented) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is greater than about 200 mg/mL.

27. (Currently Amended) An aqueous formulation comprising water and at least about 20 mg/mL a given concentration of an antibody, or an antigen-binding fragment thereof, wherein the antibody, or an, antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or an, antigen-binding fragment thereof, in a buffered solution at the same concentration.

28. (Currently Amended) The formulation of claim 27, wherein the Dh of the antibody, or antigen-binding fragment thereof, protein is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, protein in phosphate buffered saline (PBS) at the same concentration.

29. (Currently Amended) The formulation of claim 28, wherein the Dh of the protein is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, protein in PBS at the same concentration.

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30. (Currently Amended) The formulation of claim 28, wherein the Dh of the antibody, or antigen-binding fragment thereof, protein is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, protein in PBS at the same concentration.

31. (Canceled)

32. (Previously Presented) The formulation of any one of claims 1, 11, 21, and 27, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

33. (Currently Amended) The formulation of claim 1, 11, 21 or 27, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody, or an antigen-binding fragment thereof.

34. (Previously Presented) The formulation of claim 33, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® (adalimumab ), Remicade® (lnfliximab), golimumab (Centocor), Cimzia® (Certolizumab pegol), and CNTO 1275 (ustekinumab ).

35-36. (Canceled)

37. (Currently Amended) An aqueous formulation comprising an antibody, or an antigen-binding fragment, at a concentration of at least about 10 mg/mL.,_ and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm ..§. tfflr.

38. (Currently Amended) The formulation of claim 37, wherein the antibody has a Dh of less than about 3 nm ttffi.

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39. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

40. (Currently Amended) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody, or antigen-binding fragment thereof.

41. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® (adalimumab ), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (lmciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliximab), ReoPro® (Abciximab), Rituxan® (Rituximab), Simulect® (Basiliximab), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab­ CD3), Panorex® (Edrecolomab), and Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab) and Avastin® (bevacizumab ).

42. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, further comprising a non-ionizable excipient.

43. (Previously Presented) The formulation of claim 42, wherein the non- ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

44. (Original) The formulation of claim 43, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

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45. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

46-47. (Canceled)

48. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

49. (Canceled)

50. (Previously Presented) The formulation of any one of claims 1, 11, 21, 27, and 37, wherein the formulation is suitable for in vitro or in vivo use.

51. (Canceled)

52. (Previously Presented) The formulation of claim 50, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

53. (Withdrawn) Use of the formulation of claim 50 in the treatment of a disorder in a subject.

54. (Previously Presented) A device comprising the formulation of any one claims 1, 11, 21, 27, and 37.

55. (Canceled) 9 MEI 12104094v.1

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56. (Previously Presented) An article of manufacture comprising the formulation of any one of claims 1, 11, 21, 27, and 37.

57. (Withdrawn) A method of preparing an aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, and water, the method comprising: a) providing the antibody, or antigen-binding fragment thereof, in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved to thereby prepare the aqueous formulation.

58. (Withdrawn) A method of preparing an aqueous formulation of an antibody, or an antigen-binding fragment thereof, the method comprising: a) providing the antibody, or an antigen-binding fragment thereof, in a first solution; b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five-fold volume exchange with the water has been achieved to thereby prepare a diafiltered antibody, or antigen-binding fragment thereof, solution; and c) concentrating the diafiltered antibody, or an antigen-binding fragment thereof, solution to thereby prepare the aqueous formulation of the antibody, or antigen-binding fragment thereof.

59. (Withdrawn) The method of claim 58, wherein the concentration of the diafiltered antibody, or antigen-binding fragment thereof, solution is achieved via centrifugation.

60. (Withdrawn) The method of claim 57 or 58, wherein the diafiltration medium consists of water.

61. (Withdrawn) The method of claim 57 or 58, wherein the first solution is subjected to diafiltration with water until at least about a six-fold volume exchange or at least about a seven­ fold volume exchange is achieved.

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62. (Canceled)

63. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation has a final concentration of excipients which is at least about 95% less or 99% less than the first solution.

64. (Canceled)

65. (Withdrawn) The method of claim 57 or 58, wherein the first antibody, or antigen- binding fragment thereof, solution is obtained from a mammalian cell expression system and has been purified to remove host cell proteins (HCPs).

66. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, has a Mw greater than about 1 kDa.

67. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, has a molecular weight (Mw) selected from the group consisting of greater than about 10 kDa, greater than about 47 kDa, reater than about 57 kDa, greater than about 100 kDa, greater than about 150 kDa, greater than about 200 kDa, and greater than about 250 kDa.

68.-73. (Canceled)

74. (Withdrawn) The method of claim 57 or 58, further comprising adding an excipient to the aqueous formulation.

75. (Withdrawn) The method of claim 74, wherein the aqueous formulation is a pharmaceutical formulation.

76. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation is a pharmaceutical formulation.

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77. (Withdrawn) The method of claim 76, further comprising loading the aqueous formulation into a device suitable for administering the aqueous formulation to a subject.

78-79. (Canceled)

80. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

81. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

82. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, is selected from the group consisting of Humira® (adalimumab), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (lmciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliximab ), ReoPro® (Abciximab ), Rituxan® (Rituximab ), Simulect® (Basiliximab ), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab-CD3), Panorex® (Edrecolomab), Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab), and Avastin® (bevacizumab).

83.-85. (Canceled)

86. (Previously Presented) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, the antibody, or antigen-binding fragment thereof, prepared by a) providing the antibody, or antigen-binding fragment thereof, in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration

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medium until at least a five fold volume exchange with the water has been achieved.

87.-101. (Canceled)

102. (Previously Presented) The formulation of claim 43, wherein the polyol is mannitol or sorbitol.

103. (Currently Amended) An aqueous formulation comprising water and at least about 50 mg/ml a given concentration of an antibody, or an antigen-binding fragment thereof, wherein the antibody, or antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% [[-80% ]] less than the Dh of the antibody, or ffil antigen-binding fragment thereof, in a buffered solution at the same concentration.

104. (Previously Presented) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 150 mg/mL.

105. (Previously Presented) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is greater than about 200 mg/mL.

106. (Previously Presented) The formulation of claim 103, further comprising a non-ionizable excipient.

107. (Previously Presented) The formulation of claim 106, wherein the non- ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

108. (Previously Presented) The formulation of claim 107, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

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109. (Previously Presented) The formulation of claim 103, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

110. (Previously Presented) The formulation of claim 103, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

111. (Previously Presented) The formulation of claim 103, wherein the formulation is suitable for in vitro or in vivo use.

112. (Previously Presented) The formulation of claim 111, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

113. (Previously Presented) Use of the formulation of claim 111 in the treatment of a disorder in a subject.

114. (Previously Presented) A device comprising the formulation of claim 103.

115. (Previously Presented) An article of manufacture comprising the formulation of claim 103.

116. (Previously Presented) The formulation of claim 21 or 27, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® (adalimumab), Campath® (Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (lmciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliximab), ReoPro® (Abciximab), Rituxan® (Rituximab), 14 MEI 12104094v.1

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Simulect® (Basiliximab), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (Ibritumomab Tiuxetan), Orthoclone OKT3® (Muromonab-CD3), Panorex® (Edrecolomab), Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), Vectibix® (panitumumab), Bexxar® (tositumomab and 1131 tositumomab), and Avastin® (bevacizumab ).

117. (New) An aqueous formulation comprising water and an antibody, or antigen­ binding fragment thereof, at a concentration of at least about 50 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

118. (New) The formulation of claim 117, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 75 mg/mL.

119. (New) The formulation of claim 117, wherein the formulation has a conductivity of less than about 2 mS/cm.

120. (New) The formulation of claim 117, further comprising a non-ionizable excipient.

121. (New) The formulation of claim 120, wherein the non-ionizable excipient is a polyol. 15 MEI 12104094v.1

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122. (New) The formulation of claim 121, wherein the polyol is mannitol.

123. (New) The formulation of claim 120, wherein the non-ionizable excipient is a non­ ionic surfactant.

124. (New) The formulation of claim 123, wherein the non-ionic surfactant is polysorbate 80.

125. (New) The formulation of claim 120, wherein the non- ionizable excipient is sucrose.

126. (New) The formulation of claim 117, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

127. (New) The formulation of claim 117, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

128. (New) An aqueous formulation comprising water and an antibody, or an antigen­ binding fragment thereof, at a concentration of at least about 50 mg/mL, wherein the antibody, or an antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a 16 MEI 12104094v.1

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single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

129. (New) The formulation of claim 128, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in phosphate buffered saline (PBS) at the same concentration.

130. (New) The formulation of claim 129, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

131. (New) The formulation of claim 130, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

132. (New) The formulation of claim 128, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

133. (New) The formulation of claim 128, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

134. (New) The formulation of any one of claim 128, further comprising a non-ionizable excipient.

135. (New) The formulation of claim 134, wherein the non-ionizable excipient is a polyol.

136. (New) The formulation of claim 135, wherein the polyol is mannitol. 17 MEI 12104094v.1

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137. (New) The formulation of claim 134, wherein the non-ionizable excipient is a non­ ionic surfactant.

138. (New) The formulation of claim 137, wherein the non-ionic surfactant is polysorbate 80.

139. (New) The formulation of claim 134, wherein the non-ionizable excipient is sucrose.

140. (New) The formulation of claim 128, further comprising mannitol and polysorbate 80.

141. (New) The formulation of claim 128, further comprising sucrose and polysorbate 80.

142. (New) The formulation of claim 140 or 141, wherein the antibody, or antigen­ binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

143. (New) The formulation of claim 140 or 141, wherein the antibody, or antigen­ binding portion thereof, is adalimumab.

144. (New) An aqueous formulation comprising water and an antibody, or an antigen­ binding fragment, at a concentration of at least about 50 mg/mL, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 18 MEI 12104094v.1

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domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

145. (New) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, has a Dh of less than about 3 nm.

146. (New) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

147. (New) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

148. (New) The formulation of any one of claims 144, further comprising a non-ionizable excipient.

149. (New) The formulation of claim 148, wherein the non-ionizable excipient is a polyol.

150. (New) The formulation of claim 149, wherein the polyol is mannitol.

151. (New) The formulation of claim 148, wherein the non-ionizable excipient is a non­ ionic surfactant.

152. (New) The formulation of claim 151, wherein the non-ionic surfactant is polysorbate 80.

153. (New) The formulation of claim 148, wherein the non-ionizable excipient is sucrose. 19 MEI 12104094v.1

IPR Page 1883 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

154. (New) The formulation of claim 144, further comprising mannitol and polysorbate 80.

155. (New) The formulation of claim 144, further comprising sucrose and polysorbate 80.

156. (New) The formulation of claim 154 or 155 wherein the antibody, or antigen­ binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

157. (New) The formulation of claim 154 or 155, wherein the antibody, or antigen­ binding portion thereof, is adalimumab.

158. (New) Use of the formulation of any one of claims 117, 128, or 144 in the treatment of a disorder in a subject.

159. (New) A device comprising the formulation of any one of claims 117, 128, or 144.

160. (New) An article of manufacture comprising the formulation of any one of claims 117, 128, or 144.

161. (New) An aqueous formulation comprising mannitol, polysorbate 80, an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified 20 MEI 12104094v.1

IPR Page 1884 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

162. (New) The formulation of claim 161, wherein the formulation has a conductivity of less than about 1.0 mS/cm.

163. (New) The formulation of claim 161, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

164. (New) The formulation of claim 161, wherein the antibody concentration is at least about 50 mg/mL.

165. (New) The formulation of claim 161, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

166. (New) The formulation of claim 161, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

167. (New) An aqueous formulation comprising mannitol, polysorbate 80, an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, 21 MEI 12104094v.1

IPR Page 1885 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

wherein the antibody, or an antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

168. (New) The formulation of claim 167, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in phosphate buffered saline (PBS) at the same concentration.

169. (New) The formulation of claim 168, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

170. (New) The formulation of claim 168, wherein the Dh of the antibody, or antigen­ binding fragment thereof, is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

171. (New) The formulation of claim 167, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

22 MEI 12104094v.1

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172. (New) The formulation of claim 167, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

173. (New) The formulation of claim 167, wherein the antibody concentration is at least about 50 mg/mL.

174. (New) An aqueous formulation comprising mannitol, polysorbate 80, an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

175. (New) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, has a Dh of less than about 3 nm.

176. (New) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2. 23 MEI 12104094v.1

IPR Page 1887 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

177. (New) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

178. (New) The formulation of claim 174, wherein the antibody concentration is at least about 50 mg/mL.

179. (New) Use of the formulation of any one of claims 161, 167, and 17 4 in the treatment of a disorder in a subject.

180. (New) A device comprising the formulation of any one of claims 161, 167, and 174.

181. (New) An article of manufacture comprising the formulation of any one of claims 161, 167, and 174.

24 MEI 12104094v.1

IPR Page 1888 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

REMARKS Claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52-54, 56-61, 63, 65- 67, 74-77, 79-82, 85, and 102-116 were pending in the application (following entry of the June 2, 2011 Amendment). Claims 11, 27-30, 33, 37, 38, 40, 103 have been amended. New claims 117- 181 have been added. Claims 53, 57-61, 63, 65-67, 74-77, and 79-84 have been withdrawn by the Examiner as being directed to non-elected subject matter. Accordingly, following entry of this amendment, claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52-54, 56-61, 63, 65-67, 74-77, 79-82, 85, and 102-181 will be pending in the application.

Support for the amendments to the claims and the new claims may be found throughout the specification and claims as originally filed. Claims 11, 27-30, 33, 40 have been amended for clarity and/or in view of the previous amendments. Additional support for the amendment to claim 37 and 38 can be found throughout the specification, including at least at paragraph 176 of the published application (US 2009/0291062); additional support for the amendment to claim 103 can be found throughout the specification, including at least at paragraph 176 of the published application; additional support for the amendments to claims 27 and 103 may be found at, for example, paragraph 164 of the published application (US 2009/0291062); support for new claims 117, 118, and 161-164 may be found in, for example, originally filed claims 1, 7, and 10, and at, for example, [0164] and [0188] of the published application (US 2009/0291062); support for new claims 128-131 and 167-170, and 173 may be found in, for example, originally filed claim 28 and at, for example, [0020], [0164], [0177], and [0188] of the published application (US 2009/0291062); support for new claim 144, 174, 175, and 178 may be found in, for example, originally filed claim 37, and at, for example, [0164], [0176], and [0188] of the published application (US 2009/0291062); support for new claims 120-125, 134-141, 148-155 and additional support for new claims 161, 167, and 174 may be found at, for example, [0023], [0169], and [0170] of the published application (US 2009/0291062); support for new claims 127, 133, 143, 147, 157, 166, and 177 may be found at, for example, [0022] of the published application (US 2009/0291062); and support for new claims 158- 160 and 179-181 may be found in, for example, originally filed claims 53, 54, and 56 of the published application (US 2009/0291062). No new matter has been added.

25 MEI 12104094v.1

IPR Page 1889 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

Any amendments to the claims are not to be construed as an acquiescence to any of the rejections set forth in the instant Office Action, and were done solely to expedite prosecution of the application. Applicants hereby reserve the right to pursue the subject matter of the claims as originally filed in this or a separate application(s). Entry of the foregoing claim amendments is respectfully requested.

Support for the amendments to the specification and additional support for new claims 117, 126, 128, 132, 142, 144, 146, 156, 161, 165, 167, 171, 174, and 176 can be found in the published application (US 2009/0291062) at paragraph 133, page 10, where Applicants have expressly incorporated by reference US Patent No. 6,090,382, and at paragraph 231, page 21, where the entire contents of all the references, patents and published patent applications cited throughout the specification were expressly incorporated into the present application by reference. No new matter has been added.

Applicants respectfully submit that as required by 37 CPR§ l.75(d)(l) and MPEP § 608.0l(o), the present specification has been amended to recite the specific sections from U.S. Patent No. 6,090,382 which provide support for the amended claims.

Based on the amendments to the specification described above, Applicants also submit herewith a computer-readable form of the "Sequence Listing" for the above-identified patent application.

The Sequence Listing text file accompanying this paper is a compliant ASCII text file, and, thus, will serve as both the paper copy required by 37 C.F.R. § l.82l(c) and the C.R.F required by 37 C.F.R. § l.82l(e).

Also submitted herewith is the required statement under 37 C.F.R. § l.82l(f).

No new matter has been added.

As the Examiner has expressly requested that Applicants submit this Supplemental Response, Applicants should not be subject to a reduction of Patent Term when this application issues as a patent in accordance with 37 C.F.R. § l.704(c)(8).

26 MEI 12104094v.1

IPR Page 1890 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

Interview Summary

Inventor Wolfgang Fraunhofer and Applicants' attorneys, Tara Seshadri and Cristin Cowles, wish to thank Examiners Bruce Hissong and Robert Landsman for the courtesy of a personal interview on July 12, 2011. During the interview, the state of the art with respect to the instant invention and the Konstantinov and Matheus references were discussed.

With respect to an exemplary conductivity measure, Applicants provide the enclosed chemical product description from Fisher Scientific for PBS (submitted herewith as Appendix A). As noted in Appendix A, PBS includes sodium chloride, potassium chloride, sodium phosphate dibasic, and potassium phosphate mono basic . As indicated in Appendix A, PBS has a conductivity of 14,000 to 17,800 µmhos/cm (or 14-17.8 mS/cm).

Rejection of Claims 1-4, 7, 9-11, 13, 15-18, 20, 31-36, 42-45, 48, 50, 52, 54, 56, 86, and 102

Under 35 U.S.C. § 103(a)

The Examiner has rejected claims 1-4, 7, 9-11, 13, 15-18, 20, 31-35, 42, 43, 45, 48, 50, 52, 54, 56, 86, and 102 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475). The Examiner has rejected claim 44 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475), and further in view of Schusching (U.S. Patent Application No. U.S. 2007 /0202051). The Examiner has rejected claim 36 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475), and further in view of Giles-Komar, et al. (U.S. Patent Application No. U.S. 2003/0143603). Applicants respectfully traverse each of the aforementioned obviousness rejections, and provide the following supplemental comments regarding the combined teachings of the Konstantinov and Matheus references.

As described in Applicants' response of June 2, 2011, Konstantinov et al. teach methods for

27 MEI 12104094v.1

IPR Page 1891 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

concentrating a macromolecule, and provide a working example using IL2SA, which is less than 18 kDa in molecular weight. While Konstantinov et al. teach that that methods disclosed therein may be used to concentrate a macromolecule by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov et al.), the working examples of Konstantinov et al. teach, for example, that the methods disclosed therein concentrate proteins at a concentration of at most 12 mg/mL (see, e.g., Example 3 at page 5 and Figure 6 of Konstantinov et al.). Accordingly, Konstantinov et al. fail to teach or suggest compositions comprising antibodies, or antigen-binding fragments thereof, at a concentration of at least 20 mg/mL.

Applicants respectfully submit that the teachings of Matheus do not make up for the deficiencies of Konstantinov et al. and that one of ordinary skill would not be motivated to combine the teachings of Konstantinov and Matheus or have an expectation of success. In particular, Matheus explicitly teaches that there are difficulties in achieving high antibody concentrations in liquid formulations. For example, Matheus teaches that it was known in the art that high protein concentration formulations were difficult to achieve, stating, for example, at paragraph [007] that

.. .it is clear that the preparation of liquid highly concentrated antibody formulations which are stable for a sufficiently long time is proving to be extremely difficult for the person skilled in the art. In addition, the preparation of a highly concentrated liquid formulation was unattractive to the person skilled in the art since the greatly pronounced aggregation tendency of proteins and in particular of antibodies, even in low concentrations, was sufficiently known ...

In addition to acknowledging known difficulties with respect to high concentration antibody liquid formulations, Matheus also teaches at paragraph [011] that "due to the difficulties to be expected, already established protein formulations generally cannot be applied to new protein active ingredients, [and therefore] the object of the present invention was to find novel, stable, highly concentrated liquid formulations for therapeutic proteins." In an attempt to solve these problems and achieve stable, liquid formulations having high protein concentrations, Matheus provides working examples describing high concentration formulations containing anti-EGFR antibodies having specific excipients, e.g., NaCl and phosphate buffer (see Examples 1-3). Matheus teaches that in order to achieve stable, liquid formulations having high protein concentrations, specific excipients are required, thus, teaching away from the current invention which is directed to

28 MEI 12104094v.1

IPR Page 1892 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

formulations having little to no excipients.

Furthermore, whereas the Matheus reference is focused on antibody formulations (antibodies having an average size of 150 kDa), the Konstantinov reference provides working examples for a much smaller molecule, i.e., IL-2SA, having a size of about 18 kDa. Therefore, even though Konstantinov mentions larger macromolecules (see, e.g., paragraph 0041]), there is no evidence that the methods described therein would work for larger molecules, such as antibodies. Thus, one of ordinary skill would have no expectation of success that the methods described in Konstantinov would even work for larger macromolecules, such as antibodies, described in Matheus.

In sum, Matheus teaches that high concentration antibody formulations present numerous challenges and also teaches that these challenges may be overcome by using certain excipients, in contrast to Applicants' discovery. Applicants submit that absent the teachings in the present application, one of ordinary skill in the art would have no expectation of success or be motivated to combine the teachings of Matheus with Konstantinov to arrive at the invention of independent claims 1 and 11 (and claims that depend therefrom). Accordingly, Applicants respectfully request that the foregoing rejections of the claims be reconsidered and withdrawn.

29 MEI 12104094v.1

IPR Page 1893 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

SUMMARY

If a telephone conversation with Applicant's Attorney would expedite the prosecution of the above-identified application, the Examiner is urged to call the undersigned at (617) 449-6500.

The Commissioner is hereby authorized to charge any fees associated with the filing of this communication or any subsequent filing to our Deposit Account No. 50-4876, under Order No. 117813-26902 from which the undersigned is authorized to draw.

Dated: August 9, 2011 Respectfully submitted,

Electronic signature: / Cristin Cowles /

Cristin H. Cowles, Ph.D. Registration No.: 55,281 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6500 (617) 607-9200 Attorney For Applicants

30 MEI 12104094v.1

IPR Page 1894 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Filer: Cristin E. Howley

Attorney Docket Number: 117813-26902

Filed as Large Entity

Utility under 35 USC 111 (a) Filing Fees

Sub-Total in Description Fee Code Quantity Amount USO($)

Basic Filing:

Pages:

Claims:

Claims in excess of 20 1202 63 52 3276

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Patent-Appeals-and-Interference:

Post-Allowance-and-Post-Issuance:

IPR Page 1895 of 2482 Sub-Total in Description Fee Code Quantity Amount USO($)

Extension-of-Time:

Miscellaneous:

Total in USD ($) 4596

IPR Page 1896 of 2482 Electronic Acknowledgement Receipt

EFSID: 10689285

Application Number: 12325049

International Application Number:

Confirmation Number: 1766

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Customer Number: 87501

Filer: Cristin E. Howley

Filer Authorized By:

Attorney Docket Number: 117813-26902

Receipt Date: 09-AUG-2011

Filing Date: 28-NOV-2008

Time Stamp: 23:48:40

Application Type: Utility under 35 USC 111 (a)

Payment information:

Submitted with Payment yes

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Payment was successfully received in RAM $4596

RAM confirmation Number 5902

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File Listing:

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This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents, characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a Post Card, as described in MPEP 503.

New A~~lications Under 35 U.S.C. 111 If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR 1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this Acknowledgement Receipt will establish the filing date of the application.

National Stage of an International A~~lication under 35 U.S.C. 371 If a timely submission to enter the national stage of an international application is compliant with the conditions of 35 U.S.C. 371 and other applicable requirements a Form PCT/DO/E0/903 indicating acceptance of the application as a national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt, in due course.

New International A~~lication Filed with the USPTO as a Receiving Office If a new international application is being filed and the international application includes the necessary components for an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number and of the International Filing Date (Form PCT/R0/1 OS) will be issued in due course, subject to prescriptions concerning national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of the application.

IPR Page 1898 of 2482 fisherSci.com - Chemical Product Specs Display Page 1 of 1

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[7647·14·5 (Sodium Chloride)], [7447·40·7 (Potassium Chloride)], [7558·79·4 (Sodium Phosphate Dibasic)], [7778·77·0 (Potassium Phosphate Monobasic)]

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http://www.fishersci.com/ecomm/servlet/chemicalproductspecsview?catalogld=-l&produc...IPR 7Page /27/2011 1899 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Dated: August 9, 2011 !=l,=,rtrnnir ~inn~ti in=• fnr r:ric:tin 1--l r:nwl,::,c: Ph n · /("'.ric:tin 1--l r:nwl,::,c: Ph n / DocketNo.: 117813-26902 (PATENT)

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Application of: Wolfgang Fraunhofer

Attorney Docket No.: 117813-26902

Application No.: 12/325,049 Group Art Unit: 1646

Filed: November 28, 2008 Examiner: Hissong, Bruce D.

For: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

STATEMENT PURSUANT TO 37 C.F.R. § 1.821

Dear Sir:

Submitted herewith for filing in connection with the above-referenced patent application is a computer readable copy of the Sequence Listing included in the application. The sequence listing text file is a compliant ASCII text file, and, thus, will serve as both the paper copy required by 37 C.F.R. § 1.821 ( c) and the CRF required by 37 C.F.R. § 1.82l(e). No new matter has been added.

I hereby state that I have reviewed the paper copy of the Sequence Listing contained on pages 1 to 3 of said Sequence Listing, as required by 37 C.F.R. § 1.82l(c), and have reviewed the computer readable form of the Sequence Listing, as required by 37 C.F.R. § 1.821 ( e ), and that the content of the paper and computer readable copies for the above-referenced Patent application are the same as required by 37 C.F.R. § 1.82l(f).

Dated: August 9, 2011 Respectfully submitted,

Electronic signature: / Cristin H. Cowles, Ph.D./

Cristin H. Cowles, Ph.D. Registration No.: 55,281

McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6500 (617) 607 -9200 Attorney/Agent For Applicant

ME1 11769806v.1

IPR Page 1900 of 2482 DocCode - SEQ.TXT

SCORE Placeholder Sheet for IFW Content

Application Number: 12325049 Document Date: 08/09/2011

The presence of this form in the IFW record indicates that the following document type was received in electronic format on the date identified above. This content is stored in the SCORE database. • Sequence Listing

Since this was an electronic submission, there is no physical artifact folder, no artifact folder is recorded in PALM, and no paper documents or physical media exist. The TIFF images in the IFW record were created from the original documents that are stored in SCORE.

To access the documents in the SCORE database, refer to instructions developed by SIRA.

At the time of document entry (noted above): • Examiners may access SCORE content via the eDAN interface. • Other USPTO employees can bookmark the current SCORE URL (http://es/ScoreAccessWeb/). • External customers may access SCORE content via the Public and Private PAIR interfaces.

Form Revision Date: February 8, 2006

IPR Page 1901 of 2482 PTO/SB/06 (07-06) Approved for use through 1/31/2007. 0MB 0651-0032 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid 0MB control number. PATENT APPLICATION FEE DETERMINATION RECORD Application or Docket Number Filing Date Substitute for Form PT0-875 12/325,049 11/28/2008 D To be Mailed

APPLICATION AS FILED - PART I OTHER THAN (Column 1) (Column 2) SMALL ENTITY D OR SMALL ENTITY FOR NUMBER FILED NUMBER EXTRA RATE($) FEE($) RATE($) FEE($)

D BASIC FEE N/A N/A N/A N/A (37 CFR 1.16(a), (b), or (cl)

D SEARCH FEE N/A N/A N/A N/A (37 CFR 1.16(k), (i), or (ml)

D EXAMINATION FEE N/A N/A N/A N/A (37 CFR 1.16(0), (p), or (q)) TOTAL CLAIMS X $ OR X $ (37 CFR 1.16(i)) minus 20 = * = = INDEPENDENT CLAIMS X $ X $ (37 CFR 1.16(h)) minus 3 = * = = If the specification and drawings exceed 100 sheets of paper, the application size fee due 0APPLICATION SIZE FEE is $250 ($125 for small entity) for each (37 CFR 1.16(s)) additional 50 sheets or fraction thereof. See 35 U.S.C. 41 (a)(1 )(G) and 37 CFR 1.16(s). D MULTIPLE DEPENDENT CLAIM PRESENT (37 CFR 1.16(j)) * If the difference in column 1 is less than zero, enter "O" in column 2. TOTAL TOTAL

APPLICATION AS AMENDED - PART II OTHER THAN (Column 1) (Column 2) (Column 3) SMALL ENTITY OR SMALL ENTITY CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) f-- 08/09/2011 AFTER PREVIOUSLY EXTRA FEE($) FEE($) z AMENDMENT PAID FOR w Total (37 CFR Minus X $ = OR X $52= ~ 1.16(i)) • 166 ** 839 = 0 0 0 Independent Minus X $ OR X $220= z (37 CFR 1 .16(h)) • 15 ***9 = 6 = 1320 w ~ D Application Size Fee (37 CFR 1.16(s)) <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L 1320 FEE FEE

(Column 1) (Column 2) (Column 3) CLAIMS HIGHEST REMAINING NUMBER PRESENT ADDITIONAL ADDITIONAL RATE($) RATE($) AFTER PREVIOUSLY EXTRA FEE($) FEE($) AMENDMENT PAID FOR f-- Total (37 CFR z * Minus ** X $ = OR X $ = w 1.16(i)) = Independent ~ Minus X $ OR X $ 0 (37 CFR 1 .16(h)) * *** = = = z w D Application Size Fee (37 CFR 1.16(s)) ~ <( D FIRST PRESENTATION OF MULTIPLE DEPENDENT CLAIM (37 CFR 1.16(j)) OR TOTAL TOTAL ADD'L OR ADD'L FEE FEE * If the entry in column 1 is less than the entry in column 2, write "O" in column 3. Legal Instrument Examiner: ** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 20, enter "20". /PAUL STANBACK/ *** If the "Highest Number Previously Paid For" IN THIS SPACE is less than 3, enter "3". The "Highest Number Previously Paid For" (Total or Independent) is the highest number found in the appropriate box in column 1. This collection of 1nformat1on 1s required by 37 CFR 1.16. The 1nformat1on 1s required to obtain or retain a benefit by the public which 1s to file (and by the US PTO to process) an application. Confidentiality is governed by 35 U.S.C. 122 and 37 CFR 1 .14. This collection is estimated to take 12 minutes to complete, including gathering, preparing, and submitting the completed application form to the US PTO. Time will vary depending upon the individual case. Any comments on the amount of time you require to complete this form and/or suggestions for reducing this burden, should be sent to the Chief Information Officer, U.S. Patent and Trademark Office, U.S. Department of Commerce, P.O. Box 1450, Alexandria, VA 22313-1450. DO NOT SEND FEES OR COMPLETED FORMS TO THIS ADDRESS. SEND TO: Commissioner for Patents, P.O. Box 1450, Alexandria, VA 22313-1450. If you need assistance in completing the form, ca/11-800-PT0-9199 and select option 2.

IPR Page 1902 of 2482 Sequence Listing was accepted. See attached Validation Report. If you need help call the Patent Electronic Business Center at (866) 217-9197 (toll free). Reviewer: Saleem, Syed (ASRC) Timestamp: [year=2011; month=8; day=16; hr=7; min=31; sec=23; ms=534;

IPR Page 1903 of 2482 Validated By CRFValidator v 1.0.3

Application No: 12325049 Version No: 1.0

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IPR Page 1904 of 2482 SEQUENCE LISTING

<110> FRAUNHOFER, Wolfgang BARTL, Annika KRAUSE, Hans-Jeurgen TSCHOPPE, Markus KALETA, Katharina

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<400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro 80 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 2 <211> 121 <212> PRT <213> Artificial Sequence

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IPR Page 1905 of 2482 <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser 50 55 60 Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 65 70 75 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 80 85 90 95 Cys Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

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IPR Page 1907 of 2482 IPR Page 1908 of 2482 UNITED STA1ES p A IBNT AND TRADEMARK OFFICE UNITED STA TES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O. Box 1450 Alexandria., Virginia 22313-1450 www.uspto.gov

APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.

12/325,049 11/28/2008 Wolfgang FRAUNHOFER 117813-26902 1766

87501 7590 01/04/2012 EXAMINER McCarter & English, LLP / Abbott Laboratories Ltd. 265 Franklin Street HISSONG, BRUCE D Boston, MA 02110 ART UNIT PAPER NUMBER

1646

MAIL DATE DELIVERY MODE

01/04/2012 PAPER

Please find below and/or attached an Office communication concerning this application or proceeding.

The time period for reply, if any, is set in the attached communication.

PTOL-90A (Rev. 04/07) IPR Page 1909 of 2482 Application No. Applicant(s)

12/325,049 FRAUNHOFER ET AL.

Office Action Summary Examiner Art Unit

Bruce D. Hissong, 1646 -- The MAILING DA TE of this communication appears on the cover sheet with the correspondence address -­ Period for Reply A SHORTENED STATUTORY PERIOD FOR REPLY IS SET TO EXPIRE ;J. MONTH(S) OR THIRTY (30) DAYS, WHICHEVER IS LONGER, FROM THE MAILING DATE OF THIS COMMUNICATION. Extensions of time may be available under the provisions of 37 CFR 1.136(a). In no event, however, may a reply be timely filed after SIX (6) MONTHS from the mailing date of this communication. If NO period for reply is specified above, the maximum statutory period will apply and will expire SIX (6) MONTHS from the mailing date of this communication. Failure to reply within the set or extended period for reply will, by statute, cause the application to become ABANDONED (35 U.S.C. § 133). Any reply received by the Office later than three months after the mailing date of this communication, even if timely filed, may reduce any earned patent term adjustment. See 37 CFR 1.704(b). Status

1 )0 Responsive to communication(s) filed on 6/2/2011 and 8/9/2011. 2a)0 This action is FINAL. 2b)~ This action is non-final. 3)0 An election was made by the applicant in response to a restriction requirement set forth during the interview on __; the restriction requirement and election have been incorporated into this action. 4)0 Since this application is in condition for allowance except for formal matters, prosecution as to the merits is closed in accordance with the practice under Ex parte Quayle, 1935 C.D. 11, 453 O.G. 213. Disposition of Claims

5)~ Claim(s) See Continuation Sheet is/are pending in the application. 5a) Of the above claim(s) 53.57-61.63.65-67.74-77.80-82. 113. 158 and 179 is/are withdrawn from consideration. 6)0 Claim(s) __ is/are allowed. 7)~ Claim(s) 1-4. 6-7. 9-11. 15-18. 20-22. 24. 26-30. 32-34. 37-45. 48. 50. 52. 54. 56. 86. 102-112. 114-157. 159-178. and 180-181 is/are rejected. 8)0 Claim(s) __ is/are objected to. 9)0 Claim(s) __ are subject to restriction and/or election requirement.

Application Papers

10)0 The specification is objected to by the Examiner. 11 )0 The drawing(s) filed on __ is/are: a)O accepted or b)O objected to by the Examiner. Applicant may not request that any objection to the drawing(s) be held in abeyance. See 37 CFR 1.85(a). Replacement drawing sheet(s) including the correction is required if the drawing(s) is objected to. See 37 CFR 1.121 (d). 12)0 The oath or declaration is objected to by the Examiner. Note the attached Office Action or form PT0-152. Priority under 35 U.S.C. § 119

13)0 Acknowledgment is made of a claim for foreign priority under 35 U.S.C. § 119(a)-(d) or (f). a)O All b)O Some * c)O None of: 1.0 Certified copies of the priority documents have been received. 2.0 Certified copies of the priority documents have been received in Application No. __. 3.0 Copies of the certified copies of the priority documents have been received in this National Stage application from the International Bureau (PCT Rule 17.2(a)). * See the attached detailed Office action for a list of the certified copies not received.

Attachment{s) 1) 0 Notice of References Cited (PT0-892) 4) 0 Interview Summary (PT0-413) 2) 0 Notice of Draftsperson's Patent Drawing Review (PT0-948) Paper No(s)/Mail Date. __ . 3) ~ Information Disclosure Statement(s) (PTO/SB/08) 5) 0 Notice of Informal Patent Application PaperNo(s)/Mail Date 6/2111 7/6/11 7/9/11. 6) ~ Other: Sequence Comparisons 1-8.

U.S. Patent and Trademark Office PTOL-326 (Rev. 03-11) Office Action Summary Part of Paper No./Mail Date 20111115

IPR Page 1910 of 2482 Continuation Sheet (PTOL-326) Application No. 12/325,049

Continuation of Disposition of Claims: Claims pending in the application are 1-4,6,7,9-11, 15-18,20-22,24,26-30,32-34,37- 45,48,50,52-54,56-61,63,65-67,74-77,80-82,86 and 102-181.

2

IPR Page 1911 of 2482 Application/Control Number: 12/325,049 Page 2 Art Unit: 1646

DETAILED ACTION

Formal Matters 1. Applicants' response to the office action mailed on 3/2/2011, including arguments/remarks and amended claims, was received on 6/2/2011 and is made of record. Applicants' supplemental response was received on 8/9/2011 and has been entered into the record.

2. In the response received on 6/2/2011, Applicants cancelled claims 13, 31, 35-36, 79, 83, and 84, and added new claims 103-116. Additionally, Applicants added new claims 117-181 in the supplemental response received on 8/9/2011.

3. Claims 1-4, 6-7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52-54, 56-61, 63, 65-67, 74-77, 80-82, 86, and 102-181 are currently pending. Claims 53, 57-61, 63, 65-67, 74-77, and 80-82 are withdrawn as discussed in the previous office action. Additionally, new claims 113, 158, and 179 are also withdrawn as being directed to the subject matter of non -elected Group III of the requirement for restriction mailed on 10/18/2010.

4. claims 1-4, 6-7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52, 54, 56, 86, 102-112, 114-157, 159-178, and 180-181 are presently under examination.

Information Disclosure Statement The information disclosure statements received on 6/2/2011, 7/6/2011, and 7/9/2011 have been fully considered.

Specification Applicants' amendments to the specification on 6/2/2011 and 8/9/2011 are noted.

IPR Page 1912 of 2482 Application/Control Number: 12/325,049 Page 3 Art Unit: 1646

Claim Ob;ections 1. Objections to claims 27-30 and 86, for reasons set forth on page 3 of the office action mailed on 3/2/2011, are withdrawn in view of Applicants' amendments to the claims.

2. The Examiner suggests amending claim 134 to recite "the formulation of claim 128", rather than "any one of claim 128". A similar amendment is suggested for claim 148.

Reiections Withdrawn

Claim Reiections - 35 USC§ 112, first paragraph - written description The following is a quotation of the first paragraph of 35 U.S.C. 112: The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.

Rejection of claims 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52, 54, 56, 86 and 102 under 35 USC § 112, first paragraph, regarding lack of written description for the genus of proteins which can be formulated as presently claimed, as set forth on pages 3-4 of the office action mailed on 3/2/2011, is withdrawn in response to Applicants' amendments to the claims to recite formulations comprising an antibody, or an antigen-binding fragment thereof.

Claim Reiections - 35 USC§ 112, second paragraph The following is a quotation of the second paragraph of 35 U.S.C. 112: The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.

Rejection of claims 34 and 41 under 35 USC § 112, second paragraph, as being indefinite regarding recitation of trademarks, as set forth on page 4 of the office action mailed on 3/2/2011, is withdrawn in response to Applicants' amendments to the claims to properly identify recited trademarks.

Reiections Maintained/New Grounds o(Reiection

IPR Page 1913 of 2482 Application/Control Number: 12/325,049 Page 4 Art Unit: 1646

Claim Reiections - 35 USC § 103 The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action: (a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.

1. Claims 1-4, 7, 9-11, 15-18, 20, 32-34, 42-43, 45, 48, 50, 52, 54, 56, 86, and 102 remain re;ected, and claims 6, 21-22, 24, 26-30, 37-41, 103-112, and 114-116 are also rejected under 35 USC§ 103(a) as being obvious in view of the combination of Konstantinov et al (US 20060149042) and Matheus et al (US 20070172475), as set forth on pages 5-6 of the office action mailed on 3/2/2011. The claims of the instant invention are directed to an aqueous formulation comprising an antibody, or antigen-binding fragment thereof, and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and the protein has a molecular weight greater than 47 kDa. The claims further recite formulations comprising proteins having molecular weights greater than 57 kDa, 100 kDa, or 150 kDa, and wherein the conductivity of the formulation is less than about 2 mS/cm, 1 mS/cm, or 0.5 mS/cm, and wherein the concentration of the protein is at least about 10 mg/ml, 100 mg/ml, 150 mg/ml, or greater than 200 mg/ml. The claims also recite aqueous formulations wherein the antibody or antigen - binding fragment thereof is a chimeric, human, humanized, or domain antibodies, and specifically Humira® (adalimumab). Also claimed are formulations comprising a non-ionizable excipient, including polyols such as mannitol, and formulations which are stable in liquid form for at least about 3 months or at least about 12 months, and a device or article of manufacture comprising said formulation. Konstantinov discloses a method for concentration of proteins, wherein the resulting formulation has a conductivity of less than 2.5 mS/cm (paragraph 0013; claims 4 and 5). Specifically Konstantinov shows that a method of concentrating proteins using ultrafiltration followed by diafiltration against water can be used to lower the conductivity of the solution to less than 6 mS/cm, and preferably, from 0.5 to 5 mS/cm. Konstantinov is silent regarding formulation of a protein having a molecular weight of at least 47 kDa, or any specific concentration of protein. However, Matheus teaches formulations of antibodies and water (paragraph 0034), wherein the antibody concentration is at least 1 mg/ml, and up to 250 mg/ml (paragraph 0061). Matheus also suggests formulation of Humira® ( also defined as adalimumab in the instant application) (paragraph 0062),

IPR Page 1914 of 2482 Application/Control Number: 12/325,049 Page 5 Art Unit: 1646 formulations comprising mannitol (paragraph 0051), and formulations intended for in vivo administration via routs of administration such as intravenous and subcutaneous (paragraph 0064). In the responses received on 6/2/2011 and 8/9/2011, the Applicants argue that the claimed invention is not obvious in view of the combination of Konstantinov and Matheus because the claims have been amended to recite formulations which comprise an antibody, or an antigen-binding fragment thereof, at a concentration of at least 20 mg/ml, wherein the antibody, or antigen-binding fragment thereof has a molecular weight greater than 47 kDa. In contrast, Konstantinov teaches concentration of IL-2SA, which has a molecular weight of less than 18 kDa, and does not teach concentration of this or any other protein at a concentration of at least 20 mg/ml. The Applicants assert that while Konstantinov teaches up to 100-fold concentration of proteins, it only discloses examples of proteins (IL-2SA) concentrated to 12 mg/ml, and therefore does not provide motivation to formulate proteins at a higher concentration. Additionally, the Applicants argue that the teachings of Matheus do not make up for the deficiencies of Konstantinov, as Matheus teaches that formulations containing a high protein concentration are known to be difficult to achieve due to the tendency of proteins, and in particular antibodies, to aggregate even in low concentrations. Furthermore, although Matheus does teach formulations comprising antibodies at a high concentration, Matheus teaches that the presence of excipients such as NaCl and phosphate buffer is required for such formulation, and formulations which comprise phosphate buffered saline have a higher conductivity that what is required by the claims, as evidenced by the description of PBS from Fisher Scientific (submitted as Appendix A). The Applicants also assert that while Matheus is focused on antibody formulations, Konstantinov provides working examples of a much smaller molecule (IL-2SA having a molecular weight of 18 kDa), and therefore there is no evidence that the methods described therein would work for larger molecules such as antibodies, and therefore one of ordinary skill in the art would have no expectation of success that the methods described in Konstantinov would work for larger molecules, such as antibodies. These arguments have been fully considered and are not persuasive. As discussed above and in the previous office action, Konstantinov teaches a method of concentrating proteins using ultrafiltration followed by diafiltration against water, wherein the conductivity of the resulting solution is lowered to less than 6 mS/cm, and preferably 0.5 - 5 mS/cm. While Konstantinov does not specifically exemplify using this method to concentrate antibodies or antigen-binding fragments thereof, or other proteins having a molecular weight greater than 47 kDa, it does teach that antibodies such as infliximab and abciximab, which are greater than 47 kDa, can be concentrated in this manner (paragraph 0041). Therefore, a person of ordinary skill in the art would have the motivation to concentrate antibodies in this manner.

IPR Page 1915 of 2482 Application/Control Number: 12/325,049 Page 6 Art Unit: 1646

Regarding Applicants' arguments that Matheus teaches away from formulating antibodies at a high concentration without the use of excipients, it is noted that the method of Konstantinov provides a suitable way to concentrate proteins such as antibodies, and thus a person of ordinary skill in the art would know, via Konstantinov, of a way to solve the solubilty/aggregation problem discussed in Matheus. It is also noted that the language of the instant claims specifies a formulation "comprising" an antibody, or antigen-binding fragment thereof, and are thus not limited to a formulation comprising only an antibody and water. Therefore, although the Applicants argue that it would have not been obvious to include excipients in the claimed formulations, the recitation of "comprising" in the claims allows for the obvious use of excipients, and it would be obvious to a person of ordinary skill in the art to include excipients, such as the Pluronic F68 taught by Konstantinov, as long as the conductivity remains low. Furthermore, while Konstantinov exemplifies a protein (IL-2SA) formulation having a concentration of only 12 mg/ml, rather than 20 mg/ml, Konstantinov does suggest that the method of ultrafiltration/diafiltration with water can be used to concentrate proteins 100-fold. Therefore, because Konstantinov suggests high concentrations of proteins, such as antibodies, and Matheus teaches that antibodies can be formulated up to 250 mg/ml, a person of ordinary skill in the art would have the motivation to formulate antibodies such as Humira®/adalimumab at high concentrations such as 20 mg/ml. With regards to Applicants' arguments that the instant invention, such as in claim 10, requires a conductivity less than 0.5 mS/cm, it is noted that the claims recite a conductivity of less than "about" 0.5 mS/cm, and therefore the conductivity of 0.5 mS/cm taught by Konstantinov would be less than 0.55, or 0.6 mS/cm, both of which could be considered to be "about" 0.5 mS/cm. It is also noted that because Konstantinov teaches the general concept of concentration of proteins by lowering the conductivity, a person of ordinary skill in the art would have both the motivation, and the ability to optimize the conductivity of the formulation in order to create the most effectively stabilized composition. MPEP 2144.05 states: "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 454, 105 USPQ 223,235, (CCPA 1955).

Similarly, while Konstantinov does not specifically teach ultrafiltration and diafiltration of proteins wherein a five-fold volume exchange of water has been achieved, as is recited in claim 86, a person of ordinary skill in the art would have the motivation to optimize the exchange volume of water in the method of Konstantinov in order to create a formulation with suitably low conductivity.

IPR Page 1916 of 2482 Application/Control Number: 12/325,049 Page 7 Art Unit: 1646

Regarding claims 6, 21-22, 24, 26-30, 37-41, 103-112, and 114-116, these claims are directed to formulations comprising an antibody, or antigen-binding fragment thereof, wherein the formulation has an osmolality of no more than 30 mOsmol/kg, or wherein the antibody or antigen-binding fragment thereof has a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the antibody in a buffered solution at the same concentration, or alternatively, less than about 4 nm. Although neither Konstantinov nor Matheus specifically teach formulations with a specific osmolality, or teach that the formulated protein must have a specific hydrodynamic diameter, it is noted that the method employed by Konstantinov, namely ultrafiltration followed by diafiltration against water, is the same method employed to prepare the presently-claimed formulations. In absence of evidence to the contrary, it would be expected that the formulations suggested by Konstantinov and Matheus would inherently exhibit an osmolality of no more than 30 mOsmol/kg, or 15 mOsmol/kg, and/or would inherently comprise an antibody molecule with a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the same antibody in a buffered solution, or less than about 4 nm. Because the USPTO does not have the facilities for testing the formulations/methods of Konstantinov and Matheus, the burden is on the Applicants to show a novel and unobvious difference between the claimed formulations and those suggested by the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and Ex parte Gray, 10 USPQ 2d 1922 1923 (PTO Bd. Pat. App. & Int.). In the instant case, although neither Konstantinov nor Matheus specifically suggest formulations with the recited values for osmolality or hydrodynamic diameters, the combination of Konstantinov and Matheus provides the motivation, for the reasons discussed above, to formulate antibody molecules using a method of ultrafiltration followed by diafiltration against water, wherein this method would be expected to inherently produce formulations with the recited osmolality and/or hydrodynamic diameters.

2. Claim 44 remains rdected under 35 USC § 103(a) as being obvious in view of the combination of Konstantinov et al (US 20060149042), Matheus et al (US 20070172475), and Schurschnig (US 20070202051 ), as set forth on page 7 of the office action mailed on 3/2/2011. In the response received 6/2/2011, the Applicants argue that the subject matter of the instant invention is not obvious in view of the combination of Konstantinov and Matheus for the reasons discussed above, and therefore claim 44 is also not obvious in view of any combination based on this combination.

IPR Page 1917 of 2482 Application/Control Number: 12/325,049 Page 8 Art Unit: 1646

These arguments have been fully considered and are not persuasive for reasons set forth above. Therefore, the rejection is maintained for reasons of record.

3. Claims 117-122, 125-136, 139, 144-150, 153, 159-160, and 180-181 are rejected under 35 U.S.C. 103(a) as being unpatentable over Konstantinov et al (US 20060149042), in view of Matheus et al (US 20070172475), and further in view of Salfeld et al (US 6,090,382, cited in the IDS received on 10/19/2010). The subject matter of claims 117-122, 125-127, and 180-181 is directed to an aqueous formulation comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 50 mg/ml, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7, or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 8. The claims also recite said formulation comprising the antibody at a concentration of at least about 75 mg/ml, a conductivity of less than 2 mS/cm, and wherein said formulation comprises a non-ionizable excipient, such as a polyol, and specifically, mannitol or sucrose. Also claimed is a formulation comprising an antibody, or antigen­ binding portion thereof, which has a LCVR comprising the amino acid sequence of SEQ ID NO: 1, and a HCVR comprising the amino acid sequence of SEQ ID NO: 2, and specifically, wherein said antibody is adalimumab. Konstantinov and Matheus teach as discussed above, but are silent regarding a formulation comprising an antibody which comprises LCVR and HCVR domains of SEQ ID NOs 1 and 2, respectively, and which comprise various CDR domains as recited in claim 117. However, Salfeld discloses an anti-TNF-a antibody which comprises a LCVR region comprising SEQ ID NO: 1, and a HCVR comprising SEQ ID NO: 2, wherein said SEQ ID NOs 1-2 of Salfeld are identical to SEQ ID NOs 1-2 of the instant application. Furthermore, Salfeld teaches that said antibody comprises a LCVR which comprises CDR3, CDR2, and CDRl domains of SEQ ID NOs 3, 5, and 7,

IPR Page 1918 of 2482 Application/Control Number: 12/325,049 Page 9 Art Unit: 1646 respectively, and a LCVR which comprises CDR3, CDR2, and CDRl domains of SEQ ID NOs 4, 6, and 8, respectively, wherein SEQ ID NOs 3-8 of Salfeld are identical to SEQ ID NOs 3-8 of the instant application (see accompanying Sequence Comparisons 1-8). Therefore, a person of ordinary skill in the art, at the time the instant invention was conceived, would have been motivated to create an aqueous formulation comprising an antibody, or antigen binding fragment thereof, wherein said formulation has an antibody concentration of at least 50 mg/ml, and a conductivity of less than about 2.5 mS/cm, and wherein said antibody comprises a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7, or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 8. The motivation to do so comes from the combination of Konstantinov and Matheus, which provide the motivation to create a formulation comprising antibodies, wherein said formulation has a conductivity of less than 6 mS/cm, and preferably 0.5 to 5 mS/cm, and wherein said formulation may comprise antibodies which may be concentrated 100-fold. As discussed above and in the previous office action, Konstantinov teaches that proteins, including antibodies, can be concentrated via ultrafiltration and diafiltration against water, wherein the conductivity is lowered, allowing the protein to be concentrated up to 100-fold. Matheus teaches that antibodies, when properly stabilized, can be formulated at concentrations up to 250 mg/ml, and also suggests formulation of antibodies, including adalimumab (Humira®) with excipients including mannitol and sucrose (paragraph 0046). Thus, given the guidance provided by Konstantinov and Matheus, a person of ordinary skill in the art would have had the motivation to use the method of Konstantinov to concentrate an antibody by ultrafiltration and diafiltration against water, resulting in lowering the conductivity to between 0.5 and 5 mS/cm. Furthermore, because Salfeld teaches a specific anti-TNF antibody which comprises a LCVR identical to SEQ ID NO: 1 of the instant invention, and a HCVR identical to SEQ ID NO: 2 of the instant invention, a person of ordinary skill in the art would have been motivated to formulate this antibody using the method suggested by Konstantinov and Matheus because a skilled artisan would have known that this method would result in a stabilized formulation of said antibody, wherein said antibody could be concentrated up to 100-fold. While Konstantinov does not explicitly suggest formulation of antibodies at

IPR Page 1919 of 2482 Application/Control Number: 12/325,049 Page 10 Art Unit: 1646

50 mg/ml, or 75 mg/ml, Matheus teaches that once properly stabilized, antibodies can be formulated at such concentrations, and therefore a skilled artisan would be motivated to create formulations comprising the antibody of Salfeld at these concentrations. Furthermore, by suggesting antibody formulations comprising mannitol and/or sucrose, Matheus would provide a skilled artisan with the motivation to incorporate these excipients into said formulation. Regarding claims 128-136, 139, 144-150, 153, and 159-160, these claims are directed to formulations comprising an antibody having the CDR sequences discussed above, or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the antibody in a buffered solution at the same concentration, or alternatively, having a hydrodynamic diameter of less than 4 nm. Although none of Konstantinov, Matheus, or Salfeld specifically teach formulations with a specific osmolality, or teach that the formulated protein must have a specific hydrodynamic diameter, it is noted that the method employed by Konstantinov, namely ultrafiltration followed by diafiltration against water, is the same method employed to prepare the presently-claimed formulations. In absence of evidence to the contrary, it would be expected that the formulations suggested by Konstantinov, Matheus, and Salfeld would inherently exhibit an osmolality of no more than 30 mOsmol/kg, or 15 mOsmol/kg, and/or would inherently comprise an antibody molecule with a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the same antibody in a buffered solution, or less than about 4 nm. Because the USPTO does not have the facilities for testing the formulations/methods of Konstantinov, Matheus, and Salfeld, the burden is on the Applicants to show a novel and unobvious difference between the claimed formulations and those suggested by the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and Ex parte Gray, 10 USPQ 2d 1922 1923 (PTO Bd. Pat. App. & Int.). In the instant case, although none Konstantinov, Matheus, or Salfeld specifically suggest formulations with the recited values for osmolality or hydrodynamic diameters, the combination of Konstantinov, Matheus, and Salfeld provides the motivation, for the reasons discussed above, to formulate antibody molecules using a method of ultrafiltration followed by diafiltration against water, wherein this method would be expected to inherently produce formulations with the recited osmolality and/or hydrodynamic diameters.

4. Claims 123-124, 137-138, 140-143, 151-152, 154-157, 161-178, and 180-181 are rejected under 35 U.S.C. 103(a) as being unpatentable over Konstantinov et al (US 20060149042), in view of

IPR Page 1920 of 2482 Application/Control Number: 12/325,049 Page 11 Art Unit: 1646

Matheus et al (US 20070172475), and further in view of Salfeld et al (US 6,090,382), and further in view of Schuschnig (US 20070202051). The subject matter of the instant invention is discussed above. Claims 123-124 are further drawn to the formulation of claim 117 (discussed above), further comprising a non-ionizable surfactant, and specifically, polysorbate 80. Similarly, claims 161-166 are drawn to an aqueous formulation comprising mannitol, polysorbate 80, and an antibody or antigen-binding fragment thereof at a concentration of at least about 20 mg/ml, having a conductivity of less than about 2.5 mS/cm, and wherein said antibody comprises a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5, 7, or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 8. Konstantinov, Matheus, and Salfeld teach as discussed above, and provide the motivation to create an aqueous formulation comprising an antibody which comprises a LCVR having CDR3, CDR2, and CDRl domains of SEQ ID NOs 3, 5, and 7, respectively, and a HCVR having CDR3, CDR2, and CDRl domains of 4, 6, and 8, respectively, and wherein said formulation has a conductivity from 0.5 to 5 mS/cm, and further comprises excipients such as mannitol. Konstantinov, Matheus, and Salfeld are silent regarding formulations comprising polysorbate 80. However, Schuschnig teaches formulation of various therapeutic proteins, including antibodies (paragraphs 0118, 0124), wherein said formulation can comprise non-ionic surfactants such as polysorbate 80 (paragraph 0140). Therefore, it would have been obvious to a person of ordinary skill in the art, at the time the instant invention was conceived, to create a formulation comprising an antibody as set forth in claims 117 and/or 161, wherein said formulation comprises said antibody at a concentration of at least 50-75 mg/ml, and has a conductivity of less than 2.5 mS/cm. The motivation to do so comes from the combination of Konstantinov, Matheus, and Salfeld, which as discussed above, provides the motivation to create antibody formulations having a low conductivity, and wherein said formulations comprise antibodies which have been concentrated 100-fold. Further motivation comes from Schurschnig, teach suggests formulation

IPR Page 1921 of 2482 Application/Control Number: 12/325,049 Page 12 Art Unit: 1646 with polysorbate 80, and thus a skilled artisan would have found it obvious to incorporate polysorbate 80 into the formulation suggested Konstantinov, Matheus, and Salfeld. Regarding claims 137-138, 140-143, 151-152, 154-157, and 167-178, these claims are directed to formulations comprising an antibody, or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the antibody in a buffered solution at the same concentration, or alternatively, has a hydrodynamic diameter of less than about 4 nm. Although none of Konstantinov, Matheus, Salfeld, and Schuschnig specifically teach formulations with an antibody with a specific hydrodynamic diameter, it is noted that the method employed by Konstantinov, namely ultrafiltration followed by diafiltration against water, is the same method employed to prepare the presently-claimed formulations. In absence of evidence to the contrary, it would be expected that the formulations suggested by Konstantinov, Matheus, Salfeld, and Schurschnig would inherently comprise an antibody molecule with a hydrodynamic diameter which is at least about 50% less than the hydrodynamic diameter of the same antibody in a buffered solution, or less than about 4 nm. Because the USPTO does not have the facilities for testing the formulations/methods of Konstantinov, Matheus, Salfeld, and Schurschnig, the burden is on the Applicants to show a novel and unobvious difference between the claimed formulations and those suggested by the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and Ex parte Gray, 10 USPQ 2d 1922 1923 (PTO Bd. Pat. App. & Int.). In the instant case, although none Konstantinov, Matheus, Salfeld, or Schurschnig specifically suggest formulations with the recited values for osmolality or hydrodynamic diameters, the combination of Konstantinov, Matheus, Salfeld, and Schurschnig provides the motivation, for the reasons discussed above, to formulate antibody molecules using a method of ultrafiltration followed by diafiltration against water, wherein this method would be expected to inherently produce formulations with the recited osmolality and/or hydrodynamic diameters.

Conclusion No claim is allowable.

Any inquiry concerning this communication or earlier communications from the examiner should be directed to Bruce D. Hissong, whose telephone number is (571)272-3324. The examiner can normally be reached M-F from 8:30 am - 5:00 pm. If attempts to reach the examiner by telephone are

IPR Page 1922 of 2482 Application/Control Number: 12/325,049 Page 13 Art Unit: 1646 unsuccessful, the examiner's supervisor, Gary B. Nickol, can be reached at (571) 272-0835. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair­ direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.

Bruce D. Hissong Art Unit 1646

/Robert Landsman/ Primary Examiner, Art Unit 1647

IPR Page 1923 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: July 9, 2011 Electronic Signature for Cristin Howley Cowles, Ph.D.: /Cristin Cowles/

ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name B. Hissong

Sheet I 1 I of I 2 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Pages, Columns, Lines, Examiner Document Number Cite Publication Date Name of Patentee or Where Relevant Passages or Initials* 2 No. 1 Number-Kind Code ( if MM-DD-YYYY Applicant of Cited Relevant known) Document Fiqures Appear A1 us 4877608 10-31-1989 Lee et al. A2 us 6436397 08-20-2002 Baker et al. A3 US20040197324 10-07-2004 Liu et al. A4 US200602694 79 11-30-2006 Colton et al. A5 US20070065440 03-22-2007 Tomlinson et al. A6 US20070122402 05-31-2007 Balli et al.

Examiner Date Signature iBruce Hissong/ Considered 1i/15i2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 11968784v.1 IPR Page 1924 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name B. Hissong

Sheet I 2 I of I 2 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS

Foreign Patent Documet Pages, Columns, Publication Name of Patentee or Lines, Applicant of Cited Document Where Relevant 3 4 Date TB Examiner Cite Country Code -Number - Passages Or 1 MM-DD-YYYY Initials* No. Kind Code5 (if known) Relevant Figures Appear D NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Examiner Cite 1 the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T2 Initials No. number(s), publisher, citv and/or countrv where published. C1 LIU et al. "Reversible Self-Association Increases the viscosity of a Concentrated Monoclonal Anitbody in Aqueous Solution", J Pharma Sci ,Vol. 94, No. 9, pp. 1928-1940 (2005) C2 WANG et al. "Opalescence of an lgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients", BioPharm lnternatl, Vol. 22(4) (2009)

Examiner Date Signature /Bruce Hissong/ Considered 11/15/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 11968784v.1 IPR Page 1925 of 2482 Application/Control No. Applicant(s)/Patent Under Reexamination Search Notes 12325049 FRAUNHOFER ET AL.

Examiner Art Unit

Bruce D Hissong, Ph.D. 1646

SEARCHED

Class I Subclass I Date I Examiner None I None I 11/15/2011 I BDH

SEARCH NOTES

Search Notes Date Examiner WEST, STN: update 2/26/2011 search 11/15/2011 BDH PALM: inventor search 11/15/2011 BDH STIC Sequence search: SEQ ID NOs 1-8 (search results in SCORE) 11/15/2011

INTERFERENCE SEARCH

Class I Subclass I Date I Examiner I I I

U.S. Patent and Trademark Office Part of Paper No. : 20111115

IPR Page 1926 of 2482 Sequence Comparision 8 - 12/325,049 (comparison of SEQ ID NO: 8 with SEQ ID NO: 8 of US 6,090,382)

US-08-599-226-8 Sequence 8, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-8

Query Match 100.0%; Score 30; DB 2; Length 5; Best Local Similarity 100.0%; Matches 5; Conservative O; Mismatches 0., Indels O; Gaps 0.,

Qy 1 DYAMH 5

I 1111 Db 1 DYAMH 5

IPR Page 1927 of 2482 IPR Page 1928 of 2482 Sequence Comparision 2 - 12/325,049 (comparison of SEQ ID NO: 2 with SEQ ID NO: 2 of US 6,090,382)

US-08-599-226-2 Sequence 2, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 121 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-2

Query Match 100.0%; Score 635; DB 2; Length 121; Best Local Similarity 100.0%; Matches 121; Conservative 0., Mismatches 0., Indels 0., Gaps 0.,

Qy 1 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY 60

I 1111111111111111111111111111111111111111111111111111111111 I Db 1 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY 60

IPR Page 1929 of 2482 Qy 61 ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS 120

I 1111111111111111111111111111111111111111111111111111111111 I Db 61 ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS 120

Qy 121 s 121

I Db 121 S 121

IPR Page 1930 of 2482 Sequence Comparision 3 - 12/325,049 (comparison of SEQ ID NO: 3 with SEQ ID NO: 3 of US 6,090,382)

US-08-599-226-3 Sequence 3, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal FEATURE: NAME/KEY: Modified-site LOCATION: 9 OTHER INFORMATION: /note= "Xaa is Thr or Ala" US-08-599-226-3

Query Match 100.0%; Score 46; DB 2; Length 9; Best Local Similarity 100.0%; Matches 8; Conservative O; Mismatches 0., Indels O; Gaps 0.,

IPR Page 1931 of 2482 Qy 1 QRYNRAPY 8

I 111111 I Db 1 QRYNRAPY 8

IPR Page 1932 of 2482 Sequence Comparision 4 - 12/325,049 (comparison of SEQ ID NO: 4 with SEQ ID NO: 4 of US 6,090,382)

US-08-599-226-4 Sequence 4, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal FEATURE: NAME/KEY: Modified-site LOCATION: 12 OTHER INFORMATION: /note= "Xaa is Tyr or Asn" US-08-599-226-4

Query Match 100.0%; Score 50; DB 2; Length 12; Best Local Similarity 100.0%; Matches 11; Conservative 0., Mismatches 0., Indels 0., Gaps 0.,

IPR Page 1933 of 2482 Qy 1 VSYLSTASSLD 11

I 1111111111 Db 1 VSYLSTASSLD 11

IPR Page 1934 of 2482 Sequence Comparision 5 - 12/325,049 (comparison of SEQ ID NO: 5 with SEQ ID NO: 5 of US 6,090,382)

US-08-599-226-5 Sequence 5, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 5: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-5

Query Match 100.0%; Score 30; DB 2; Length 7; Best Local Similarity 100.0%; Matches 7; Conservative O; Mismatches 0., Indels O; Gaps 0.,

Qy 1 AASTLQS 7

I 111111 Db 1 AASTLQS 7

IPR Page 1935 of 2482 IPR Page 1936 of 2482 Sequence Comparision 6 - 12/325,049 (comparison of SEQ ID NO: 6 with SEQ ID NO: 6 of US 6,090,382)

US-08-599-226-6 Sequence 6, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-6

Query Match 100.0%; Score94; DB2; Lengthl7; Best Local Similarity 100.0%; Matches 17; Conservative 0., Mismatches 0., Indels 0., Gaps 0.,

Qy 1 AITWNSGHIDYADSVEG 17

I 1111111111111111 Db 1 AITWNSGHIDYADSVEG 17

IPR Page 1937 of 2482 IPR Page 1938 of 2482 Sequence Comparision 7 - 12/325,049 (comparison of SEQ ID NO: 7 with SEQ ID NO: 7 of US 6,090,382)

US-08-599-226-7 Sequence 7, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-7

Query Match 100.0%; Score 54; DB 2; Length 11; Best Local Similarity 100.0%; Matches 11; Conservative 0., Mismatches 0., Indels 0., Gaps 0.,

Qy 1 RASQGIRNYLA 11

I 1111111111 Db 1 RASQGIRNYLA 11

IPR Page 1939 of 2482 IPR Page 1940 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with § 1.6(a)(4). Dated: July 6, 2011 Electronic Signature for Deborah L. Nagle, Ph.D.: /Deborah L. Nagle/ PTO/SB/08b (11-08) Approved for use through 12/31/2008. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid 0MB control number.

Complete if Known Substitute for form 1449/PTO Application Number 12/325,049-Conf.# 1766 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1646 (Use as many sheets as necessary) Examiner Name Hissong, Bruce D. Sheet I 1 I 01 I 7 Attorney Docket Number 117813-26902

Pages, Columns, Document Number Publication Date Name of Patentee or Examiner Cite Lines, Where Relevant Initials' No. 1 Number-Kind Code2 ( if known) MM-DD-YYYY Applicant of Cited Document Passages or Relevant Fi~ u res Appear

A1* US-20070184045 08-09-2007 Doctor et al

A2* US-20070269463 11-22-2007 Donavan et al

A3* US-20060142549 06-29-2006 Takeda et al

A4* US-20070237762 10-11-2007 Winter

Examiner Date Signature iBruce Hissong/ Considered 11i15/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901 .04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 11955346v. 1 ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ IPR Page 1941 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4).

Dated: June 2, 2011 Electronic Signature for Cristin Howley Cowles, Ph.D.: /Deborah L. Nagle/

ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned

Sheet I 1 I of I 2 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Pages, Columns, Lines, Examiner Document Number Cite Publication Date Name of Patentee or Where Relevant Passages or Initials* 2 No. 1 Number-Kind Code ( if MM-DD-YYYY Applicant of Cited Relevant known) Document Fiqures Appear A1 us 20100160894 06-24-2010 Julian et al. A2 us 6,693,173 02-17-2004 Mamidi et al. A3 us 6,171,586 01-09-2001 Lam et al. A4 us 5,608,038 03-04-1997 Eibl et al. A5 us 2004-0170623 09-02-2004 Arvinte et al. A6 us 2006-0115472 06-01-2006 Li et al. A7 us 20090148406 06-11-2009 Jezek et al. A8 us 20040038878 02-26-2004 Tanikawa et al. A9 us 20050214278 09-25-2005 Kakuta et al. A10 us 200401 05889 06-03-2004 Ryde et al. A11 us 20060115472 06-01-2006 Li et al. A12 us 20060210557 09-21-2006 Luisi et al. A13 us 20080311078 12-18-2008 Gokarn et al. A14 us 20080139792 06-12-2008 Sek et al. A15 us 20080200655 08-21-2008 Sek et al. A16 us 20110054414 03-03-2011 Shanget al. A17 us 20100278822 11-04-2010 Fraunhofer et al.

Examiner iBruce Hissong/ Date Signature Considered ii/15/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 11784332v.1 IPR Page 1942 of 2482 ALTERNATIVE TO PTO/SB/OSA/B (Based on PTO 01-08 version) Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1636 (Use as many sheets as necessary) Examiner Name Not yet assigned Sheet I 2 I 01 I 2 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS Foreign Patent Pages, Columns, Documet Publication Name of Patentee or Lines, 3 Applicant of Cited Document Country Code - Date Where Relevant Te Examiner Cite Number4-Kind Code5 MM-DD- Passages Or 1 yyyy Initials* No. (if known) Relevant Figures Appear

B1 WO 95/03826 02-09-1995 Pasteur Merieux Serums ET Vaccins D B2 WO 98/044948 10-19-1998 Cangene Corporation D B3 WO 00/67789 11-16-2000 CSL Limited D NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Exami~er Cite 1 the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T' Initials No. number(s), publisher, city and/or country where published. C1 Li et al., "Resurrecting Abandoned Proteins with Pure Water: CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water," biophysj 91 :4201-4209 (2006)

C2 Manning et al., "Stability of Protein Pharmaceuticals," Pharm res 6:903-918 (1989)

C3 Shire et al., "Challenges in the Development of High Protein Concentration Formulations," J Pharm Sci 93:1390-1402 (2004)

C4 Tian et al., "Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations," Intl J Pharm 355:20- 31 (2007)

Examiner /Bruce Hissong/ Date Signature Considered 11i15/2011

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Oftice that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached. ALL REFERENCES CONSIDERED EXCEPT WHERE LINED THROUGH. /BH/ ME1 11784332v.1 IPR Page 1943 of 2482 Sequence Comparision 1 - 12/325,049 (comparison of SEQ ID NO: 1 with SEQ ID NO: 1 of US 6,090,382)

US-08-599-226-1 Sequence 1, Application US/08599226 Patent No. 6090382 GENERAL INFORMATION: APPLICANT: Salfeld, Jochen G. APPLICANT: Allen, Deborah J. APPLICANT: Hoogenboom, Hendricus R.J.M. APPLICANT: Kaymakcalan, Zehra APPLICANT: Labkovsky, Boris APPLICANT: Mankovich, John A. APPLICANT: McGuinness, Brian T. APPLICANT: Roberts, Andrew J. APPLICANT: Sakorafas, Paul APPLICANT: Schoenhaut, David APPLICANT: Vaughan, Tristan J. APPLICANT: White, Michael APPLICANT: Wilton, Andrew J. TITLE OF INVENTION: Human Antibodies that Bind Human TNFa NUMBER OF SEQUENCES: 37 CORRESPONDENCE ADDRESS: ADDRESSEE: LAHIVE & COCKFIELD STREET: 60 State Street, suite 510 CITY: Boston STATE: Massachusetts COUNTRY: USA ZIP: 02109-1875 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release #1.0, Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US/08/599,226 FILING DATE: 08-FEB-1996 CLASSIFICATION: 424 ATTORNEY/AGENT INFORMATION: NAME: Deconti, Giulio A., Jr. REGISTRATION NUMBER: 31,503 REFERENCE/DOCKET NUMBER: BBI-043 TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 227-7400 TELEFAX: (617)227-5941 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide FRAGMENT TYPE: internal US-08-599-226-1

Query Match 100.0%; Score 553; DB 2; Length 107; Best Local Similarity 100.0%; Matches 107; Conservative 0., Mismatches 0., Indels 0., Gaps 0.,

Qy 1 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS 60

I 1111111111111111111111111111111111111111111111111111111111 I Db 1 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS 60

IPR Page 1944 of 2482 Qy 61 RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK 107

I 1111111111111111111111111111111111111111111111 Db 61 RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK 107

IPR Page 1945 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with§ 1.6(a)(4). Dated: May 21, 2012 Electronic Signature for Cristin H. Cowles / Cristin Cowles/

Docket No.: 117813-26902 (PATENT) IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Application of: Wolfgang Fraunhofer et al.

Attorney Docket No.: 117813-26902

Application No.: 12/325,049 Group Art Unit: 1646

Filed: November 28, 2008 Examiner: Hissong, Bruce D.

For: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

AMENDMENT AND RESPONSE TO NON-FINAL OFFICE ACTION

Dear Sir:

This communication is responsive to the Office Action dated January 4, 2012. An appropriate extension of time is being submitted herewith. Responsive to the Office Action, please amend the above-identified application as follows.

Amendments to the Claims begin on page 2 of this paper.

Remarks/Arguments begin on page 26 of this paper.

MEI 13469622v.1

IPR Page 1946 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

AMENDMENTS TO THE CLAIMS

Please amend the claims as follows:

1. (Previously Presented) An aqueous formulation comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm and the antibody, or antigen­ binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa.

2. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 57 kDa.

3. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 100 kDa.

4. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 150 kDa.

5. (Canceled)

6. (Previously Presented) The formulation of claim 1, wherein the antibody, or antigen-binding fragment thereof, has a Mw greater than about 250 kDa.

7. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 2 mS/cm.

8. (Canceled)

2

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9. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 1 mS/cm.

10. (Original) The formulation of claim 1, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

11-14. (Canceled)

15. (Previously Presented) The formulation of claim l_-l-l-, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 100 mg/mL.

16. (Previously Presented) The formulation of claim l_-l-l-, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 150 mg/mL.

17. (Previously Presented) The formulation of claim l_-l-l-, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 200 mg/mL.

18. (Previously Presented) The formulation of claim l_-l-l-, wherein the concentration of the antibody, or antigen-binding fragment thereof, is greater than abem 200 mg/mL.

19-20. (Canceled)

21. (Currently Amended) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, at a concentration of at least about 50 mg/mL, and water, wherein the formulation has an osmolality of no more than about 30 mOsmol/kg and wherein the antibody, or antigen-binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa.

3

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22. (Original) The formulation of claim 21, wherein the osmolality of the formulation is no more than about 15 mOsmol/kg.

23. (Canceled)

24. (Previously Presented) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is at least about 150 mg/mL.

25. (Canceled)

26. (Previously Presented) The formulation of claim 21, wherein the concentration of the antibody, or an antigen-binding fragment thereof, is greater than about 200 mg/mL.

27. (Currently Amended) An aqueous formulation comprising water and at least about 20 mg/mL of an antibody, or an antigen-binding fragment thereof, wherein the antibody, or antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa ..

28. (Previously Presented) The formulation of claim 27, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in phosphate buffered saline (PBS) at the same concentration.

29. (Previously Presented) The formulation of claim 28, wherein the Dh of the protein is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

4

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30. (Previously Presented) The formulation of claim 28, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

31. (Canceled)

32. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, and 27, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

33. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, and 27, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody, or an antigen-binding fragment thereof.

34. (Currently Amended) The formulation of claim 33, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of Humira® fadalimumab[[)]], infliximabRemicade® (lnfliJcimab), golimumab, certolizumab pegol, and ustekinumab. (Centocor), Cimzia® (Certolizumab pegol), and CNTO 1275 (ustekinumab).

35-36. (Canceled)

37. (Currently Amended) An aqueous formulation comprising an antibody, or an antigen-binding fragment, at a concentration of at least about -l-0 20 mg/mL.,_ and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm.,_ and wherein the antibody, or antigen-binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa.

5

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38. (Previously Presented) The formulation of claim 37, wherein the antibody has a Dh of less than about 3 nm.

39. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

40. (Previously Presented) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody, or antigen-binding fragment thereof.

41. (Currently Amended) The formulation of claim 37, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab pentetate, capromab pendetide, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromonab-CD3, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab pegol, eculizumab, ustekinumab, panitumumab, tositumomab, 1131 tositumomab, and bevacizumab. Humira@ (adalimumab), Campath@ (Alemtuzumab), CEA Scan Arcitumomab (fab fragment), Erbitux@ (Cetuximab), Herceptin@ (Trastuzumab), Myoscint@ (Imciromab Pentetate), Prosta8cint@ (Capromab Pendetide), Remicade@ (lnfliximab), ReoPro@ (AbciJcimab), RituJcan@ (RituJcimab), 8imulect@ (Basifocimab), 8ynagis@ (Palivizumab), Verluma@ (Nofetumomab), Xolair@ (Omalizumab), ZenapaJc@ (Daclizumab), Zevalin@ (lbritumomab Tiuxetan), Orthoclone OKT3@ (Muromonab CD3), Panorex@ (Edrecolomab), and Mylotarg@ (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia@ (Certolizumab pegol), 8oliris@ (Eculizumab), CNTO 1275 (ustekinumab), VectibiJc@ (panitumumab), BeJocar@ (tositumomab and F tositumomab) and Avastin@ (bevacizumab).

6

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42. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, 27, and 37, further comprising a non-ionizable excipient.

43. (Previously Presented) The formulation of claim 42, wherein the non- ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

44. (Original) The formulation of claim 43, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

45. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, 27, and 37, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

46-47. (Canceled)

48. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, 27, and 37, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

49. (Canceled)

50. (Currently Amended) The formulation of any one of claims 1, -l-1-, 21, 27, and 37, wherein the formulation is suitable for in vitro or in vivo use.

51. (Canceled)

7

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52. (Previously Presented) The formulation of claim 50, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

53. (Withdrawn) Use of the formulation of claim 50 in the treatment of a disorder in a subject.

54. (Currently Amended) A device comprising the formulation of any one claims 1, -l-1-, 21, 27, and 37.

55. (Canceled)

56. (Currently Amended) An article of manufacture comprising the formulation of any one of claims 1, -l-1-, 21, 27, and 37.

57. (Withdrawn) A method of preparing an aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, and water, the method comprising: a) providing the antibody, or antigen-binding fragment thereof, in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved to thereby prepare the aqueous formulation.

58. (Withdrawn) A method of preparing an aqueous formulation of an antibody, or an antigen-binding fragment thereof, the method comprising: a) providing the antibody, or an antigen-binding fragment thereof, in a first solution; b) subjecting the first solution to diafiltration using water as a diafiltration

8

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medium until at least a five-fold volume exchange with the water has been achieved to thereby prepare a diafiltered antibody, or antigen-binding fragment thereof, solution; and c) concentrating the diafiltered antibody, or an antigen-binding fragment thereof, solution to thereby prepare the aqueous formulation of the antibody, or antigen-binding fragment thereof.

59. (Withdrawn) The method of claim 58, wherein the concentration of the diafiltered antibody, or antigen-binding fragment thereof, solution is achieved via centrifugation.

60. (Withdrawn) The method of claim 57 or 58, wherein the diafiltration medium consists of water.

61. (Withdrawn) The method of claim 57 or 58, wherein the first solution is subjected to diafiltration with water until at least about a six-fold volume exchange or at least about a seven­ fold volume exchange is achieved.

62. (Canceled)

63. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation has a final concentration of excipients which is at least about 95% less or 99% less than the first solution.

64. (Canceled)

65. (Withdrawn) The method of claim 57 or 58, wherein the first antibody, or antigen- binding fragment thereof, solution is obtained from a mammalian cell expression system and has been purified to remove host cell proteins (HCPs).

66. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, has a Mw greater than about 1 kDa.

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67. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, has a molecular weight (Mw) selected from the group consisting of greater than about 10 kDa, greater than about 47 kDa, reater than about 57 kDa, greater than about 100 kDa, greater than about 150 kDa, greater than about 200 kDa, and greater than about 250 kDa.

68.-73. (Canceled)

74. (Withdrawn) The method of claim 57 or 58, further comprising adding an excipient to the aqueous formulation.

75. (Withdrawn) The method of claim 74, wherein the aqueous formulation is a pharmaceutical formulation.

76. (Withdrawn) The method of claim 57 or 58, wherein the aqueous formulation is a pharmaceutical formulation.

77. (Withdrawn) The method of claim 76, further comprising loading the aqueous formulation into a device suitable for administering the aqueous formulation to a subject.

78-79. (Canceled)

80. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, is selected from the group consisting of a chimeric antibody, a human antibody, a humanized antibody, and a domain antibody (dAb).

81. (Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen- binding fragment thereof, is an anti-TNFa or an anti-IL-12 antibody.

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82. (Currently Amended; Withdrawn) The method of claim 57 or 58, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab pentetate, capromab pendetide, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromonab-CD3, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab pegol, eculizumab, ustekinumab, panitumumab, tositumomab, 1131 tositumomab, and bevacizumab. Humira® (adalimumab), Campath® (Alemtuzumab), CEA Scan Arcitumomab (fab fragment), Erbitux® (Cetuximab), Herceptin® (Trastuzumab), Myoscint® (Imciromab Pentetate), ProstaScint® (Capromab Pendetide), Remicade® (lnfliJcimab), ReoPro® (AbciJcimab), RituJcan® (RituJcimab), Simulect® (Basifocimab), Synagis® (Palivizumab), Verluma® (Nofetumomab), Xolair® (Omalizumab), Zenapax® (Daclizumab), Zevalin® (lbritumomab Tiuxetan), Orthoclone OKT3® (Muromonab CD3), Panorex® (Edrecolomab), and Mylotarg® (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia® (Certolizumab pegol), Soliris® (Eculizumab), CNTO 1275 (ustekinumab), VectibiJc® (panitumumab), BeJocar® (tositumomab and F tositumomab) and Avastin® (bevacizumab).

83.-85. (Canceled)

86. (Previously Presented) An aqueous formulation comprising an antibody, or an antigen-binding fragment thereof, the antibody, or antigen-binding fragment thereof, prepared by a) providing the antibody, or antigen-binding fragment thereof, in a first solution; and b) subjecting the first solution to diafiltration using water as a diafiltration medium until at least a five fold volume exchange with the water has been achieved.

87.-101. (Canceled)

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102. (Previously Presented) The formulation of claim 43, wherein the polyol is mannitol or sorbitol.

103. (Currently Amended) An aqueous formulation comprising water and at least about 50 mg/ml of an antibody, or an antigen-binding fragment thereof, wherein the antibody, or antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a molecular weight (Mw) greater than about 47 kDa ..

104. (Previously Presented) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 150 mg/mL.

105. (Currently Amended) The formulation of claim 103, wherein the concentration of the antibody, or antigen-binding fragment thereof, is greater than abettt 200 mg/mL.

106. (Previously Presented) The formulation of claim 103, further comprising a non-ionizable excipient.

107. (Previously Presented) The formulation of claim 106, wherein the non- ionizable excipient is selected from the group consisting of a polyol, a non-ionic surfactant, sucrose, trehalose, raffinose, and maltose.

108. (Previously Presented) The formulation of claim 107, wherein the non-ionic surfactant is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

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109. (Previously Presented) The formulation of claim 103, wherein the formulation is stable in a liquid form for at least about 3 months or at least about 12 months.

110. (Previously Presented) The formulation of claim 103, wherein the formulation does not comprise an agent selected from the group consisting of a tonicity modifier, a stabilizing agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking agent, a lyroprotectant, a basic component, and an acidic component.

111. (Previously Presented) The formulation of claim 103, wherein the formulation is suitable for in vitro or in vivo use.

112. (Previously Presented) The formulation of claim 111, wherein the formulation is suitable for administration to a subject via a mode of administration selected from the group consisting of subcutaneous, intravenous, inhalation, intradermal, transdermal, intraperitoneal, and intramuscular.

113. (Withdrawn) Use of the formulation of claim 111 in the treatment of a disorder in a subject.

114. (Previously Presented) A device comprising the formulation of claim 103.

115. (Previously Presented) An article of manufacture comprising the formulation of claim 103.

116. (Currently Amended) The formulation of claim 21 or 27, wherein the antibody, or antigen-binding fragment thereof, is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab pentetate, capromab pendetide, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab,

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daclizumab, ibritumomab tiuxetan, muromonab-CD3, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab pegol, eculizumab, ustekinumab, panitumumab, tositumomab, 1131 tositumomab, and bevacizumab. Humira@ (adalimumab), Campath@ (Alemtuzumab), CEA Scan Arcitumomab (fab fragment), Erbitux@ (Cetuximab), Herceptin@ (Trastuzumab), Myoscint@ (Imciromab Pentetate), Prosta8cint@ (Capromab Pendetide), Remicade@ (lnfliximab), ReoPro@ (AbciJcimab), RituJcan@ (RituJcimab), 8imulect@ (Basifocimab), 8ynagis@ (Palivizumab), Verluma@ (Nofetumomab), Xolair@ (Omalizumab), ZenapaJc@ (Daclizumab), Zevalin@ (lbritumomab Tiuxetan), Orthoclone OKT3@ (Muromonab CD3), Panorex@ (Edrecolomab), and Mylotarg@ (Gemtuzumab ozogamicin), golimumab (Centocor), Cimzia@ (Certolizumab pegol), 8oliris@ (Eculizumab), CNTO 1275 (ustekinumab), VectibiJc@ (panitumumab), BeJocar@ (tositumomab and F tositumomab) and Avastin@ (bevacizumab).

117. (Previously Presented) An aqueous formulation comprising water and an antibody, or antigen-binding fragment thereof, at a concentration of at least about 50 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQ ID N0:8.

118. (Previously Presented) The formulation of claim 117, wherein the concentration of the antibody, or antigen-binding fragment thereof, is at least about 75 mg/mL.

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119. (Previously Presented) The formulation of claim 117, wherein the formulation has a conductivity of less than about 2 mS/cm.

120. (Previously Presented) The formulation of claim 117, further comprising a non-ionizable excipient.

121. (Previously Presented) The formulation of claim 120, wherein the non- ionizable excipient is a polyol.

122. (Previously Presented) The formulation of claim 121, wherein the polyol is mannitol.

123. (Previously Presented) The formulation of claim 120, wherein the non- ionizable excipient is a non-ionic surfactant.

124. (Previously Presented) The formulation of claim 123, wherein the non-ionic surfactant is polysorbate 80.

125. (Previously Presented) The formulation of claim 120, wherein the non- ionizable excipient is sucrose.

126. (Previously Presented) The formulation of claim 117, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

127. (Previously Presented) The formulation of claim 117, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

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128. (Previously Presented) An aqueous formulation comprising water and an antibody, or an antigen-binding fragment thereof, at a concentration of at least about 50 mg/mL, wherein the antibody, or an antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

129. (Previously Presented) The formulation of claim 128, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in phosphate buffered saline (PBS) at the same concentration.

130. (Previously Presented) The formulation of claim 129, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

131. (Previously Presented) The formulation of claim 130, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

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132. (Previously Presented) The formulation of claim 128, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

133. (Previously Presented) The formulation of claim 128, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

134. (Currently Amended) The formulation of any one of claim 128, further comprising a non-ionizable excipient.

135. (Previously Presented) The formulation of claim 134, wherein the non- ionizable excipient is a polyol.

136. (Previously Presented) The formulation of claim 135, wherein the polyol is mannitol.

137. (Previously Presented) The formulation of claim 134, wherein the non- ionizable excipient is a non-ionic surfactant.

138. (Previously Presented) The formulation of claim 137, wherein the non-ionic surfactant is polysorbate 80.

139. (Previously Presented) The formulation of claim 134, wherein the non- ionizable excipient is sucrose.

140. (Previously Presented) The formulation of claim 128, further comprising mannitol and polysorbate 80.

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141. (Previously Presented) The formulation of claim 128, further comprising sucrose and polysorbate 80.

142. (Previously Presented) The formulation of claim 140 or 141, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

143. (Previously Presented) The formulation of claim 140 or 141, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

144. (Previously Presented) An aqueous formulation comprising water and an antibody, or an antigen-binding fragment, at a concentration of at least about 50 mg/mL, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

145. (Previously Presented) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, has a Dh of less than about 3 nm.

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146. (Previously Presented) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

147. (Previously Presented) The formulation of claim 144, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

148. (Currently Amended) The formulation of any one of claims claim 144, further comprising a non-ionizable excipient.

149. (Previously Presented) The formulation of claim 148, wherein the non- ionizable excipient is a polyol.

150. (Previously Presented) The formulation of claim 149, wherein the polyol is mannitol.

151. (Previously Presented) The formulation of claim 148, wherein the non- ionizable excipient is a non-ionic surfactant.

152. (Previously Presented) The formulation of claim 151, wherein the non-ionic surfactant is polysorbate 80.

153. (Previously Presented) The formulation of claim 148, wherein the non- ionizable excipient is sucrose.

154. (Previously Presented) The formulation of claim 144, further comprising mannitol and polysorbate 80.

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155. (Previously Presented) The formulation of claim 144, further comprising sucrose and polysorbate 80.

156. (Previously Presented) The formulation of claim 154 or 155 wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

157. (Previously Presented) The formulation of claim 154 or 155, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

158. (Withdrawn) Use of the formulation of any one of claims 117, 128, or 144 in the treatment of a disorder in a subject.

159. (Previously Presented) A device comprising the formulation of any one of claims 117, 128, or 144.

160. (Previously Presented) An article of manufacture comprising the formulation of any one of claims 117, 128, or 144.

161. (Previously Presented) An aqueous formulation comprising mannitol, polysorbate 80, an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, wherein the formulation has a conductivity of less than about 2.5 mS/cm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region

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(LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

162. (Previously Presented) The formulation of claim 161, wherein the formulation has a conductivity of less than about 1.0 mS/cm.

163. (Previously Presented) The formulation of claim 161, wherein the formulation has a conductivity of less than about 0.5 mS/cm.

164. (Previously Presented) The formulation of claim 161, wherein the antibody concentration is at least about 50 mg/mL.

165. (Previously Presented) The formulation of claim 161, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

166. (Previously Presented) The formulation of claim 161, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

167. (Previously Presented) An aqueous formulation comprising mannitol, polysorbate 80,

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an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, wherein the antibody, or an antigen-binding fragment thereof, has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

168. (Previously Presented) The formulation of claim 167, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in phosphate buffered saline (PBS) at the same concentration.

169. (Previously Presented) The formulation of claim 168, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 60% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

170. (Previously Presented) The formulation of claim 168, wherein the Dh of the antibody, or antigen-binding fragment thereof, is at least about 70% less than the Dh of the antibody, or antigen-binding fragment thereof, in PBS at the same concentration.

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171. (Previously Presented) The formulation of claim 167, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

172. (Previously Presented) The formulation of claim 167, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

173. (Previously Presented) The formulation of claim 167, wherein the antibody concentration is at least about 50 mg/mL.

17 4. (Previously Presented) An aqueous formulation comprising mannitol, polysorbate 80, an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, and water, wherein the antibody, or antigen-binding fragment, has a hydrodynamic diameter (Dh) of less than about 4 nm, and wherein the antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:3, or modified from SEQ ID N0:3 by a single alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid sequence of SEQ ID N0:5, and a CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID N0:4, or modified from SEQ ID N0:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDRl domain comprising the amino acid sequence of SEQIDN0:8.

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175. (Previously Presented) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, has a Dh of less than about 3 nm.

176. (Previously Presented) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, has an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1, and an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2.

177. (Previously Presented) The formulation of claim 174, wherein the antibody, or antigen-binding portion thereof, is adalimumab.

178. (Previously Presented) The formulation of claim 17 4, wherein the antibody concentration is at least about 50 mg/mL.

179. (Withdrawn) Use of the formulation of any one of claims 161, 167, and 17 4 in the treatment of a disorder in a subject.

180. (Previously Presented) A device comprising the formulation of any one of claims 161, 167, and 174.

181. (Previously Presented) An article of manufacture comprising the formulation of any one of claims 161, 167, and 174.

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REMARKS Claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52-54, 56-61, 63, 65- 67, 74-77, 80-82, 86, and 102-181 were pending in the application. Claims 21, 27, 32-34, 37, 41- 42, 45, 48, 50, 54, 56, 82, 103, 105, 116, 134, and 148 have been amended, and claims 11 and 20 have been canceled. Claims 53, 57-61, 63, 65-67, 74-77, 80-82, 113, 158, and 179 have been withdrawn by the Examiner as being directed to non-elected subject matter. Accordingly, following entry of this amendment, claims 1-4, 6, 7, 9-10, 15-18, 21-22, 24, 26-30, 32-34, 37-45, 48, 50, 52- 54, 56-61, 63, 65-67, 7 4-77, 80-82, 86, and 102-181 will be pending in the application.

Support for the amendments to the claims can be found throughout the specification and in the claims as originally filed. Additional support for the amendment to claims 21, 27, 37, and 103 can be found at least at para. [0013] and [0016] of the US publication corresponding to the instant application. Claims 32, 33, 42, 45, 48, 50, 54, 56 have been amended to correct dependencies in view of claim cancellations. No new matter has been added.

Any amendment or cancellation to the claims has been made solely to expedite the prosecution of the application. Applicants reserve the right to pursue the claims as previously pending prior to this Amendment in this or a separate application(s).

Interview Summary

Inventor Wolfgang Fraunhofer and Applicants' attorneys, Tara Seshadri and Cristin Cowles, wish to thank Examiners Vanessa Ford, Bruce Hissong, and Robert Landsman for the courtesy of a personal interview on March 28, 2012. During the interview, the state of the art with respect to the instant invention and the Konstantinov and Matheus references were discussed.

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Claim Objections

Applicants acknowledge the withdrawal of the objection of claims 27-30 and 86. With respect to the objection of claims 134 and 148, Applicants have amended these claims in accordance with the Examiner's suggestion, thus rendering the objection moot.

Withdrawn Rejections

Applicants acknowledge the Examiner's withdrawal of the rejections of claims 1-4, 7, 9-11, 13, 15-18, 20-22, 24, 26-30, 32-34, 37-45, 48, 50, 52, 54, 56, 86, and 102 under 35 U.S.C. § 112, first paragraph (written description); and claims 34 and 41 under 35 U.S.C. § 112, second paragraph.

Rejection of Claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-43, 45, 48, 50, 52, 54, 56, 86, 102-112, and 114-116 Under 35 U.S.C. § 103(a)

The Examiner has rejected claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-43, 45, 48, 50, 52, 54, 56, 86, 102-112, and 114-116 under 35 U.S.C. § 103(a) for being obvious in view of the combined teachings of Konstantinov et al. (U.S. 20060149042) and Matheus et al. (U.S. 20070172475). The Examiner suggests that given the aggregation challenges described in Matheus, one of ordinary skill would be motivated to "solve the solubility/aggregation problem discussed in Matheus" using the methods of Konstantinov. Applicants respectfully traverse the rejection and maintain that the teachings of Konstantinov and Matheus, alone or in combination, fail to render claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-43, 45, 48, 50, 52, 54, 56, 86, 102-112, and 114-116 obvious.

To establish a prima facie case of obviousness, three basic criteria must be met. First, there must be some suggestion or motivation, either in the references themselves or in the

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knowledge generally available in the art, to modify the reference or to combine reference teachings. Second, there must be a reasonable expectation of success. Finally, the prior art reference must teach or suggest all the claim limitations. The teaching or suggestion to make the claimed combination and the reasonable expectation of success must be found in the prior art and not based on applicant's disclosure. In re Vaeck, 947 F.2d 488 (Fed. Cir. 1991). MPEP 706.02U). The test for prima facie obviousness is consistent with the legal principles enunciated in KSR Int'l Co. v. Teleflex Inc., 127 S. Ct. 1727 (2007). Takeda Chem. Indus., Ltd. v. Alphapharm Pty., Ltd., 492 F.3d 1350, 1356 (Fed. Cir. 2007). "While the KSR Court rejected a rigid application of the teaching, suggestion, or motivation ("TSM") test, the Court acknowledged the importance of identifying 'a reason that would have prompted a person of ordinary skill in the relevant field to combine the elements in the way the claimed new invention does' in an obviousness determination." Id. (quoting KSR, 127 S. Ct. at 1731). Although the TSM test should not be applied in a rigid manner, it can provide helpful insight to an obviousness inquiry. KSR, 127 S. Ct. at 1731. The KSR Court upheld the secondary considerations of non-obviousness, noting that there is "no necessary inconsistency between the idea underlying the TSM test and the Graham analysis." Id. Although the prior art reference, or references when combined, need not teach or suggest all of the claim limitations, a reason must be given why the differences between the prior art and the claimed limitation would have been obvious to one of skill in the art (see Examination Guidelines for Determining Obviousness Under 35 U.S.C. 103, Federal Register, Vol. 72, No. 195).

Sub;ectmatterofclaims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-43, 45, 48, 50, 52, 54, 56, 86, 102-112, and 114-116 Independent claims 1, 21, 27, 37, and 103 each describe an aqueous formulation comprising at least about 20 mg/mL of an antibody, or an antigen-binding fragment thereof, and water, wherein the antibody or antigen binding fragment thereof, has a molecular weight greater than about 47 kDa, and wherein the formulation is defined as having a conductivity of less than about 2.5 mS/cm, an osmolality of no more than about 30 mOsmol/kg, a hydrodynamic diameter (Dh) which is at least

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about 50% less than the Dh of the antibody in a buffered solution at the same concentration, or a Dh of less than about 4 nm. Independent claim 11 has been canceled. In the Office Action, the Examiner notes that the claims specify a formulation "comprising" an antibody, or antigen-binding fragment thereof, and water. Applicants note that the claims also require specific characteristics relating to conductivity, osmolality, or Dh,

Prior to Applicants' invention, the ordinary skilled artisan could not have predicted the claimed formulations with any reasonable expectation of success given the teachings of Konstantinov and Matheus. Matheus teaches that "aggregation of proteins is described in the literature as the commonest physical instability reaction" associated with highly concentrated antibody formulations (see para. [0007]). The Examiner suggests that one of ordinary skill would solve this problem using the methods of Konstantinov to arrive at the claimed invention. Applicants submit that one of ordinary skill would have had no reasonable expectation of success in arriving at the claimed invention based on the teachings of Konstantinov and Matheus, and, furthermore, would not be motivated to combine the teachings.

State of art regarding high concentration protein formulations Applicants respectfully submit that one of ordinary skill in the art would not look to Konstantinov to solve the aggregation and stability problems described in Matheus. Matheus explicitly teaches that "the preparation of liquid highly concentrated antibody formulations which are stable for a sufficiently long time is proving to be extremely difficult for the person skilled in the art" and, moreover, that "the preparation of a highly concentrated liquid formulation was unattractive to the person skilled in the art since the greatly pronounced aggregation tendency of proteins and in particular of antibodies, even in low concentrations was sufficiently known" (see, e.g., paragraph [007] of Matheus). Matheus teaches that protein aggregation at high concentrations is a problem known in the art, and, furthermore, that formulations known in the art do not solve the problems of stable antibody formulations. Specifically, Matheus teaches that "due to the difficulties to be expected, already established protein formulations generally cannot be applied to new protein 28

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active ingredients." (see para. [0011]).

The formulation challenges described in Matheus were well known in the art at the time of filing of the instant application. In support of this assertion, Applicants provide herewith a scientific review entitled "Antibody Structure, Instability, and Formulation" (Wang et al. (2007) J of Pharma Sci 96(1): 1) (hereinafter the "Wang review"). The Wang review provides a detailed description of commercial therapeutic antibodies and their formulations, and describes challenges in formulation development, including difficulties with highly concentrated antibody formulations. For example, at page 8, 2nd col., last paragraph of the Wang review, the authors state that "[a]ntibody aggregation is a more common manifestation of physical instability. The concentration­ dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations." The challenge of formulating antibodies at high concentrations in aqueous formulations is further evidenced by Table 1 of the Wang review. Table 1 provides a list of twenty-three commercial therapeutic antibody formulations, including the formulation ingredients for each. As described in Table 1, of the fifteen aqueous formulations, only three had concentrations of 20 mg/mL or greater (see Avastin 25 mg/mL, Humira 50 mg/mL, and Tysabri 20 mg/mL). Thus, the Wang review supports the assertion that aqueous formulations having high concentrations of antibodies were known in the art to be challenging, and reiterates many of the difficulties described in Matheus.

Given the knowledge in the art with respect to the challenges of antibody formulations, evidenced by Matheus and the Wang reference, one of ordinary skill in the art would not look to the teachings of Konstantinov to solve the aggregation problems identified in Matheus as being a challenge to achieve high concentration formulations. One of ordinary skill would not have an expectation of success or be motivated to combine the teachings of Matheus and Konstantinov, because 1) Konstantinov does not teach formulations having high concentrations of proteins and 2) Konstantinov does not provide the requisite expectation of success with respect to predictably using the methods described therein for proteins greater than 18kDa.

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Konstantinov does not teach formulations having high concentrations of proteins

The Examiner maintains that Konstantinov teaches high concentrations of proteins because Konstantinov states the methods may be used to concentrate proteins up to 100-fold. Applicants respectfully note, however, that a 100-fold increase is relative to the starting concentration. Applicants further submit that Konstantinov never demonstrates actual concentrations within the range of the claimed invention, e.g., at least about 20 mg/mL. Given the challenges identified in the art with respect to high concentrations, this omission is significant.

In Applicants' previous response, it was noted that the highest concentration Konstantinov actually provides is 12 mg/mL (see Figure 6), which notably is specific to total protein obtained from a cell culture preparation. With respect to IL2SA, the exemplary protein described in Konstantinov, Figure 7 teaches that when the protein is concentrated 25 fold it results in!! concentration of 800 mg/L. 800 mg/L concentration is considerably lower than the claimed concentration of at least about 20 mg/mL. Thus, while Konstantinov teaches that that methods disclosed therein may be used to concentrate a protein by as much as 100 fold (see, e.g., paragraph [0014] of Konstantinov), Figures 6 and 7 of Konstantinov demonstrate that the actual resulting protein concentration is low.

Given the challenges known in the art with respect to high concentrations, coupled with the low concentrations exemplified in Konstantinov, especially for IL2SA, one of ordinary skill would not have an expectation of success that the methods disclosed in Konstantinov would be effective for achieving high concentrations of antibodies, including 20 mg/ml or greater as described in the pending claims. Furthermore, one of ordinary skill would not be motivated to combine the two references as Konstantinov does not demonstrate high protein concentrations and Matheus cautions regarding the challenges associated with high concentration formulations.

Konstantinov does not provide the requisite expectation of success with respect to proteins greater

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than 18kDa

The Examiner also suggests that one of ordinary skill would have an expectation of success with respect to using the methods of Konstantinov for antibodies because Konstantinov teaches antibodies, albeit not in the working examples. Applicants respectfully disagree.

Konstantinov provides working examples based on the protein IL2SA. As described in Applicants' previous response, IL2SA is a protein which is less than 18kDa in size. Applicants respectfully disagree with the Examiner's suggestion that one of ordinary skill would look to Konstantinov for concentrating proteins larger than 18 kDa, such as antibodies, given the teachings of Example 5. Example 5 teaches that by "reducing conductivity .... proteins appear to precipitate only at high Pluronic F-68 concentration." (see para. [0075]). Results from the experiments described in Example 5 are described in Figure 11, which provides a graph that compares the yield (protein solubility; see [0075] of Konstantinov) of IL2SA and total protein in original cell culture conductivity conditions and low conductivity conditions versus increased Pluronic concentration (increased Pluronic concentration resulting in precipitating proteins). As described in Figure 11, IL2SA in a "low conductivity" solution had a yield of about 100% (or more) at the highest concentration of Pluronic. In contrast, however, only about 50% of the total protein at the highest Pluronic concentration remained soluble. Thus, Figure 11 suggests that a low conductivity solution improves solubility of IL2SA, which has a molecular weight of 18 kDa, but shows far less improvement for the remaining proteins in the cell culture supernatant. Given these findings, one of ordinary skill would not have an expectation of success that the methods of Konstantinov would be effective for proteins other than those of similar size to IL2SA (18 kDa), let alone for protein concentrations greater than 800 mg/L (Figure 7). Thus, one of ordinary skill could not predictably use the methods disclosed in Konstantinov to arrive at the claimed formulations comprising antibodies, or antigen binding portions thereof, having a molecular weight of greater than about 47 kDa.

In sum, Applicants submit that one of ordinary skill would not have a reasonable expectation 31

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of success that the aggregation problems described in Matheus could be solved by the methods disclosed in Konstantinov, as Konstantinov does not teach or suggest high antibody concentrations and does not provide evidence that the methods described therein are effective for proteins other than IL2SA. Furthermore, given the challenges known in the art with respect to aqueous formulations having high, e.g., 20 mg/ml or greater, antibody concentrations, one of ordinary skill would also not be motivated to combined the teachings of the cited references. Accordingly, Applicants respectfully request that the foregoing rejection of claims 1-4, 6, 7, 9-11, 15-18, 20-22, 24, 26-30, 32-34, 37-43, 45, 48, 50, 52, 54, 56, 86, 102-112, and 114-116 be reconsidered and withdrawn.

Reiection of Claim 44 Under 35 U.S.C. § 103(a)

The Examiner has rejected claim 44 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov, et al. (U.S. Patent Application No. 2006/0149042) in view of Matheus, et al. (U.S. Patent Application No. U.S. 2007 /0172475), and further in view of Schusching (U.S. Patent Application No. U.S. 2007/0202051).

Applicants respectfully traverse this rejection and submit that the teachings of Konstantinov. Matheus, and Schusching, alone or in combination, fail to render the claimed formulations obvious Claim 44, which depends from claim 1, is directed to formulations comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm (claim 1, and claims dependent therefrom), further comprising a surfactant. As discussed in detail above, one of ordinary skill in the art would have no motivation to apply the teachings of Konstantinov to molecules larger than about 18kDa at a concentration of at least about 20 mg/mL. Matheus teaches that protein aggregation at high concentrations of antibodies is a problem known in the art. Thus, one of ordinary skill would not look to a reference that provides low concentrations of small proteins (i.e., 18 kDa) to arrive at the claimed invention, directed to proteins having a molecular weight greater than 47 kDa at a concentration of at least about 20 mg/mL. While Konstantinov mentions antibodies (see, e.g., paragraph 0041]), there is no

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evidence that the methods described therein would work for proteins over 18 kDa (see above comments with respect to the teachings of Figure 11) or that the methods described therein would work for protein concentrations over 800 mg/L (see above comments with respect to the teachings of Figure 7). Thus, one of ordinary skill would have no expectation of success that the methods described in Konstantinov would work for larger proteins, such as antibodies, described in Matheus. Based on the foregoing, it is clear that Konstantov et al. and Matheus, fail to teach or suggest the aqueous formulation of claim 44. The teachings of Schusching do not make up for the deficiencies of Konstantinov and Matheus. In particular, Schusching merely teaches aerosol formulations and that a surfactant may be added to the formulations. There is no teaching or suggestion in Schusching that the authors contemplated formulations comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm and wherein the antibody, or antigen-binding fragment thereof, has a molecular weight greater than about 47 kDa, further comprising a surfactant. Accordingly, Applicants respectfully request that the foregoing rejection of claim 44 be reconsidered and withdrawn.

Reiection of Claims 117-122, 125-136, 139, 144-150, 153, 159, 160, 180, and 181

Under 35 U.S.C. § 103(a)

The Examiner has rejected claims 117-122, 125-136, 139, 144-150, 153, 159, 160, 180, and 181 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov et al. (US 2006/0149042) in view of Matheus et al. (US U.S. 2007 /0172475), and further in view of Salfeld et al. (US 6,090,382). In particular, the Examiner is of the opinion that one of ordinary skill in the art would be motivated to use the methods of Konstantinov and Matheus to concentrate a TNFa antibody as taught by Salfeld. Applicants traverse the rejection.

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As discussed above, one of ordinary skill would not be motivated to combine Konstantinov and Matheus given the challenges known in the art known for formulating highly concentrated (e.g., 20 mg/mL) aqueous antibody formulations. Furthermore, knowing the challenges, one of ordinary skill would not have an expectation of success that the methods of Konstantinov (shown to be successful for an 18 kDa protein at a low concentration) could be used to arrive at the claimed aqueous formulations.

The teachings of Salfeld do not make up for the deficiencies of Konstantinov and Matheus. In particular, Salfeld teaches fully human antibodies and methods of use of such antibodies. Although, Salfeld provides general teachings regarding the formulation of such antibodies, Salfeld fails to teach or suggest formulations of such antibodies comprising an antibody, or antigen-binding fragment thereof, at a concentration of at least about 20 mg/mL, wherein the formulation has a conductivity of less than about 2.5 mS/cm (claim 117, and claims dependent therefrom) or wherein the formulation has a hydrodynamic diameter (Dh) which is at least about 50% less than the Dh of the antibody, or antigen-binding fragment thereof, in a buffered solution at the same concentration (claim 128, and claims dependent therefrom), or wherein the formulation has a hydrodynamic diameter (Dh) of less than about 4nm (claim 144, and claims dependent therefrom). Accordingly, Applicants respectfully request that the foregoing rejection of claims 117-122, 125-136, 139, 144-150, 153, 159, 160, 180, and 181 be reconsidered and withdrawn.

Reiection of Claims 123, 124, 137, 138, 140-143, 151, 152, 154-157, 161-157, 161-178, 180, and 181 Under 35 U.S.C. § 103(a)

The Examiner has rejected claims 123, 124, 137, 138, 140-143, 151, 152, 154-157, 161-157, 161-178, 180, and 181 under 35 U.S.C. § 103(a) as being unpatentable over Konstantinov et al. (US 2006/0149042) in view of Matheus et al. (US 2007 /0172475), and further in view of Salfeld (US 6,090,382), and further in view of Schusching (US 2007/0202051). Applicants respectfully traverse the rejection.

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For the reasons set forth above, Applicants submit that one of ordinary skill would have no expectation of success or be motivated to combine the teachings of Konstantinov and Matheus to arriveattheinventionofclaims 123,124,137,138, 140-143, 151,152, 154-157, 161-157, 161-178, 180, and 181. Secondary references Salfeld and Schusching fail to make up for the deficient teachings of Konstantinov and Matheus.

Claims 123, 124, 137, 138, 140-143, 151, 152, 154-157, 161-157, 161-178, 180, and 181 are directed to aqueous formulations comprising a human anti-TNFa antibody, or antigen-binding fragment thereof, having specific CDR sequences corresponding to the antibody adalimumab, and certain excipients, wherein the formulation has a conductivity of less than about 2.5 mS/cm or the antibody has a certain hydrodynamic diameter. Applicants respectfully submit that there is no teaching or suggestion in Konstantinov and Matheus, either alone or in combination, that would lead one of ordinary skill in the art to arrive at the claimed formulations. Indeed, Konstantinov is silent with respect to excipients (as it describes organic polymers associated with cell culture processes). Matheus teaches formulations specific to anti-EGFR antibodies and does not teach or suggest the specific combinations of antibodies and excipients described in claims 123, 124, 137, 138, 140-143, 151, 152, 154-157, 161-157, 161-178, 180, and 181, let alone formulations having low conductivity or small antibody hydrodynamic diameters.

Accordingly, Applicants respectfully request that the foregoing rejection of claims 123, 124, 137, 138, 140-143, 151, 152, 154-157, 161-157, 161-178, 180, and 181 be reconsidered and withdrawn.

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SUMMARY

If a telephone conversation with Applicant's Attorney would expedite the prosecution of the above-identified application, the Examiner is urged to call the undersigned at (617) 449-6500.

The Commissioner is hereby authorized to charge any fees associated with the filing of this communication or any subsequent filing to our Deposit Account No. 50-4876, under Order No. 117813-26902 from which the undersigned is authorized to draw.

Dated: May 21, 2012 Respectfully submitted,

Electronic signature: / Cristin Cowles /

Cristin H. Cowles, Ph.D. Registration No.: 55,281 McCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6500 (617) 607-9200 Attorney For Applicants

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IPR Page 1981 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

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Docket Number (Optional) PETITION FOR EXTENSION OF TIME UNDER 37 CFR 1.136(a) 117813-26902

Application Number 12/325,049 Filed November 28, 2008

For PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

Art Unit 1646 Examiner Hissong, Bruce D.

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IPR Page 1987 of 2482 PTO/SB/08a (07-09) Approved for use through 07/31/2012. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE u n d er t he p aoerwor k Re d uct1on Act o 1995, no oersons are reau1re d to resoon d to a co II ect1on o f.in f ormat1on un Iess 1t contains a vaI'd 1 0MB contro I num b er. Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1646 (Use as many sheets as necessary) Examiner Name B. D. Hissong

Sheet I 1 I 01 I 3 Attorney Docket Number 117813-26902

U.S. PATENT DOCUMENTS Document Number Pages, Columns, Lines, Where Publication Date Examiner Cite Name of Patentee or Relevant Passages or Relevant 1 Number-Kind Code2 ( if known) MM-DD-YYYY Applicant of Cited Document Initials' No. Figures Appear A1 US-4,597,966 07-01-1986 Zolton et al. A2 US-6,485,932 11-26-2002 McIntosh et al. A3 US-7,070,775 07-04-2006 Le et al. A4 US-7,250, 1 65 07-31-2007 Heavner et al. A5 US-7,276,239 10-02-2007 Le et al. A6 US-7,758,860 07-20-2010 Warne et al. A7 US-2006-0159653 07-20-2006 Saito et al. A8 US-2003-0180287 09-25-2003 Gombotz et al. A9 US-2003-0190316 10-09-2003 Kakuta et al. A10 US-2005-0118163 06-02-2005 Mizushima et al. A11 US-2006-01827 40 08-17-2006 Yanq et al. A12 US-2006-0246073 11-02-2006 Kniqht et al. A13 US-2007-0020255 01-25-2007 Ueno et al. A14 US-2007-0036779 02-15-2007 Bardet et al. A15 US-2007-0053906 03-08-2007 Samaritani et al. A16 US-2007-0184050 08-09-2007 Ishikawa et al. A17 US-2007-0190047 08-16-2007 Brych et al A18 US-201 0-0129379 05-27-2010 Carpenter et al. A19 US-2011-0171227 07-14-2011 Okun et al. A20 US-2011-0300151 12-08-2011 OKUN et al. A21 US-2012-0014956 01-19-2012 Kupper et al.

IExaminer I IDate Signature Considered

*EXAM INER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if possible. 6 Applicant is to place a check mark here if English language Translation is attached.

ME1 13460023v.1

IPR Page 1988 of 2482 PTO/SB/08a (07-09) Approved for use through 07/31/2012. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE u n d er t he p aoerwor k Re d uct1on Act o 1995, no oersons are reau1re d to resoon d to a co II ect1on o f.in f ormat1on un Iess 1t contains a vaI'd 1 0MB contro I num b er. Complete if Known Substitute for form 1449/PTO Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1646 (Use as many sheets as necessary) Examiner Name B. D. Hissong

Sheet I 2 I 01 I 3 Attorney Docket Number 117813-26902

FOREIGN PATENT DOCUMENTS

Foreiqn Patent Document Publication Pages, Columns, Lines, Name of Patentee or Examiner Cite Date Where Relevant Passages 1 T6 Initials' No. Country Code3 -Number4-Kind Code5 (if known) MM-DD-YYYY Applicant of Cited Document Or Relevant Figures Appear B1 W0-1989/ 11298-A 1 11-30-1989 Centocor Inc D B2 W0-1993/08837-A1 05-13-1993 Wellcome Found D B3 W0-1999/37329-A 1 07-29-1999 Astra Ab et al. D B4 EP-0486526-A 1 05-27-1992 Peptide Technoloqy Ltd D B5 EP-1174148-A1 01-23-2002 Yamanouchi Pharma Co Ltd D B6 W0-2002012502-A2 02-14-2002 Centocor Inc D Suomen Punainen Risti B7 W02005072772 08-11-2005 Veripalvelu D Arizona Board of Regents for B8 W02009015367 01-29-2009 and on behalf of Arizona State University D IExaminer I IDate Signature Considered

*EXAM INER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant. • CITE NO .. Those application(s) which are marked with an single asterisk(') next to the Cite No. are not supplied (under 37 CFR 1.98(a)(2)(iii)) because that application was filed after June 30, 2003 or is available in the IFW. 1 Applicant's unique citation designation number (optional). 2 See Kinds Codes of USPTO Patent Documents at www.uspto.gov or MPEP 901.04. 3 Enter Office that issued the document, by the two-letter code (WIPO Standard ST.3). 4 For Japanese patent documents, the indication of the year of the reign of the Emperor must precede the serial number of the patent document. 5 Kind of document by the appropriate symbols as indicated on the document under WIPO Standard ST.16 if 6 possible. Applicant is to place a check mark here if English language Translation is attached.

ME1 13460023v.1

IPR Page 1989 of 2482 PTO/SB/08b (07-09) Approved for use through 07/31/2012. 0MB 0651-0031 U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE Under the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it contains a valid 0MB control number.

Substitute for form 1449/PTO Complete if Known Application Number 12/325,049 INFORMATION DISCLOSURE Filing Date November 28, 2008 STATEMENT BY APPLICANT First Named Inventor Wolfgang Fraunhofer Art Unit 1646 (Use as many sheets as necessary) Examiner Name B. D. Hissong

Sheet I 3 I 01 I 3 Attorney Docket Number 117813-26902

NON PATENT LITERATURE DOCUMENTS Include name of the author (in CAPITAL LETTERS), title of the article (when appropriate), title of Examiner Cite the item (book, magazine, journal, serial, symposium, catalog, etc.), date, page(s), volume-issue T' No. 1 Initials number(s), publisher, city and/or country where published. Akers, et al., "Development and Manufacture of Protein Pharmaceuticals (Pharmaceutical C1 Biotechnology)", Chapter 2: "Formulation Development of Protein Dosage Forms", 2002, -.J Kluver Academic/Plenum, pub., New York. Barrera, et al., "Effects of treatment with a fully human antitumour necrosis factor alpha C2 monoclonal antibody on the local and systemic homeostasis of interleukin 1 and TNFalpha in patients with rheumatoid arthritis," Ann Rheum. Dis. 2001, 60(7):660-669 Harris, et al., "Commercial manufacturing scale formulation and analytical characterization of C3 therapeutic recombinant antibodies", Drug Development Research, 2004, vol. 61, no. 3, pg 137-154 Hillgren, et al., "Protection mechanism of Tween 80 during freeze-thawing of a model protein," C4 International Journal of Pharmaceutics, 2002, Vol 237: 57-69 Holt, et al., "Domain antibodies: proteins for therapy," Trends in Biotechnology, 2003, Vol. C5 21 ( 11) :484-490 Wang, et al., "Antibody Structure, Instability, and Formulation," J Pharmaceutical Sci, 2007, C6 96(1): 1-26 Wang, et al., "Instability, Stablization, and Formulation of Liquid Protein Pharmaceuticals," Int. C7 J. Pharmaceutics, 1999, 185:129-188 Adalimumab entry from National Library of Medicine website: www. nlm.nih.gov/cgi/mesh; C8 printed on 28 September 2009 C9 International Preliminary Examination Report for Application No. PCT/US2008/085066, 2009 C10 International Search Report for APPiication No.PCT/US2008/085066, 2009

!Examiner I IDate Signature Considered

*EXAMINER: Initial if reference considered, whether or not citation is in conformance with MPEP 609. Draw line through citation if not in conformance and not considered. Include copy of this form with next communication to applicant.

'Applicant's unique citation designation number (optional). 2Applicant is to place a check mark here if English language Translation is attached.

ME113460023v.1

IPR Page 1990 of 2482 I hereby certify that this paper (along with any paper referred to as being attached or enclosed) is being transmitted via the Office electronic filing system in accordance with 37 CFR § 1.6(a)(4).

Dated: May 22, 2012 Electronic Signature for Cristin H. Cowles, Ph.D.: /Cristin H. Cowles, Ph.D./ Docket No.: 117813-26902 (PATENT)

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE

In re Utility Application of: Wolfgang Fraunhofer et al.

Application No.: 12/325,049 Confirmation No.: 1766

Filed: November 28, 2008 Art Unit: 1646

For: PROTEIN FORMULATIONS AND Examiner: B. D. Hissong METHODS OF MAKING SAME

SUPPLEMENTAL INFORMATION DISCLOSURE STATEMENT (IDS)

MS Amendment Commissioner for Patents P.O. Box 1450 Alexandria, VA 22313-1450

Dear Madam:

Pursuant to 37 CPR 1.56, 1.97 and 1.98, the attention of the Patent and Trademark Office is hereby directed to the references listed on the attached PTO/SB/08. It is respectfully requested that the information be expressly considered during the prosecution of this application, and that the references be made of record therein and appear among the "References Cited" on any patent to issue therefrom.

This Supplemental Information Disclosure Statement (SIDS) is filed after the mailing date of the first Office Action on the merits, but before the mailing date of any of a Final Office Action, a Notice of Allowance (37 CPR 1.97(c)) or an action that otherwise closes prosecution in the application, and is accompanied by the fee set forth in § 1.17 (p ).

In addition to those commonly owned related applications and issued patents previously cited in the Information Disclosure Statement dated October 19, 2010 and June 2, 2011, Applicants

MEI 13364377v.1

IPR Page 1991 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

wish to bring to the attention of the Examiner the following commonly owned related applications and issued patents:

Patent No. or Publication No. Publication Date MM-DD-YYYY US 2011-0171227 Al 07-14-2011 USSN 13/019,810 Not Published US 2012-0014956 Al 01-19-2012 USSN 13/034,809 Not Published US 2011-0300151 Al 12-08-2011 USNN 13/280,613 Not Published

The publications referenced above are cited as references Al9-A21 in the enclosed Form SB/08. Applicants understand that the Examiner can access papers from the prosecution of the cited cases electronically. However, if the Examiner has difficulty obtaining papers from that source he is invited to call the undersigned who will be happy to supply them.

In accordance with 37 CPR 1.98(a)(2)(ii), Applicants have not submitted copies of U.S. patents and U.S. patent applications. Applicants submit herewith copies of foreign patents and non­ patent literature in accordance with 37 CPR 1.98(a)(2).

In accordance with 37 CPR 1.97(g), the filing of this Information Disclosure Statement shall not be construed to mean that a search has been made or that no other material information as defined in 37 CPR 1.56(a) exists. In accordance with 37 CPR 1.97(h), the filing of this Information Disclosure Statement shall not be construed to be an admission that any patent, publication or other information referred to therein is "prior art" for this invention unless specifically designated as such.

It is submitted that the Information Disclosure Statement is in compliance with 37 CPR 1.98 and the Examiner is respectfully requested to consider the listed references.

2 MEI 13364377v.1

IPR Page 1992 of 2482 Application No.: 12/325,049 Docket No.: 117813-26902

The Director is hereby authorized to charge any deficiency in the fees filed, asserted to be filed or which should have been filed herewith (or with any paper hereafter filed in this application by this firm) to our Deposit Account No. 50-4876, under Order No. 117813-26902.

Dated: May 22, 2012 Respectfully submitted,

Electronic signature: /Cristin H. Cowles, Ph.D./ Cristin H. Cowles, Ph.D. Registration No.: 55,281 MCCARTER & ENGLISH, LLP 265 Franklin Street Boston, Massachusetts 02110 (617) 449-6550 (617) 607-9200 (Fax) Attorney For Applicants

3 MEI 13364377v.1

IPR Page 1993 of 2482 Electronic Patent Application Fee Transmittal

Application Number: 12325049

Filing Date: 28-Nov-2008

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

Filer: Cristin E. Howley

Attorney Docket Number: 117813-26902

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Application Number: 12325049

International Application Number:

Confirmation Number: 1766

Title of Invention: PROTEIN FORMULATIONS AND METHODS OF MAKING SAME

First Named Inventor/Applicant Name: Wolfgang FRAUNHOFER

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Filer: Cristin E. Howley

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This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents, characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt similar to a Post Card, as described in MPEP 503.

New A~~lications Under 35 U.S.C. 111 If a new application is being filed and the application includes the necessary components for a filing date (see 37 CFR 1.53(b)-(d) and MPEP 506), a Filing Receipt (37 CFR 1.54) will be issued in due course and the date shown on this Acknowledgement Receipt will establish the filing date of the application.

National Stage of an International A~~lication under 35 U.S.C. 371 If a timely submission to enter the national stage of an international application is compliant with the conditions of 35 U.S.C. 371 and other applicable requirements a Form PCT/DO/E0/903 indicating acceptance of the application as a national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt, in due course.

New International A~~lication Filed with the USPTO as a Receiving Office If a new international application is being filed and the international application includes the necessary components for an international filing date (see PCT Article 11 and MPEP 181 O), a Notification of the International Application Number and of the International Filing Date (Form PCT/R0/1 OS) will be issued in due course, subject to prescriptions concerning national security, and the date shown on this Acknowledgement Receipt will establish the international filing date of the application.

IPR Page 1999 of 2482 I WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)

(51) International Patent Oassiflcation 4 : (11) International Publication Number: WO 89/11298 Al A61K 39/395, 47/00 ( 43) International Publication Date: 30 November 1989 (30.11.89)

(21) International Application Number: PCT/US89/02289 (81) Designated States: AT (European patent), BE (European patent), CH (European patent), DE (European patent), (22) International Filing Date: 25 May 1989 (25.05.89) FR (European patent), GB (European patent), IT (Euro­ .. pean patent), JP, LU (European patent), NL (European patent), SE (European patent). (30) Priority data: 199,936 27 May 1988 (27.05.88) us Published With international search report. (71) Applicant: CENTOCOR, INC. [US/US]; 244 Great Valley Before the expiration of the time limit for amending the Parkway, Malvern, PA 19355 (US). claims and to be republished in the event of the receipt of amendments. (72)Inventors: SHEALEY, David, J. ; 1322 Broadview West, Downingtown, PA 19335 (US). PHILLIPS, Christopher, P. ; P.O. Box 65, Brandamore, PA 19316 (US).

(74)Agents: DECONTI, Giulio, A., Jr. et al.; Hamilton, Brook, Smith & Reynolds, Two Militia Drive, Lexington, MA 02173 (US).

(54) Title: FORMULATION FOR ANTIBODY REAGENTS

(57) Abstract

A composition suitable for intraveneous injection is disclosed, which comprises an aqueous stabilizing buffer solution con­ taining antibody or antibody fragments and maltose. The composition is effective in stabilizing the antibody or fragments in solu­ tion, inhibiting precipitation and the formation of particulates in the final product vial, while maintaining a high level of immun­ oreactivity.

•

IPR Page 2000 of 2482 :

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AT Austria FI Fmland ML Mali AU Australia FR France MR Mauritania BB Barbadas GA. Gabon MW Malawi BE Belgium GB United Kingdom NL Netherlands BF Burkina Fasso HU Hungary NO Norway ' BG Bulgaria IT Italy RO Romani~ BJ Benin JP Japan SD Sudan - BR Brazil KP Democratic People's RepubfIC SE Sweden CF Cenual African Republic of Korea S'oi Senegal CG Congo KR Republic of Korea gJ Soviet Union CH Switzccland u Liechtenstein TD Chad CM Cameroon LK Sri Lanka TG Togo DE Germany. Federal Republic of w Luxembourg l5 United States of America DK Denmark MC Monaco fS Spain MG Madagascar

IPR Page 2001 of 2482 W089/11298 PCT/US89/02289

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FORMULATION FOR ANTIBODY REAGENTS

Background of the Invention This invention relates generally to antibody preparations and particularly to a highly stabilized 05 antibody preparation for parenteral administration. It is well known that many protein preparations intended for administration to humans require stabilizers to prevent denaturation of the proteins, agglomeration and other alterations to the proteins 10 prior to the use of the preparation. Many preparations are particularly unstable in dilute solutions. This instability is manifested in the formation of insoluble particles, and is often increased when the protein preparation is stored, or 15 shipped. This phenomenon, known as "shedding", is often increased when the protein preparation is stored at room temperature or higher, so that the preparation must often be refrigerated. "Shedding" generally refers to a visible precipitation of 20 protein molecules. Various methods for stabilizing protein preparations have been used with varying degrees of success. For example, increasing the concentration of the protein or adding another protein such as 25 human serum albumin (HSA) has been known to enhance stability in some cases. However, such preparations may not always be acceptable for therapeutic purposes. In considering an appropriate stabilizer, such factors as lack as antigenicity, the biological

IPR Page 2002 of 2482 W089/11298 PCT/US89/02289

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activity of the specific proteins being stabilized and the availability and cost of the stabilizer are important. Various carbohydrates have been used to 05 stabilize and/or enhance solubility of certain biologically active protein preparations. For example, U.S. Patent 4,186,192, to Lundblad et al. disc:loses the use of maltose to increase the stability of an immune serum globulin preparation 10 for intramuscular or intravenous administration. In U.S. Patent 2,826,533, Fewell discloses the use of dextrose to increase the solubility of a fibrinogen preparation. In U.S. Patent 4,089,944 Thomas discloses the use of a variety of carbohydrates, 15 such as dextrose, mannose, galactose, fructose, lactose, sucrose, and maltose to increase the solubility of an AHF-fibrinogen composition. It has been found that when dextrose is added to immune serum globulin to enhance stability and or 20 solubility, the globulins tend to aggregate over time, thereby increasing the optical density of the solution, and resulting in shedding. The exact nature of shedding is not fully understood. Shedding is an undesirable 25 manifestation since it is visually observable and indicates the possibility that the shedded protein may be inactive or denatured, therefore reducing the effective amount of protein available. A protein preparation in which shedding is apparent is an

IPR Page 2003 of 2482 W089/11298 PCT/US89/02289

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unsatisfactory product in terms of visual appearance. The formation of protein aggregates and , particulates has long been considered a problem in 05 the development of parenteral immunoglobulin products. The administration of immunoglobulin G, for example, was limited to the intramuscular route because of endogenous anticomplementary activity due to aggregated immunoglobulin until the recent 10 development of chemically and enzymatically treated immunoglobulin G. J.E. Pennington, Rev.Inf.Dis., ~:5371-5373 (1986). Recent modifications in immunoglobulin G formulations have also helped to alleviate this problem. J. P. Mccue et al., Rev •. 15 Inf.Dis., ~:5374-5381 (1986). However, most commercially available formulations now in use require filtration of the product prior to injection to remove these aggregates and particulates. ·The addition of maltose to protein solutions 20 for various purposes is known. Maltose is readily available in pure form and has good stability in aqueous solutions in concentrations up to 20% by weight. Preparations containing maltose can be autoclaved without browning of the solution. 25 Maltose in small quantities is practically physio­ logically inert. When administered parenterally, it is partially converted to glucose by the specific enzyme maltase found in many tissue sites in most animal species, including humans. The conversion to 30 glucose is gradual and frequently undetectable when

IPR Page 2004 of 2482 W089/11298 PCT/US89/02289

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plasma glucose is serially measured; therefore there is no apparent increase in circulating insulin levels. Since maltose is a disaccharide, a 10% solution is approximately isotonic in humans. In 05 U.S. Patent 4,499,073, Tenold discloses the use of a carbohydrate in a preparation of immune serum globulin to impart physiologically acceptable isotonicity to the preparation. Tenold specifies 10% weight to volume maltose for this purpose. 10 Lundblad et al., report in U.S. Patent 4,186,192 that a solution of immune serum globulin is stabilized when maltose is added in a concentration of between 5 and 18%- by weight. Fernandes et al. describe a preparation of intravenous gamma-globulin 15 stabilized with maltose to minimize precipitation and improve in vitro shelf stability. Vox Sang,39:101.-l.l.2(1.980).

Summary of the Invention The invention comprises an aqueous stabilizing 20 buffer containing an antibody or antibody fragments, and maltose. This buffer composition has the ability to inhibit the antibody or antibody fragments in solutions intended for intravenous administration from precipitating and forming 25 particulates in the final product vial. The antibody or antibody fragment may be derivatized with a chelating agent, such as, for example, diethylenetriamiepentaacetic acid (DTPA) for binding radiometals. The formulation of the buffer solution ,-

IPR Page 2005 of 2482 WO 89/11298 PCT/US89/02289

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contains phosphate, sodium chloride and maltose. The present composition has been successful in stabilizing monoclonal antibodies, or fragments thereof, for shipping and short-term storage at OS ambient temperatures without loss of immuno­ reactivity, and requires no refrigeration or other special handling. The invention provides a stable, liquid formulation for monoclonal antibody products without shedding, thus increasing the shelf life of 10 the antibody product.

Description of The Preferred Embodiments The aqueous stabilizing buffer of this invention minimizes the formation of protein aggregates and particulates in reagents containing 15 antibodies or antibody fragments, and insures that the antibody in solution maintains its immuno­ reactivity over time. The preparation comprises a sterile, pharmaceutically acceptable solution containing a phosphate buffer, sodium chloride, an 20 antimyosin monoclonal antibody or antibody fragment, and maltose. A preferred embodiment of this in­ vention comprises about lOmM to about lOOmM sodium phosphate (pH 6-8), about 145mM sodium chloride and about 5-20% (w/v) maltose and between about o.s-s.2 25 mg/ml antibody, preferably antimyosin. However, other antibodies or fragments, for example, antifibrin may be used. This buffer enhances the stability of immunological activity of the monoclonal antibody, and prevents the

IPR Page 2006 of 2482 W089/11298 PCT/US89/02289

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immunoglobulins in solution intended for intravenous administration to human subjects from precipitating and forming particulates in the final product vial. Another embodiment of the formulation contains pure 05 monoclonal antibody molecules, or fragments, that have been modified for diagnostic therapeutic applications; for example derivatized with a chelating agent such as diethylenetriamine­ pentaacetic acid (DTPA). The derivatized antibody 1 O can then be used as a radiopharmaceutical due to the chelator•s ability to bind a radioactive heavy metal, such as, fo.r example, Indium.-111. In a more preferred embodiment, the antibody solution includes a monoclonal antibody fragment, such as antimyosin, 15 derivatized with DTPA. The chelating agent is used for incorporating a radiometal, such as Indiun-111, into the antibody protein, forming a protein-chelate-radiometal complex. This complex is then administered to a subject to deliver the 20 radiometal to a site defined by the antigen which is the target of the antibody. The radiolabeled antibody can be used in scintigraphy, for example, in the imaging of tumors, or of disease sites, such as mycocardial infarct or blood clots. For example, 25 injection of labeled antimyosin antibody, which is specific for cardiac myosin, will result in localization of the radiometal at the site where antimyosin binds to myosin, and the site can then be scanned with a gamma camera to obtain an image of 30 the myocardium useful for diagnostic purposes.

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The present formulation exhibits superior stabilizing characteristics in terms of minimal protein particle formation, preservation of immunoreactivity and radiometal incorporation over 05 time, and under stress conditions, such as elevated temperatures, vial filling and shipping. Maltose, which is used to stabilize the antibody solution, is described in detail in, for example, the Merck Index, 10th edition, Merck and 10 Co., Inc. Rahway, NJ (1983). Maltose is· a disaccharide, (4-0-a-D-glucopyranosyl-D­ glucopyranose), which has been established as useful for maintaining pharmaceutically acceptable iso­ tonicity of immunoglobulin solutions. (See U.S. 15 Patent 4,499,073 to Tenold and U.S. Patent 4,186,192 to Lundblad et al., both discussed hereinabove). It has also been determined that maltose is not metabolized by humans when administered intra­ venously, and is excreted as maltose, with no 20 apparent elevation in blood glucose levels or release of insulin. Buffers have long been used to solubilize and stabilize antibody products for parenteral injec­ tion. They are utilized as biologically acceptable 25 carriers for proteins. Protein solubility in the buffer solution depends upon a number of factors, such as ionic strength of the solution and the isoelectric point of the protein. Buffers which have been used as antibody carriers include citrate,

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sodium chloride and phosphate. The preferred buffer for this formulation is sodium phosphate buffers. Sodium chloride is added to antibody composi­ tions to enhance stability and to render the solu- 05 tion physiologically acceptable upon injection. Other alkali metal salts, such as potassium chloride, are not physiologically acceptable when injected intraveneously. Stability studies have demonstrated that a 10 composition of-the invention has successfully maintained the following characteristics after 65 weeks: antibody solubility (determined via liquid borne particulate analysis), chelator activity (greater than 88% binding of Indium-1.11 at 10 15 minutes), antibody immunoactivity (when compared to reference standard material) and antibody molecular integrity {via high pressure liquid chromatography and SDS page electrophoresis comparisons to reference standard material.

20 The invention is further illustrated by the following examples:

Example 1.

Preparation of an Optimized Antimyosin Fab-DTPA Formulation 25 Formulations were tested with a variety of buffers, salt concentrations, pH levels, and excipients, such as, human serum albumin,

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surfactants, mandelic acid and N-acetyl tryptophanate. The test formulations were initially screened by visual inspection after incubation at 4°c, 22°c, 37°c and 45°C.

0 5 MATERIALS AND METHODS

Protein Samples

a. Antimyosin Fab-DTPA (Centocor, Inc., lot# 00745), 0.5 mg/ml in 100 mM sodium citrate, pH 5.0; manufactured 3/15/85. 10 Source of antibody: Ascites fluid.

b. Antimyosin Fab-DTPA (Centocor, Inc., lot# 03505), 5.2 mg/ml in 0.9% NaCl, manufactured 12/16/85. Source of antibody: Cell supernatant

15 Buffers Tested The following reagents were used to make the test buffers: Sodium citrate (Sigma Chemical Co., St. Louis, MO) Sodium chloride (J.T. Baker Co.) 20 Sodium phosphate Monobasic and Dibasic (Sigma Chemical Co.) Maltose (Sigma Chemical co.) Lactose (Sigma Chemical Co.) Tween 80 (Sigma Chemical Co.) 25 Dextrose (Sigma Chemical co.)

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Human Serum Albumin (25%) {Armour) Propylene Glycol (Fisher Scientific co.} Sodium acetate (Sigma Chemical Coo) Trishydroxymethylaminomethane (tris buffer) 05 (Sigma Chemical Co.) Hydroxyethyl piperazine ethane sulfonic acid (HEPES buffer) (Sigma Chemical Co.) N-acetyl tryptophanate (Sigma Chemical Co.) Mandelic acid (Sigma Chemical co.)

10 Test buffers: 1. 100 mM Na Citrate Buffer, pH 5 2. 100 mM Citrate Buffer, 0.05% Tween 20, pH 5 3. 100 mM Citrate Buffer, 0.01% Tween 20, pH 5 4. 100 mM Na Citrate Buffer, 0.2% Tween 20, pH 5 15 5. 100 mM Na Citrate Buffer, 5% Lactose, pH 5 6. 100 mM Na Citrate Buffer, 7.5% Lactose, pH 5 7. 100 mM Na Citrate Buffer, 10% Lactose, pH 5 8. 100 mM Na Citrate Buffer, 5% Dextrose, pH 5 9. 100 mM Na Citrate Buffer, 7.5% Dextrose, pH 5 20 10. 100 mM Na Citrate Buffer, 10% Dextrose, pH s 11. 100 mM Na Citrate Buffer, 5% Maltose, pH 5 12. 100 mM Na Citrate Buffer, 7.5% Maltose, pH 5 13. 100 mM Na Citrate Buffer, 10% Maltose, pH s 14. 100 mM Na Citrate Buffer, 0.5% Human Serum 25 Albumin (HSA), pH 5 15. 100 mM Na Citrate Buffer, 1% HSA, pH 5 16. 100 mM Na Citrate Buffer, 2% HSA, pH 5 17. 100 mM Na Citrate Buffer, pH 5e5 18. 100 mM Na Citrate Buffer, 5% Maltose, pH 5.5

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19. 100 mM Na Citrate Buffer, 7.5% Maltose, pH 5.5 20. 100 mM Na Citrate Buffer, 10% Maltose, pH 5.5 21. 100 mM Na Citrate Buffer, 0.5% HSA, pH 5.5 22. 100 mM Na Citrate Buffer, 1% HSA, pH 5.5 05 23. 100 mM Na Citrate Buffer; 2% HSA, pH 5.5 24. 100 mM Na citrate Buffer, pH 6 25. 100 mM Na Citrate Buffer, 5% Maltose, pH 6 26. 100 mM Na Citrate Buffer, 7.5% Maltose, pH 6 27. 100 mM Na Citrate Buffer, 10% Maltose, pH 6 10 28. 100 mM Na Citrate Buffer, 0.5% HSA, pH 6 29. 100 mM Na citrate Buffer, 1% HSA, pH 6 30. 100 mM Na Citrate Buffer, 2% HSA, pH 6 31. 100 mM Na Citrate Buffer, 100 mM Sodium Chloride, pH 5 15 32. 100 mM Na Citrate Buffer, 200 mM Sodium Chloride, pH 5 33. 100 mM Na Citrate Buffer, 0.025% Tween 20, pH 5 34. 100 mM Na Citrate Buffer, 0.01% Tween 20, pH 5 35. 100 mM Na Citrate Buffer, 0.05% Tween so, pH 5 20 36. 100 mM Na Citrate Buffer, 0.025% Tween 80, pH 5 37. 100 mM Na Citrate Buffer, 0.01% Tween 80, pH 5 38. 100 mM Na Citrate Buffer, 0.1% Propylene Glycol, pH 5 39. 100 mM Na Citrate Buffer, 0.05% Propylene 25 Glycol, pH 5 40. 100 mM Na Citrate Buffer, 0.01% Propylene Glycol, pH 5 41. 500 mM Na Acetate Buffer, pH 5 42. 10 mM Phosphate Buffer, pH 7.4 30 43. 25 mM Phosphate Buffer, pH 7.3

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44. 25 mM Tris Buffer, pH 7.3 45. 25 mM HEPES Buffer, pH 7 .. 3 46. 25 :mM Tris Buffer, 100 mM Sodium Chloride, pH 7 .. 3 OS 47. 25 mM HEPES Buffer, 100 mM Sodium Chloride, pH 7.3 48. 100 InM Phosphate Buffer, pH 7.4 49. 100 mM Phosphate Buffer, 100 mM Sodium Chloride, pH 7.4 10 50. 100 mM citrate Buffer, 20 mM N-Acetyl Tryptophanate, pH 5 51. 100 mM Na Citrate Buffer, 20 mM Mandelic Acid, pH 5 52. 200 mM Na Citrate Buffer, 20 mM N-Acetyl is Tryptophanate, 20 mM Mandelic Acid, pH 5 53. 100 mM Na Citrate Buffer, 100 mM Sodium Chloride, 20 mM N-Acetyl Tryptophanate, ·pH 5 54. 100 mM Na Citrate Buffer, 100 mM Sodium Chloride, 20 mM Mandelic Acid, pH 5 20 55. 100 mM Na Citrate Buffer; 100 mM Sodium Chloride, 20 mM Mandelic Acid, 20 mM N-Acetyl Tryptophanate, pH 5 56. 100 mM Na Citrate Buffer, 100 mM Sodium Chloride, 1% HSA, pH 5 25 57. 12.5 mM Phosphate Buffer, 100 mM Sodium Chloride, pH 7.2 58. 25 mM Phosphate Buffer, 100 mM Sodium Chloride, pH 7.2 59. 50 mM Phosphate Buffer, 100 mM Sodium Chloride, 30 pH 7.,2

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60. 100 mM Phosphate Buffer, 100 mM Sodium Chloride, pH 7.2 61. 12.5 mM Phosphate Buffer, pH 7.2 62. 25 mM Phosphate Buffer, pH 7.2 05 63. 50 mM Phosphate Buffer, pH 7~2 64. 100 mM Phosphate Buffer, pH 7.2 65. 12.5 mM Phosphate Buffer 0.1% Tween 80, pH 7.2 66. 25 mM Phosphate Buffer, 0.1% Tween so, pH 7.2 67. 50 mM Phosphate Buffer, 0.1% Tween so, pH 7.2 10 68. 100 mM Phosphate Buffer, 0.1% Tween 80, pH 7.2 69. 12.5 mM Phosphate Buffer, 100 mM Sodium Chloride, 0.1% Tween 80, pH 7.2 70. 25 mM Phosphate Buffer, 100 mM Sodium Chloride 0.1% Tween 80, pH 7.2 15 71. 50 mM Phosphate Buffer, 100 mM Sodium Chloride, 0.1% Tween so, pH 7.2 72. 100 mM Phosphate Buffer, 100 mM Sodium Chloride, 0.1% Tween 80, pH 7.2 73. 12.5 mM Phosphate Buffer, 0.1% Propylene 20 Glycol, pH 7.2 74. 24 mM Phosphate Buffer, 0.1% Propylene Glycol, pH 7.2 75. 50 mM Phosphate Buffer, 0.1% Propylene Glycol. pH 7.2 25 76. 100 mM Phosphate Buffer, 0.1% Propylene Glycol, pH 7.2 77. 12.5 mM Phosphate Buffer, 100 mM Sodium Chloride, 0.1% Propylene Glycol, pH 7.2 78. 25 mM Phosphate Buffer, 100 mM Sodium Chloride, lO 0.1% Propylene Glycol, pH 7.2

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79. 50 mM Phosphate Buffer, 100 mM Sodium Chloride, 0.1% Propylene Glycol, pH 7.2 80. 100 mM Phosphate Buffer, 100 mM Sodium Chloride, 0 .. 1% Propylene Glycol, pH 7.2 4 05 81. 100 mM Na Citrate Buffer, 1% HSA, 8x10- M Mandelic Acid, pH 5 82.- 100 mM Na Citrate Buffer, 100 mM Sodium Chloride, 1% HSA, 8x1o-4M Mandelic Acid, pH 5 4 &J •. 100 mM Na Citrate Buffer, 1% HSA, Bx10- M 10 N-Acetyl Tryptophanate, pH 5 84. 100 mM Na Citrate Buffer, 1% HSA, 100 mM Sodium -4 Chloride, axio M N-Acetyl Tryptophanate, pH 5 4 85. 100 mM Na Citrate Buffer, 1% HSA, Bx10- M Mandelic Acid, 8xl0-4M N-Acetyl Tryptophanate, 15 pH 5 86. 100 mM Na Citrate Buffer, 1% HSA, 100 mM Sodium . -4 . . -4 Chloride, BxlO M Mandelic Acid, 8x10 M N-Acetyl Tryptophanate, pH 5 87. 100 mM Na Acetate Buffer, 1% HSA, 100 mM Sodium 20 Chloride, 10% Maltose, pH 5 88. 100 mM Na Acetate Buffer, 100 mM Sodium Chloride, 10% Maltose, pH 5 89 .. 10 mM Phosphate Buffer, 145 mM Sodium Chloride, pH 7.2 25 90. l.O mM Phosphate Buffer, 145 mM Sodium Chloride, 10% Maltose, pH 7.2 91.. 10 mM Phosphate Buffer, 145 mM Sodium Chloride, 10% Maltose, pH 7.0 10 mM Phosphate Buffer, 145 mM Sodium Chloride, 30 l.0% Maltose, pH 6.75

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93. 10 mM Phosphate Buffer, 145 mM Sodium Chloride, 10% Maltose, pH 6.5 94. 10 mM Phosphate Buffer, 145 mM Sodium Chloride, 10% Maltose, pH 6.25 05 95. 10 mM Phosphate Buffer, 145 mM Sodium Chloride, 10% Maltose, pH 6.0

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Formulation Adjustment and Testing Antimyosin Fab-DTPA was dialyzed against 100 volumes of the indicated buffers using dialysis tubing (Fisher Scientific Co.), and then 0.2 micron 05 filtered. Dialysis tubing was boiled in lOmM EDTA pH 7.0, rinsed with distilled water, and stored in 70% ethanol prior to use. Dialyzed antimyosin Fab-DTPA was adjusted to 0~5 mg/ml and filled, 1 ml/vial, aseptically into 10 sterile 1 ml vials (Wheaton) and sealed with sterile rubber stoppers (West) and metal crimps. Vials were incubated at 4°C, 22°c, 37°C and 45°C for 48-96 hours. Some vials were, in addition, stressed by shaking at 37°C (formulations 1-32) or by shipping 15 in styrofoam containers from Malvern, PA to Miami, FL, then returned to Malvern, PA by Federal Express, Priority 1 mail (formulations 90-95). Shipped vials were filled using·a peristaltic pump (Paxall) through 0.123 inch (inside diameter) silicone tubing 20 at a pump speed of 500 rpm. Vials were visually inspected by inverting several times and observing against a dark and a light background. Vials containing antimyosin Fab-DTPA were compared with control vials, prepared 25 as described above, which contained buffer only. In general, all of these buffers exhibited no shedding at 4°C and 22°c over the times they were examined. Experimental parameters for formulations exposed to temperatures of 37°C and 45°C yielded the most 30 information. The degree of precipitation was graded

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as follows:+ (precipitates, cloudy);+/- (fine precipitates); - (no precipitates). The results of the visual inspection for buffers 1-89 for formula­ tions stressed at 37°C and 45°C are shown in Table 05 1.

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TABLE 1

Visual Inspection of Antimyosin Formulations Stressed at 37°C and 45°C(a)

37°C Shaken 05 (#1-#32) 45°c Formulation 37°c (#32-89) Formulation 37°c 45°c

1 46 + 2 + 47 10 3 48 + 4 49 + 5 +/- + 50 + 6 + 51 + 7 + 52 + + 15 8 + 53 + 9 + 54 + + 10 + 55 + + 11 +/- + 56 12 + 57 + + 20 13 + 58 + + 14 + 59 + + 15 + 60 + + 16 + 61 + 17 + 62 + + 25 18 + 63 + + 19 +/- + 64 + +

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20 + + 65 + + 21 + 66 + + 22 + + 67 + + 23 + + 68 + + 05 24 + 69 + + 25 + 70 + + 26 + 71 + + 27 + +/- 72 + + 28 + 73 + 10 29 + 74 + + 30 + 75 + + 31 76 + + 32 + 77 + + 33 + 78 + + 15 34 + 79 + 35 + 80 + 36 + 81 + 37 + 82 + 38 + 83 + 20 39 + 84 + 40 + 85 + + 41 + + 86 + + 42 +/- 87 + 43 +/- 88 + 25 44 + + 89 45 +

(a) Incubated at indicated temperature 48-96 hr, or or shaken at maximum rpm on rotary shaker for 18 hr.

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+ easily visible particulates, cloudy +/- fine particulates visible no particulates

The phosphate formulations shown as formula- OS tions 90-95, were shipped to Miami, FL and returned to Malvern, PA, and formation of particulates was observed. The results of the shipping study are shown in Table 2.

TABLE 2

10 Shipping Study of Phosphate Formulationsa

Forumulation E!! Particulates

90 7.2 + 91 7.0 +/- 92 6.75 15 93 6.5 94 6.25 95 6.0

(a) 10 mM phosphate, 145 mM NaC1, 10% maltose containing 0.5 mg/ml antimyosin Fab-DTPA.

20 After storage at _4°C for over 2 years, the shipped vials were analyzed for particulates by particle counting in a Climet model CI-1000 particle counter. Each vial was degassed and three 0.1 ml

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aliquots counted to determine total particles greater than or equal to 10 microns and greater than or equal to 25 microns. Replicate runs were averaged and the total counts per dose (1.15 ml) are 05 reported in Table 3.

TABLE 3 Particle counts for Antimyosin Formulationsa Shipped and Stored at 4°C for 25 Months

total particles per dose 10 Formulations ~ 10 microns 25 microns 90 7.20 830 460 91 7.00 not tested not tested 92 6.75 370 210 93 6.50 430 260 15 94 6.25 680 520 95 6.00 530 370 speci'f' ica t' ions b 10,000 1,000

(a) 10 mM sodium phosphate, 145 mM sodium chloride, 10% maltose 20 {b) USP XXI

These results confirm the results obtained by visual inspection of the shipped vials set out in Table 2. Protein precipitation during shipping or long term storage (more than 2 years) was minimized 25 by the 10 mM sodium phosphate, 145 mM sodium

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chloride, 10% maltose formulation, particularly at pH 6.75 and 6.50.

In-111 Incorporation and Immunoreactivity After screening buffers for particulate 05 formation, selected formulations which showed minimal precipitation were further evaluated for In-111 incorporation into the protein, and im.munoreactivity of the resulting In-111 labeled antimyosin Fab-DTPA. Formulations at neutral pH 10 were acidified prior to radiolabeling with an equal volume of Oc2 M sodium citrate (pH 5) in a metal-free microfuge tube (BioRad). All transfers were also performed with metal-free pipette tips (BioRad). In-111 chloride [Amersham, 370 MBq/ml (10 15 mCi/ml) at reference] was then added to the protein-citrate mixtures to a final specific activity of 148 MBq (4 mCi) per milligram. After incubation for 15 minutes at room temperature, 10 ul was spotted 1.s cm from one end of a 1 x 10 cm 20 ITLC-SG paper strip (Gelman) and developed in 0.1 M sodium citrate (pH 5.0). The strip was cut in half and both halves measured in a dose calibrator set for In-111. Under these conditions, all In-111 that was protein bound remained at the origin. ZS In order to test for immunoreactivity, a 1000-fold dilution of the radiolabeled antimyosin Fab-DTPA was made in 0.01 M sodium phosph~te (pH 7.2), 0.15 M NaCl, 1% (w/v) bovine serum albumin (PBS-1% BSA). One hundred microliters of this

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diluted sample were applied to a 1 ml column of myosin-Sepharose Cl-4B (Pharmacia). This affinity column was prepared by the attachment of myosin purified from dog heart tissue to cyanogen-bromide 05 activated Sepharose C1-4B (Pharmacia). The column was eluted with eight 1 ml aliquots of PBS-1% BSA, followed by eight 1 ml aliquots of O.lM glycine pH z,.s, o. 01% thimerosal. The collected fractions were counted in a gamma counter set for In-111, the 10 percentage eluting with the glycine buffer representing active radiolabeled antibody. This percentage was divided by the fraction of In-111 protein bound (from ITLC-SG chromatography) to correct for unbound In-111. 15 Those formulations showing the least amount of precipitation were examined to see if the excipients would adversely effect In-111 incorporation or activity of the antibody. Table 4 shows an example with the excipient human serum albumin, where it 20 appears that a portion of the In-111 binds to sites on the albumin rather than to the antimyosin antibody. TABLE 4

Comparison of Shipped Formulations With and Without 25 Human Serum Albumin {HSA)

% % Formulation Incorporation Immunoreactivity 100 mM Citrate pH s.o

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(control) 88.9 83.3

100 mM Citrate pH 5.0, 100 NaC1, 10% maltose 96.5 71.8

100 mM citrate pH 5.0, 05 100 mM NaC1, 10% maltose, 1.% HSA 92.4 55.2

It was discovered that the majority of excipients either did not prevent precipitation, or as in the case of HSA, interfered with In-111 10 binding to the antimyosin antibody. The best formulations were at neutral pH in HEPES (N-(2-hydroxyethy1)piperazine-N 1 -(2-ethanesulfonic acid)] or phosphate buffer. Neutral pH formulations require acidification 15 prior to In-111 labeling. This was accomplished by adding an equal volume of a citrate buffer. citrate buffers ranging from 100 mM to 500 mM and from pH 3.4 to 5.2 were equally effective. A 200 mM citrate pH 5.0 buffer was chosen for all future studies. 20 Previous stability studies of antimyosin Fab-DTPA in 100 mM citrate pH 5.0 indicated no loss of immunoreactivity up to 66 weeks at 5ec. The best formulation was formulation 90, which has the composition 10 mM phosphate, 145 mM NaCl., 25 10% maltose and containing 0.5 mg/ml antimyosin Fab-DTPA. This formulation, at pH 7.2, was pumped into vials and compared with the previous 100 mM

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citrate pH 5.0 formulation. The results are shown in Table 5: TABLE 5

Comparison of Citrate and Phosphate pH 7.2 05 Formulations

% In-III Formulation Conditions Particulates Incorp. Immunoreactivity

I 37°C shaken 90 hr +/- 95.5 90.l

10 shipped + 97.6 90.3

II 37°C, shaken 90 hr 95.9 90.4

shipped 95.1 90.4

I = 0.5 mg/mL antimyosin Fab-DTPA in 100 mM citrate 15 pH 5.0

II = 0.5 mg/mL antimyosin Fab-DTPA in 10 mM phosphate pH 7.2, 145 mM NaCl, 10% maltose

The phosphate formulation (Formulation II) showed no particulates under these conditions, 20 still bound In-111, and maintained a high level of immunoreactivity.

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Example 2

stress Testing of Antimyosin Fab - DTPA Formulations

MATERIALS AND METHODS Vials of antimyosin Fab-DTPA (Centocor, Inc.) 05 were stored under the following conditions:

4°C for 12 months ambient temperature for 12 months 37°C for 12 months

and analyzed for particulates by particle counting 10 and visual inspection. Two vials each of antimyosin Fab-DTPA were stored at 4°C, and then subj~cted to the following stresses immediately prior to analyis:

24 hours at -20°c 15 24 hours at 45°c 24 hours at -20°c followed by 24 hours at 450.c e- ~ .... shipment at ambient temperature from Malvern, PA, to Miami, FL, and back zo (Federal Express Priority 1) untreated control stored at 4°C.

All vials of antimyosin Fab-DTPA were filled at 1.15 ml/vial and contained o.s mg protein per ml of buffer solution. The buffer solution was composed

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of 100 mM sodium phosphate (pH 6.5), 145 mM NaCl, 10% maltose.

Visual Inspection All vials were inspected for visible 05 particulates prior to analysis and the results recorded on a scale of 1 to 5, with 1 meaning no visible particles and 5 meaning that numerous, easily visible particulates were present. Table 6 summarizes the results of visual inspection and 10 particle counting after stressing.

TABLE 6

Visual Inspection and Particle Counting

Visual (1) Particle Counting (2) Treatment Inspection 10 microns 25 microns

15 24 hr., -2o·c 3 465 177 24 hr, 4s•c 3 840 318 24 hr, -2o·c;24 hr. 4s·c 2 686 253 .. . .. shipping at ambient temp. 2 648 295 storage at 4"C 1 625 226 20· 12 mo. a.t 4 ·c 2 571 288 12 mo. at ambient 3 587 280 12 mo. at 37•c 3 575 261

(1) Average of observations of two vials, graded on a scale of 1 to 5, with 1 meaning no visible

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particles, 5 meaning numerous visible particles ? or fibers. (2) Average of analysis of three 0.1 ml aliquots, and expressed as total particles per 1.15 ml 05 (one dose). In order to avoid diluting samples and possibly dissolvin~ particles, three 0.1 ml aliquots were analyzed, then averaged and converted to total particles per dose (1.15 ml). No differences were lOobserved by particle counting and all samples met USP XXI requirements. However, differences were seen by visual inspection. By this subjective evaluation, the numbers of visible particles increased in all groups relative to the untreated 15control stored at 4°C.

Full Scale Labeling The contents of one stressed vial of antimyosin Fab-DTPA were transferred to a vial containing 1 ml of 200 mM citrate pH s.o buffer, (Centocor, Inc.). 20Indium-111 chloride (Amersham, cat. no. INS-1PA) was diluted with expired, unopened Indium-111 chloride to a concentration of 1 mCi/ml, and 0.25 ml was added to the citrate buffered antimyosin Fab-DTPA. After incubation for 10 minutes at room temperature, 2sprotein bound Indium-111 was determined by ITLC-SG chromatography by the following method: Ten microliters were spotted 1.5 cm from one end of a 1 x 10 cm ITLC-SG paper strip {Gelman cat. no. 61885) and developed in 100 mM sodium citrate (pH 5.0).

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The strip was cut in half and both halves measured in a dose calibrator (Capintec CRC-5) set for In-111. Under these conditions, all In-111 that was protein bound remained at the origin. The product OS specification is greater than 90% protein bound at 10 minutes. The contents were then withdrawn by syringe through an 0.2 um filter (Millipore Millex-GV cat no. SLGV025LS). The syringe/filter/needle assembly 10 and the filter/needle were weighed before and after filtration in order to measure the weight of the entire dose and the weight retained in the filter/needle. The uci of Indium-111 in the syringe/filter/needle and in the filter/needle was 15 measured in a dose calibrator (Capintec CRC-5) after filtration~ The filtrate was then analyzed for immunoreactivity and HPLC gel filtration: Gel filtration was performed using high performance liquid chromatography (HPLC) by injecting 10 ul of 20 each sample onto a Dupont Zorbax GF-250 (0.94 x 25 cm) column at a flow rate of 1 ml/min. The mobile phase was 200 mM sodium phosphate pH 6.8, and the eluate was monitored for absorbance at 214 nanometers. The absorbance signal was integrated, 25 and elution time and integrated area of each peak determined. The product specification is greater than 98.0% of all protein elutes as monomer Fab-DTPA. Full scale clinical labelings were carried out 30 on the remaining unopened vial from each stress

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condition, using In-111 diluted to 1 mCi/ml. The loss of mass and radioactivity were measured to ? determine if the stress caused radioactivity to be preferentially held up by the filter. The results 05 show in in Table 7, indicated that the losses range from 7.0 to 11.5% and were not substantially dif­ ferent from the control.

TABLE 7

Loss of Mass and Radioactivity on Filter Unit

10 10 % Total Dose (g) % Total In-111 Samele Retained by"Filter Retained By Filter

24 hr., -2o·c 9. l. 7.8 24 hr., 4s•c 9.7 8.4 IS 24 hr., -2o·c;24 hr. 4s·c 9.0 7.0 shipping at ambient temp. l.1.5 8.9 control stored at 4•c 10.4 8.0

These losses are attributed to the expected holdup of solution in the filter. The radioactivity 20 retained by the filter averaged 1.8% less than the percent mass retained. Each full scale labeling was also evaluated for In-111 incorporation and immuno­ reactivity as shown in Table 8. All of the samples were within specifications for these two lots.

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TABLE 8

Evaluation of Full Scale Labelings

% In-111 % sample Protein Bound Immunoreactivity os 24 hr., -2o·c 94.6 95.1 24 hr., 45•c 93.6 96.4 24 hr., -20°C/24 hr. 45•c 93.0 98.0 shipping at ambient 94.8 95.4 control stored at 4•c 94.9 97.3

10 Isolation of Particulates The second stressed vial of ant~myosin Fab-DTPA was opened in a laminar flow hood and analyzed on the_particle counter. Each vial was opened and analyzed in a horizontal laminar flow hood. Three 15 0.1 ml aliquots were counted in a Climet model CI-1000 particle analyzer set to count all particles greater than or equal to 10 microns and all particles greater than or equal to 25 microns. USP XXI specifies that a single dose must contain 10,000 20 or fewer particles greater than or equal 10 microns in size and 1,000 or fewer particles greater than or equal to 25 microns in size. The remaining contents (about 0.5ml) were transferred to a centrifugal filter unit (Rainin cat. no 38-120 assembled with a

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6 mm diameter 0.2 micron polyvinylidene difluoride membrane punched from 47 mm stock (Gelman, cat. no FP-200). The unit was centrifuged at 1000 rpm for 30 min. in a Sorvall model GLC-2B centrifuge with a OS HL4 rotor. Particles collected on the membrane were resuspended in 50 ul of distilled, deionized water, transferred to a microfuge tube and dissolved by vortexing. The dissolved particles were analyzed by HPLC gel filtration, SOS-PAGE chromatography and 10 isoelectric focusing IEF as described below. The filtrate was analyzed by optical density at 280 nanometers (OD280)v HPLC gel filtration, SOS-PAGE chromatography, and IEF.

Protein Concentration 15 The optical density at 280 nanometers was measured using a UV spectrophotometer (Milton Roy model 1.201). An 0.1 ml aliquot of sample was diluted with 0.4 ml of buffer lacking protein, and the instrument was blanked with the same buffer. 20 The 00280 reading was converted to mg protein per ml assuming. E0.1.% = 1 . 4 f or murine. immunog. lb o u 1·ins. Product specification is 0.45-0.55 mg/ml.

SDS PAGE Sodium dodecyl sulfate polyacrylamide gel 25 electrophoresis (SOS-PAGE) using gradient gels and a discontinuous buffer system was carried out on the Pharmacia Phastsystem using PhastGel gradient 1.0-15 precast gels and PhastGel sos buffer strips. The

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0.5 mm thick gels have a 4.5% T, 3% c stacking gel above a continuous 10 to 15% gradient gel (2% C) and a buffer system of 0.112 M acetate, 0.112 M Tris, pH 6.4. The sos buffer strips contain 0.20M tricine, OS 0.20 M Tris, 0.55% SOS at pH 7.5 in a 2% agarose gel. Equal volumes of sample and 2x sample buffer (5% w/v sodium dodecyl sulfate, 0.02% bromophenol blue, with or without 10% v/v 2-mercaptoethanol) 10 were heated for 5 minutes in a boiling water bath and 1 ul of each applied to a sample lane. Low molecular weight markers (Bio-Rad cat. no. 161-0304) were included on each gel. The gels were run for 60-65 Vhr with the limiting conditions of 250V, 10 15 mA and 3 • O W. The gels were stained in the Phastsystem development unit first with Coomassie Blue, then with silver nitrate using the PhastGel silver stain kit (Pharmacia). Gels were stained for 8 min. in 20 0.1% w/v Phast Gel Blue R in 30% v/v methanol, 10% v/v acetic acid for 5 min., then switched to fresh destain for 8 min., then switched again to fresh destain for 10 min. Gels were preserved in 5% v/v glycerol, 10 v/v acetic acid for 5 min. Each gel 25 was then washed for 2 min. in 10% v/v ethanol, 5% v/v acetic acid, then fresh wash solution added and washed for an additional 4 min. (50°C). The gel was treated with 5% v/v glutaraldehyde for 6 min. at 50°c, followed by two washes with 10% v/v ethanol, 30 5% v/v acetic acid for 3 min. and 5 min., then two

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washes with distilled water for 2 min. each, all at so 0 c. Gels were stained with 0.4% w/v silver nitrate for 6.5 min. at 40°C, then washed twice in distilled water each for o.s min. at 30°C. OS Developer (2.5% w/v sodium carbonate, Oc013% v/v formaldehyde) was added twice for 0.5 min. and 4 min. at 30°c. The gels were treated to reduce b:a:ekground for 2 min. at 30°C with 2.5% w.v sodium tfii:osu:Ifater 3. 7% w/v Tris-HCL. The gel was 10 preserved by washing for 5 min. at S0°C in 5% v/v glycerol. Gels were photographed and allowed to air dry. The product specification is that the sample must conform to standard, in this case, the rs untreated control.

Isoelectric Focusing Isoelectric focusing (IEF) was performed using Pharmacia PhastGel IEF 3-9 precast gels of range 3 to 9 pH units. The gels are approximately 0.5mm 20 thick homogeneous polyacrylamide gels {5% T, 3%C} containing Pharmalyte carrier ampholytes. The gels were prefocused for 75 Vhr using the limiting conditions of 2000V, 2.5 mA and 3.5 W. The gels were then run for 410 Vhr using the same limiting 2S: conditions as during prefocusing. IEF gels were stained in the PhastSystem development module as described under SOS-PAGE. The product specification is that the sample must conform to standard, in this case the untreated control.

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Column Immunoreactivity Assay Aliquots of the In-111 labeled antimyosin used to determine In-111 incorporation were also used to measure the immunoreactivity. At the same time that 05 the ITLC-SG chromatography was performed, a 1000-fold dilution of the radiolabeled antimyosin Fab-DTPA was made in 0.01 M sodium phosphate pH 7.2, 150 mM NaCl, 1%(w/v) bovine serum albumin (PBS-1% BSA). One hundred microliters of this diluted 10 sample was applied to al ml column of myosin-Sepharose Cl-4B. This affinity column was prepared by the attachment of myosin purified from dog heart tissue to cyanogen-bromide activated Sepharose Cl-4B (Pharmacia). The column was eluted 15 with ten l ml aliquots of PBS-1% BSA, followed by ten 1 mL aliquots of 0.1 M glycine pH 2.5, 0.01% thimerosal. The collected fractions were counted in a gamma counter (LI

25 The antimyosin Fab-DTPA remaining in each vial after particle counting (0.5 ml) was filtered by centrifugation through an 0.2 micron polyvinylidene difluoride filter. This method was chosen in order

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to allow evaluation of both the filtrate and particulates. Both the filtrate and the redissolved particulates were compared by HPLC gel filtration, 05 SOS-PAGE and IEF. No aggregates could be seen in any of the gel filtration profiles. Essentially no Fab-DTPA could be detected in any of the redissolved particulates. Similarly, all of the treated samples appeared equivalent to the untreated control in both 10 the SDS-PAGE and IEF analyses. No bands were present in the lanes containing the redissolved particulates. The 00 the filtrate was measured after 280 of 5-fold dilution, then converted to mg/ml. No 15 significant differences from the control sample were observed. The-results are shown in Table 9.

TABLE 9

Protein Concentration After Filtration

Protein (1) 20 Sample OD280 Concentration (mg/ml) 24 hr., -2o•c 0.113 0.40 24 hr.,_ 45•c O.l.22 0.44 24 hr., -2o·c;24 hr 45•c 0.121 0.43 shipping at ambient 0.116 0.41 25 storage at 4•c 0.114 0.41

(1)00280 X 5 = mg/ml 1.4

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Although slight increases in the number of visible particles were observed after vials were stressed, changes were not detectable by any other means of evaluation. When the particulates and 05 filtrate were separated, no aggregates could be seen in the HPLC gel filtration profile. The redissolved particulates did not contain detectable amounts of Fab-DTPA by HPLC gel filtration, SOS-PAGE or IEF. The protein concentration measurements of the 10 filtrates were all similar to the control, and all were below specification (0.45-0.55 mg/ml). The stress conditions also had no effect on the results of the full scale labelings. In-111 incorporation and immunoreactivity were within 15 specifications.

Example 3

Antifibrin Fab-DPTA Formulation Using the same analytical techniques described in Example 1, formulation development was also 20 performed using a second murine monoclonal antibody Fab fragment, antifibrin, conjugated to the metal chelator DTPA. The formulations which showed the best results were based on sodium phosphate, sodium chloride and maltose, and had a pH in the range of 25 6 • o to 7 • 2. Antifibrin Fab-DTPA (Centocor, Inc.) was formulated at a protein concentration of 0.5 mg/ml

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into buffer solutions containing 10 mM sodium phosphate, 145 mM sodium chloride and 10% w/v maltose at pH values of 7.20, 7.00, 6050, 6.25, 6.00 by equilibrium. dialysis as described in Example 1, 05 and vialed at 0.8 ml per vial. These vials were shipped at ambient temperature and visually inspected. The resu1ts, shown in Table 10, indicate that the formation of particulates in this formulation appeared to be pH dependent.

10 TABLE 10 Shipping Study of Antifibrin Formulationa

E!! Particulates 7.20 + 7.00 + 15 6.75 +/- 6.50 +/- 6025 6.00 (a) 10 mM sodium phosphate, 145 mM sodium 20 chloride, 10% w/v maltose

The best formulations were at pH 6.00 and 6.25, which showed no visible particles. These lower pH vials were also analyzed for In-III incorporation, and shown to be equivalent to standard untreated 25 antif ibrin Fab-DTPA.

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After storage at 4°C for 2 years, the shipped vials were analyzed by particle counting as des­ cribed in Example 2. The results are summarized in Table 11.

05 TABLE 11 Particle Counts for Antifibrin Formulationa Shipped and stored at 4°C for 25 Months

total particles per dose E!! 10 microns 25 microns 10 7. 20 1810 910 7.00 not tested not tested 6.75 280 170 6.50 600 360 6.25 470 250 15 6.00 840 430 spec1"f. 1ca t' ions b 10,000 1,000

(a) 10 mM sodium phosphate, 145 mM sodium chloride, 10% w/v maltose (b) USP XXI

20 These particle counts comport with the shipping study results, indicating that the higher pH formula­ tions are less stable. The data indicate that the formulation comprising 10 mM sodium phosphate, 145 mM sodium chloride, 10% w/v maltose, when optimized 25 for pH, prevented protein preciptiation from stress

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induced by shipping and long term storage(~ 2 years).

Equivalents Those skilled in the art will recognize, or be 05 able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

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CLAIMS

What is claimed is:

1. An aqueous buffer solution for monoclonal antibodies or antibody fragments, comprising: 05 (a) a buffer (b) sodium chloride (c) maltose.

2. An aqueous buffer solution of Claim l wherein the buffer comprises phosphate buffer.

10 3. An aqueous buffer of Claim 2 in wherein the phosphate buffer comprises sodium phosphate having a concentration between about 10 mM and about 100 mM and having a pH between about 6 - a.

15 4. An aqueous buffer of Claim 3 which comprises between about 5 to about 20 percent weight per volume maltose.

5. An aqueous buffer of Claim 4 which comprises about 10 percent weight per volume maltose.

20 6. An aqueous buffer of Claim 1 wherein the monoclonal antibodies are antimyosin antibody molecules, or fragments, that have been conjugated with a chelating agent.

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7. An aqueous buffer of Claim 6 wherein the chelating agent is DTPA.

8. An aqueous buffer of Cl.aim 6 wherein the monocl.ona1 antibody fragment is a Fab fragment 05 specific for cardiac myosin that has been conjugated with DTPA.

9'. An aqueous buffer of Claim 1 wherein the monoclonal antibodies are antifibrin antibody molecules, or fragments, that have been 10 conjugated with a chelating agent.

10. An aqueous buffer of Claim 9 wherein the chelating agent is DTPA.

11. An aqueous buffer of Claim 10 wherein the monoclonal antibody fragment is a Fab fragment I5 specific for fibrin that has been conjugated with DTPA.

12. An improved aqueous solution of antimyosin or antifibrin containing a buffer and a salt, wherein the improvement comprises incorporating ZO" between about 5 to about 20 percent by weight maltose into the solution whereby the stability of the antibody in solution is enhanced by the presence of the maltose and wherein the buffer is sodium phosphate and the salt is sodium 25 chloride. ..

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13. An aqueous buffer solution for monoclonal antibodies, which comprises: (a) about 10 mM to about 100 mM sodium phosphate, with a pH between about 6 - 8; 05 (b) about 145 mM sodium chloride; (c) about 5-10% weight per volume maltose; and (d} about .5 to about 5.2 mg/ml of a monoclonal antibody Fab fragment that has been conjugated with a metal chelator.

10 14. An aqueous buffer of Claim 13 wherein the antibody is specific for cardiac myosin or fibrin.

15. An aqueous buffer of Claim 14 wherein the metal chelator is DTPA.

15 16. An aqueous buffer of Claim 13 which comprises: (a) 10 mM sodium phosphate; (b) 145 mM sodium chloride; (c) 10% weight per volume maltose, and (d) .s mg/ml Fab-DTPA specific for 20 cardiac myosin

17. An aqueous buffer of Claim 16 wherein the sodium phosphate has a pH of about 6.5-6.75.

18. An aqueous buffer of Claim 13 which comprises: (a) 10 mM sodium phosphate; 25 (b) 145 mM sodium chloride;

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(c) 10% weight per volume maltose; and (d) e5 mg/ml Fab DTPA specific for fibrin. ...

19. An aqueous buffer of Claim 17 wherein the 05 sodium phosphate has a pH of between about 6.0-6.25.

"

IPR Page 2045 of 2482 (

INTERNATIONAL SEARCH REPORT International Application No PCT/US 89/02289 I. CLASSIFICATION OF SUBJECT MATTER (if several classific:s!ion aymbols 11pply, Indicate all)• According to lnlernational Patent ClaHlfication UPC) or to botll National Clasaificatlon and IPC 4 IPC : A 61 K 39/395, A 61 K 47/00 II. FIELDS SEARCHED

Minimum Doc:umentation Searched 7 Classification System I Claulfieation Symbols 4 IPC I A 61 K I Documentation Searched other than Minimum Documentation to the Extent that 1ueh Document• are lneluded In the Fields Searched •

Ill. DOCUMENTS CONSIDERED TO BE RELEVANT' Category• I Citation of Doeument, " with Indication, where appropriate, of the relevant pusaoea 12 I Relevant to Claim No. 1l

A EP, A, 0124018 (ARMOUR PHARMACEUTICAL co.) 7 November 1984 -- A CH, A, 645537 (THE GREEN CROSS CORP.) 15 October 1984

------

I i

I • Special categoriea of cited documenta: 10 "T" later document published after the International filing date or priority date and not in conflict with the appllc:at,on but "A" doeument defining the general state of the art which la not cited to understand the principle or theory underlying the considered to be of particular relevance invention M£H earlier document but publiahed on or after the International "X" document cf particular relevance; the elaimed invention filing date cannot be considered novel or cannot be considered to .. L.. document which may throw doubts on priority clalm(s) or Involve an inventive atep which is eited to establish the publleatlon date of another "Y" document of particular relevance;· the claimed Invention citation or other 1pec1al reason (as specified) cannot be considered to Involve an Inventive atei:, when the "0" document referring to an oral di1clo1ure, use, exhibition or document ia combined with one or more other auch docu- other means ments, auch combination being obvioua to a paraon skilled "P" document published prior to the international flllng date but in the art. later than the priority date claimed "&" document member of the same patent family

IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Melllng of thla International Search Report 14th September 1989 0 6 OCT 1989 I ~ - International Searching Authority Si - cer ---...... EUROPEAN PATENT OFFICE :::::------T.K. WILLIS ) Form PCT/ISA/210 (second sheet) {January tUS)

IPR Page 2046 of 2482 .--.:- r \

ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. us 8902289 SA 29322

This annex lists the patent ramify members relating to the patent documents cited in the above-mentioned international searcll report. The members are as contained in the European Patent Office EDP file on 29/09{89 The European Patent Office is in no way liable for these particulars which are merely given for the purpose or information.

!

I Patent document Publication Patent fan ;:y Publication cited in search report I date I member(s) I date " EP-A- 0124018 07-11-84 AU-8- 561590 14-05-87 AU-A- 2740284 01-11-84 CA-A- 1214102 18-11-86 DE-A- 3467706 07-01-88 JP-A- 59206312 22-11-84 us...;A- 4478829 23-10-84 CH-A- 645537 15-10-84 None

... i :;;-; =.c :...... __c.: ______J

~For more details about this annex : see Official Journal of the European Patent Office, :--;o. 12/82

IPR Page 2047 of 2482 WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)

(51) International Patent Classification 5 : (11) International Publication Number: WO 93/08837 A61K 39/395, GOlN 33/577 Cl2P 21/08 II C07K 3/28 Al (43) International Publication Date: 13 May 1993 (13.05.93) G01N21/31

(21) International Application Nnmber: PCT/GB92/01970 (81) Designated States: AU, CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, (22) International Filing Date: 27 October 1992 (27.10.92) NL, SE).

(30) Priority data: Published 9122820.5 28 October 1991 (28.10.91) GB With international search report.

(71) Applicant (for all designated States except US): THE WELL­ COME FOUNDATION LIMITED [GB/GB]; Unicorn House, 160 Euston Road, London NWI 2BP (GB).

(72) Inventors; and (75) Inventors/Applicants (for US only): SMITH, Marjorie [GB/ GB]; RIVEROS-ROJAS, Valentina [CL/GB]; Langley Court, Beckenham, Kent BR3 3BS (GB).

(74) Agent: BAKER-MONTON, N., J.; The Wellcome Founda­ tion Limited, Langley Court, Beckenham, Kent BR3 3BS (GB).

(54)Title: STABILISED ANTIBODIES

(57) Abstract

The invention relates to a stabiJised immunoglobulin composition comprising at least one immunoglobulin together with a stabilising amount of a chelator of copper ions such as EDTA or citrate. Preferably the immunoglobulin is an antibody, for exam­ ple a recombinant CDR-grafted antibody against the CDw52 antigen, most preferably CAMPATH-IH. The invention also re­ lates to a process for enhancing the stability of an immunoglobulin which comprises subjecting the imrnunoglobulin to a purifica- I tion procedure capable of removing copper ions therefrom. Preferably the irnmunoglobulin is rendered substantially free from detectable copper ions, for example on atomic absorption spectroscopy.

IPR Page 2048 of 2482 FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCr on the front pages of pamphlets publishing international applications under the PCT.

AT Au,,!ria FR 1:--'rancu. MR Mauritania. AU Australia GA Gabon MW Malawi BB Barbados GB Um!c:d Kingdom NL Netherlands BE Belgium GN Guinea NO Norway BF Burkina Faso GR Greece NZ New Zealand BG Bulgaria HU Hungary PL Poland BJ Benin IE Ireland PT Portugal BR Braisil IT i!aly RO Romania CA Canada JP Japan RU Rus:.ian Federation CF Central Arrican Republic KP Democratic PL-oplc', Republic SD Sudan cc Congo of Korea SE Swcdcn CH Swit:u:rland KR Republic of Korea SK Slovak Republic 11 Cl Cote d'Ivoire KZ. K,u.akhs!an SN Senegal CM Cameroon I.I Llcchtcns!cin SU Soviet Union cs C2.cchoslovakia LK Sri Lanka TD Chad CZ e,.cch Republic I.U Luxembourg: TG Togo DE Germany MC Monaco UA Ukraine OK Denmark MG Mad.igascar us United SI.ales of America ES Spain ML Mali VN Viet Nam Fl Finlanr.l MN Mongolia

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STABILISED ANTIBODIES

The present invention relates to the stabilisation of 5 immunoglobulins against degradation, in particular on storage and processing prior to use. Antibodies or immunoglobulins are proteinaceous bifunctional molecules. One part, which is highly variable between different antibodies, is responsible for binding to 10 an antigen, for example the many different infectious agents that the body may encounter, whilst the second, constant, part is responsible for binding to the Fe receptors of cells and also activates complement. In this way, antibodies represent a vital component of the immune 15 response of mammals in destroying foreign microorganisms and viruses. The immunisation of an animal with an antigen results in the production of different antibodies with different specificities and affinities. An antiserum obtained from 20 the immunised animal will, therefore, be heterogeneous and contain a pool of antibodies produced by many different lymphocyte clones. Antibodies thus obtained are referred to as polyclonal antibodies and this polyclonal nature has been a major drawback in the use of antibodies in 25 diagnostic assays and in therapeutic applications. A major step forward occurred in 1975 when Kohler and Milstein (Nature, 1975, 256, 495-497) reported the successful fusion of· spleen cells from mice immunized with an antigen with cells of a rnurine rnyeloma line. The 30 resulting hybrid cells, termed hybridomas, have the properties of antibody production derived from spleen cells and of continuous growth derived from the rnyeloma cells. Each hybridoma synthesizes and secretes a single antibody to a particular determinant of the original antigen. To 35 ensure that all cells in a culture are identical, i.e. that they contain the genetic information required for the

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2 synthesis of a unique antibody species, the hybridomas resulting from cell fusion are cloned and subcloned. In this way, the cloned hybridomas produce homogeneous or monoclonal antibodies. 5 The advantages of hybridoma technology are profound. Because many hybrids arising from each spleen are screened for their potential to produce antibodies to the antigen of interest and only a few are selected, it is possible to immunize with impure antigens and yet obtain specific 10 antibodies. The immortality of the cell line assures that an unlimited supply of a homogeneous, well-characterised antibody is available for use in a variety of applications including in particular diagnosis and immunotherapy of pathological disorders. Unfortunately, the usefulness of 15 such monoclonal antibodies in a clinical setting can be severely hampered by the development of human anti-mouse antibodies an anti-globulin response which may interfere with therapy or cause allergic or immune complex hypersensitivity. This has led to the development of 20 humanised antibodies. An antibody molecule is composed of two light chains and two heavy chains that are held together by interchain disulphide bonds. Each light chain is linked to a heavy chain by disulphide bonds and the two heavy chains are 25 linked to each other by disulphide bonds. Each heavy chain has at one end a variable domain followed by a number of constant domains, and each light chain has a variable domain at one end and a constant domain at the other end. The light chain variable domain is aligned with the 30 variable domain of the heavy chain. The light chain constant domain is aligned with the first constant domain of the heavy chain. The remaining constant domains of the heavy chains are aligned with each other. The constant domains in the light and heavy chains are not involved " 35 directly in binding the antibody to the antigen. The variable domains of each pair o~ light and heavy

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chains form the antigen binding site. They have the same general structure with each domain comprising a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions 5 (CDRs) . The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in . some cases comprising part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, 10 contribute to the formation of the antigen binding site. In the use of murine monoclonal antibodies, the induction of an human anti-mouse antibody response is due to the murine origin of the constant domains and four framework regions. This problem has therefore been 15 addressed by the development of modified antibodies of two basic types. The first type, referred to as chimeric antibodies, is where the murine constant domains only are replaced by equivalent domains of human origin (Morrison et al, P.N.A.S., 1984, 81, 6851-6855; Boulianne et al, 20 Nature, 1985, 314, 268-270; and Neuberger et al, Nature, 1985, 314, 268-270). The second type is where the murine constant domains and the murine framework regions are all replaced by equivalent domains and regions of human origin. This second type of modified antibody is referred to as a 25 humanised or CDR-grafted antibody (Jones et al, Nature, 1986, 321, 522-525; and Riechmann et al, Nature, 1988, 332, 323-327). To generate sufficient quantities of antibody for full clinical investigation, it is desirable to utilize an 30 efficient recombinant expression system. Since myeloma cells represent a natural host specialized for antibody production and secretion, cell lines derived from these have been used for the expression of recombinant antibodies. Often, complex vector design, based around 35 immunoglobulin gene regulatory elements, is required, and final expression levels have been reported which are highly

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...,'

variable (Winter et al, Nature, l988, 332, 323-327; Weidle et al, Gene, l987, 60, 205-216; Nakatani Bio/Technology, 1989, 2, 805-810; and Gillies Bio/Technology, 1989, 2, 799-804). 5 Other types of expression systems which have been proposed for antibodies include immortalised human B cells (Rice et al, Proc. Natl. Acad. sci. USA, (1982) 79 7862-7865) , however yields. are generally low and it is difficult to establish stable cell lines. b_ coli has been 10 used to express Fv fragments (Skerra & Plukthun, Science, ( 1988} 240, 1038-1041) or single chain antigen binding molecules (Bird et al, Science, (1988) 242, 423-426) but entire immunoglobulins have so far not been produced in the system. Antibodies have, however, been successfully 15 produced in mammalian cell expression systems which are already known for the production of recombinant proteins such as Chinese hamster ovary (CHO) cells. In the production of purified antibodies whether for therapeutic or diagnostic use, it is important that the 20 antibody is sufficiently stable on storage and various chemical entities may have an adverse effect on the stability of the antibody. The present invention is based on the surprising discovery that trace amounts of copper (Cu++) have a destabilising effect on immunoglobulin 25 molecules on storage and that this effect can be eliminated by formulating the immunoglobulin molecule with a suitable chelator of copper ions. - It has also surprisingly been found that the presence of a chelator of copper ions may have a stabilising effect 30 on the immunoglobulinmolecule even when the immunoglobulin does not contain amounts of copper which are detectable by conventional techniques such as atomic absorption spectroscopy. Whilst not wishing to be bound by any particular theory, it may be that the presence of copper 35 ions in amounts below the detection limits of techniques such as atomic absorption spectroscopy still has a

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destabilising effect on the immunoglobulin molecule which can be eliminated by the addition of a suitable chelating agent. The present invention provides a stabilised 5 immunoglobulin composition comprising at least one immunoglobulin together with a stabilising amount of a chelator of copper ions. The invention also provides the use of a chelator of copper ions to stabilise an immunoglobulin against 10 degradation on storage, for example degradation resulting from the effect of copper ions. The fact that trace amounts of copper ions have a destabilising effect on immunoglobulins means that there may be an advantage in terms of stability in ensuring that 15 immunoglobulins contain the minimum possible amount of copper ions. According to a further aspect the present invention provides a purified immunoglobulin substantially free from copper ions. In particular the invention provides an immunoglobulin in which no copper can be 20 detected by the use of conventional techniques such as atomic absorption spectroscopy. The invention also provides a process for enhancing the stability of an immunoglobulin which comprises subjecting the immunoglobulin to a purification procedure 25 capable of removing copper ions therefrom. In particular the procedure should be such that the no copper can be detected in the immunoglobulin by the use of conventional procedures such as atomic absorption spectroscopy. Copper can be removed from immunoglobulins by conventional 30 procedures known in the field of protein purification such as dialysis versus potassium cyanide containing phosphate buffer followed by gel filtration to remove copper as copper cyanide (see for example Baker and Hultquist, J. Biol. Chem., 253, 844-845 (1978)). 35 The present invention is applicable to the stabilisation of immunoglobulins of all classes, i.e IgM,

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IgG, IgA, IgE and IgD, and it also extends to the stabilisation of Fab fragments and bispecific antibodies. The invention is preferably applied to the stabilisation of immunoglobulins of the class IgG, which includes the

sub-classes IgG IgG2...,, IgG , IgG and IgG • The invention 5 11 28 3 4 • is more preferably applied to the stabilisation of immunoglobulins of the class IgGi- The invention finds particular application in the stabilisation of recombinant antibodies, most particularly 10 chimeric antibodies or humanised (CDR-grafted) antibodies. Particular examples of these include chimeric or humanised antibodies against CD2, CD3, CD4, CDS, CD7, CD8, CDlla,b, CDl.8, CD19, CD25, CD33, CD54 and especially humanised antibodies against the CDw52 antigen, such as CAMPATH-1.H 15 (CAMPATH is a Trade Mark of the Wellcome group of companies). Further examples include chimeric or humanised antibodies against various tumour cell marker antigens. The immunoglobulin will generally be formulated with the metal ion chelating agent at an early stage, for 20 example during or immediately following purification. The production procedure for an immunoglobulin will generally involve purification by means of chromatography and/or gel filtration columns. The chelating agent can be added at any convenient stage of the purification procedure, for 25 example at the stage of the final column, so that the chelating agent remains in the immunoglobulin at the end of the purification procedure. Alternatively, the chelating agent may be added at a suitable stage following purification. In the case of a lyophilised immunoglobulin 30 the chelating agent will generally be added prior to lyophilisation. .; The level at which the chelating agent is added to the irnmunoglobulin will be such as to ensure that any copper present is bound by the chelating agent and thus 35 rendered ineffective in destabilising the immunoglobulin. The invention is applicable irrespective of the intended

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end use of the irnrnunoglobulin although the chelating agent which is used should be chosen in such a way that it will not have an adverse effect on the intended end use of the immunoglobulin. For example in the case of antibodies 5 intended for therapeutic use, the chelating agent should • not show any toxic effects at the level in which it will be present. A particularly preferred metal ion chelating agent is ethylenediamine tetraacetic acid (EDTA) which may typically 10 be added to the immunoglobulin at levels of 0.05mM to SmM, preferably 0.1mM to 3mM. A level of O.lmM EDTA will often be sufficient to stabilise an immunoglobulin but levels up to 2mM or higher do not present any problem physiologically in the case of an immunoglobulin intended for 15 administration to humans. An alternative metal ion chelating agent is citrate ion, preferably used in the form of an alkali metal citrate, e.g. sodium citrate. Immunoglobulins intended for therapeutic use will generally be administered to the patient in the form of a 20 pharmaceutical formulation. Such formulations preferably include, in addition to the immunoglobulin, a physiologically acceptable carrier or diluent, possibly in admixture with one or more other agents such as other immunoglobulins or drugs, such as an antibiotic. Suitable 25 carriers include, but are not limited to, physiologic saline, phosphate buffered saline, phosphate buffered saline glucose and buffered saline. Alternatively the immunoglobulin may- be lyophilised (freeze dried) and reconstituted for use when needed by the addition of an 30 aqueous buffered solution as described above. Routes of administration are routinely parenteral, including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery. The chelating agent may be incorporated into any type of immunoglobulin 35 formulation intended either for storage and distribution or ultimate use. The pharmaceutical formulation will

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generally containr or in the case of a lyophilised preparation will be reconstituted to contain, an effective therapeutic dose of the immunoglobulin per unit dose. In the case of the humanised antibody CAMPATH-1H, liquid 5 formulations or reconstituted Iyophilised formulations • preferably contain o. 5 to 20 mg/ml of the antibody, preferably 2 mg/ml or 10 mg/ml. The invention is illustrated by the following examples: 10 EXAMPLE 1 The effect of various additives on the stability of a recombinant antibody was studied at 37°C. The antibody was CAMPATH l.H, a humanised antibody against the CDw52 15 antigen (Riechmann et al, Nature, 322, ·323-327 (1988)), which had been produced by expression in a recombinant CHO cell line transfarmed with DNA encoding the heavy and light chains of the antibody molecule. The antibody was extracted from the cell culture medium and purified and was 20 then stored as a solution (lmg/ml) in phosphate buffered saline at +4°C. Vials containing 0.5ml of the solution of CAMPATH 1H referred to above together with the additive specified were incubated at +37°C for 4 weeks under sterile conditions. 25 At the end of this period the samples were analysed by size exclusion HPLC, the stability of the sample being assessed by the extent of the formation of "peak C" (a peak formed by the major degradation product of the antibody which has a molecular weight of about SOK} based on the total eluted 30 protein. The results are set out in the following Table 1.

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9

Tl\BLE 1

~ I ADDITIVE I () PEAK C I None 12%

None (storage at +4°C) 2%

5 cu++ (lOppm) 28%

EDTA (2mM) <1%

1,10-phenanthroline (lOmM) 3%

The copper was added as and the 10 1,10-phenanthroline as a solution in water containing 2% (v/v} ethanol. These results demonstrate that copper enhances the degree of degradation of the antibody relative to the control. The addition of EDTA virtually eliminates 15 degradation whilst the other metal ion chelator 1,10-phenanthroline reduces degradation to a considerable extent.

EXAMPLE 2 20 This example also used CAMPATH lH produced in CHO ·cells of the type referred to in Example 1 ( 11. 3mg /ml in phosphate buffered saline) and the batch having been measured as containing 0.04µg cu2+/ml. In this and following examples, the copper content of antibody samples 25 was measured by atomic absorption spectroscopy using a Philips PU9400X atomic absorption spectrophotometer. The detection limit of this method was about 0.03 µg cu/ml so that samples stated to have "no detectable copper" contain less than 0.03 µg Cu/ml. Samples of this Campath lH were 30 diluted to 1 mg/ml in phosphate buffered saline and dialysed exhaustively versus 0.2M sodium phosphate buffer at pH 6.0, pH 6.4 and pH 6.8, CAMPATH lH previously having been determined to be most stable against degradation by

IPR Page 2058 of 2482 W093/08837 PCT/GB92/01970

10

heat at about pH 6. The following was added to 300µ1 samples at each pH:

(i) 30µ1 lOmM CuC12 .2H2D in wateri II (ii) 30µ1 lOmM EDTA in water; 5 (iii) 30µ1 buffer; and the samples incubated at 62 °C for 24 hours. 50 µl aliquots were analysed as described in Example 1 with degradation being assessed by size exclusion chromatography and measured as the extent of formation of "Peak C" based 10 on the total eluted protein. The results for% Peak Care given in Table 2 below: TABLE 2

pH % Peak C

Cu EDTA Buffer

15 6.0 1.75 0.38 0.69

6.4 2.94 0.34 0.72

6.8 5.31. 0.51 l..12

The results indicate that as pH increases, the effect of 20 copper on the degradation of CAMPATH 1H increases. In the absence of added copper an increase in% Peak C is also seen with increasing pH. In the presence of EDTA the degradation of CAMPATH 1H is suppressed.

25 EXAMPLE 3 This example used two different batches of CAMPATH lH produced in CHO cells of the type referred to in Example 1 (10mg/ml in phosphate buffered saline): Batch 1 contained no detectable cu2+ as determined by atomic absorption 30 spectroscopy and Batch 2 contained 0.04µg Cu2+/ml. Samples of both batches were diluted to 1 mg/ml in phosphate buffered saline and dialysed extensively for 24 hours at +4°C against SOmM ammonium hydrogen carbonate and lmM EDTA

IPR Page 2059 of 2482 PCT/GB92/01970 W093/08837

11

was added to the Batch 2 to eliminate any effect of the copper. 200µ1 aliquots of both batches were incubated for 24 hours at 4, 10, 20, 30, 40, 50 and 62°C and degradation was assessed as described in Example 1 by size exclusion 5 chromatography and measured as the extent of formation of "Peak C" based on the total eluted protein. The results for% Peak Care given in Table 3 below: TABLE 3

.!'.,- Temperature 0 Peak C

10 Batch 1 Batch 2 + EDTA

4°C 0 0

10°C 0 0

20°c 0 0

30°c Q.47 0

15 40°c 2.71 0

50°C 60.1 0

62°c 72.36 1.12

Although no detectable cu2+ was found in Batch 1, some 20 degradation was apparent on incubation at 30 and 40°C with extensive degradation at 50 and 62°C. In the case of Batch 2 2 which contained detectable cu +, minimal degradation was seen even at elevate~ tem~erature in the presence of EDTA. These results suggest the possibility that subdetectable 2 25 levels of cu + may accelerate the degradation of CAMPATH lH.

EXAMPLE 4 The results of Example 1 were confirmed by a timed incubation at 62°C over a period of 24 hours using the same 30 CAMPATH lH antibody produced in CHO cells. The batch used was determined to contain 0.03µg Cu 2+/~l by atomic

IPR Page 2060 of 2482 W093/08837 PCT/GB92/01970

12 absorption spectroscopy and 3ml of this batch containing 3.7mg/ml CAMPATH lH in phosphate buffered saline was dialysed at +4°C for 24 hours against 3 x 2 litres SOmM ammonium hydrogen carbonate. 100 µl aliquots were incubated 5 at 62°C with the following additions:

( i) 5µ1 0. OlM EDTA in water + 10µ1 0. lM CuC12 • 2H20 in water; (ii) 5µ1 0.01M EDTA in water; (iii) none. 10 The amount of EDTA added should have been sufficient to chelate any residual transition metal ions in the antibody but not sufficient to chelate the copper which is added in Sample (i). 50µ1 samples were withdrawn for analysis at the 1.5 fallowing times: O, 1, 2, 3, 4, 5 and 24 hours. The samples were analysed as in Example 1 by size exclusion HPLC with the extent of formation of Peak c again being taken as a measure of the extent to which the antibody had been degraded. The results are shown in the following 20 Table 4: TABLE 4

~ Time 0 Peak C

hours EDTA + cu EDTA None

0 a a 0

25 1 2.49 0 1.13 - 2 9.20 0 1.82

3 39.24 a 3.27

4 44.83 0 5.13

5 49.42 0 6.89

30 24 1.00 2.25 22 .12

IPR Page 2061 of 2482 PCT/GB92/01970 W093/08837

13

EXAMPLE 5 This example also used CAMPATH lH produced in CHO cells of the type referred to in Example 1 (10.0mg/ml in phosphate buffered saline) and the batch having no 5 detectable copper as measured by atomic absorption spectroscopy. A sample of this Campath lH was dialysed at +4 °c versus 50mM ammonium hydrogen carbonate and 100µ1 aliquots were incubated at 62°C for 24 hours with 10µ1 of

increasing concentrations of CuC12 • 2H20 in water. The 10 samples were analysed as in Example 1 by size exclusion HPLC with the extent of formation of "Peak C 11 again being taken as a measure of the extent to which the antibody had been degraded. The results are shown in the following Table 5: 15 TABLE 5

9,- nMoles cu per 0 Peak c I nMole CAMPATH lH I I 0 1. 61

0.018 8.09

20 0.037 11. 41

0.074 13.61

0.145 17.59

0.293 22.84

25 The extent of degradation was found to increase with increasing molar ratio of Cu2+/CAMPATH lH. At ratios above o. 3 (data not shown), aggregation was seen with lower recovery of total protein.

30 EX.AMPLE 6 This example also used CAMPATH lH produced in CHO cells of the type referred to in Example 1 ( 1. orng/ml in

IPR Page 2062 of 2482 W093/08837 PCT/GB92/01970

14 phosphate buffered saline), the batch having been found to

contain o .19µg Cu2+ /ml as measured by atomic absorption spectroscopy. The sample thus had a high copper content (copper/CAMPATH 1H molar ratio 449 pMol Cu2+/nMol CAMPATH 5 1H) and early stability studies showed that this batch was subject to substantial degradation on storage at 37°C. The effect of incubation of this sample for up to four weeks at 37°C with and without the presence of 2mM EDTA is shown below in Table 6. The samples were analysed 10 as in Example 1 by size exclusion HP~C with the extent of formation of "Peak C" again being taken as a measure of the extent to which the antibody had been degraded. TABLE 6

~ Time 0 Peak C

15 {weeks) 2 mM EDTA No EDTA

1 0.72 2.86

2 1.26 6.59

3 1.24 9.24

4 l..44 10.18

20 4 at +4°C 0.95 l. 02

2 mM EDTA substantially decreases the decomposition of the CAMPATH 1H but does not totally inhibit it. A sample of th~ same Campath 1H was dialysed at +4°C 25 versus SOmM ammonium hydrogen carbonate and 100µ1 aliquots were incubated at 62°C for 24 hours with varying concentrations of EDTA. Again the samples were analysed as in Example 1 by size exclusion HPLC with the extent of formation of "Peak C" being taken as a measure of the 30 extent to which the antibody had been degraded. The results of two separate experiments are shown in Tables 7 and 8 below:

IPR Page 2063 of 2482 W093/08837 PCT/GB92/01970

15

TABLE 7

~ I mM EDTA I 0 Peak C I 0 6.86

0.1 1. 03

5 1 1. 38

2 1.12

3 1. 26

4 1. 04

10 1.20 10 TABLE 8

!?,, I mM EDTA I 0 Peak C I 0 7.47

0.0001 8.43

15 0.001 7.28

0.01 1.83

0.04 1.68

0.1 1.63

20 These results show that as little as O.OlmM EDTA effectively inhibits decomposition of CAMPATH 1H.

EXAMPLE 7 The effect of cu2+ and of EDTA on the decomposition of 25 various antibodies is shown in Table 9 below. All samples were incubated at 4 °c and at 62 °c for 24 hours in the absence· of any additives and at 62°C for 24 hours in the 2 presence of either cu + ( lmM CuC1 2 • 2H20 + O. SmM EDTA) or EDTA

IPR Page 2064 of 2482 W093/08837 PCT/GB92/01970

16

(1mM EDTA). TABLE 9

~ 0 Peak C

Antibody 4°c 62°c 62°c 62°c

5 No EDTA No EDTA + cu2 + + EDTA

IgGl. 0.54 1.58 5.59 1.1

C1H 0 2.49 27.98 0

CD4 0.4 1.91 21.52 1. 84

IgG2 0 1.81 3.77 0 10

IgG1 = mouse monoclonal IgG1 antibody, l.mg/ml in phosphate buffered saline; Cl.H = CAMPATH 1H of the type described in Example l., l.mg/ml in phosphate buffered saline; 15 CD4 = Humanised anti-CD4 monoclonal antibody having the same framework region as CAMPATH lH and produced in CHO cells, lmg/ml in phosphate buffered saline;

IgG2 = Mouse IgG2 monoclonal antibody I-4139 20 commercially available from Sigma, supplied lyophilised from phosphate buffer and redissolved with water to lmg/ml. All samples show little or no decomposition at 4°C whereas there is some decomposition at 62°C which is increased by 25 varying degrees by the presence of copper. Decomposition at 62°C is suppressed by EDTA.

EXAMPLE 8 A comparison between the effect of 2mM EDTA in 30 phospha~e buffered saline (pH 7.2) and 50 mM citrate (pH 6.0) on the stability of Campath-lH was carried out at various levels of copper. Campath-1.H produced in CHO cells

IPR Page 2065 of 2482 W093/08837 PCT/GB92/01970

17

of the type referred to in Example 1, the batch having no detectable copper as measured by atomic absorption spectroscopy, was diluted 1 in 10 by volume with phosphate buffered saline. 1 ml aliquots were dialysed against 1 t; 5 litre of the following buffers: (i) phosphate buffered saline, pH ,.2; (ii) 2mM EDTA in phosphate buffered saline, pH 7.2; (iii) 50 mM sodium citrate, pH 6.0. Dialysis was carried out at 4°C with three changes over 16 10 hours. Protein concentration was then determined for the three samples by scanning between 340 and 200 nm using a

buffer blank and taking the extinction coefficient A280 (0.1%) as 1.32. Protein concentrations of: (i) 1. 32 mg/ml 15 (ii) 1.20 mg/ml (iii) 1.27 mg/ml were determined. 200 µl aliquots of the antibody in the above buffers were then incubated with increasing concentrations of

20 CuC12 • 2H20 (up to 20 mM) at 62 °C for 24 hours ( 62 °C being the optimal temperature for copper-induced cleavage of Campath-lH.) Samples (50 µl aliquots) were then analysed by size exclusion HPLC in the manner described in Example 1 and the various fractions integrated by cutting and

25 weighing chromatograms of the A280- absorbing peaks eluted from the column. In this case, results were recorded as% "peak B11 (whole Campath-lH). The results are set out in the following Table 10.

IPR Page 2066 of 2482 W093/08837 PCT/GB92/01970

- 18 -

TABLE 10

Added % Peak B Cu (mM) PBS only PBS+2mM EDTA SOmM Citrate

0 42.92 100 100

1 21.47 98.95 94.71

2.5 18. 72 36.96 94.66

5.0 0 0 93.43

7.5 0 0 92.82

.10 0 0 92.57

12.5 0 0 84.85

15 0 0 32.53 - 20 0 0 15.48

Cleavage of Campath-lH ln phosphate buffered saline alone at pH 7.2 is relatively rapid on incubation for 24 hours at 62°c even in the absence of added copper. In phosphate buffered saline plus 2mM EDTA, pH7.2, cleavage is induced when greater than lmM - copper is added. In _ SOmM - citrate, pH 6.0, cleavage takes place when copper in excess of lOmM is added.

IPR Page 2067 of 2482 W093/08837 PCT/GB92/01970

- 19 -

EXAMPLE 9 • A similar experiment to Example 8 also investigated the effece of varying the pH. Campath-lH produced in CHO cells of the type referred to in Example 1 in phosphate buffered saline, the batch having no detectable copper as measured by atomic absorption spectroscopy, was diluted 1: 20 in phosphate buffered saline pH 7.2. Protein concentration was then determined as described in Example 8 and the samples diluted to a protein concentration of 2mg/ml with phosphate buffered saline pH 7.2 or phosphate buffered saline pH 6.0 and the pH was checked. Either 4µ1 O.lM - trisodium citrate, pH 7.0 or 4µ1 0.1 M-EDTA, pH 7.0 was added to 200µ1 aliquots of each of the Campath-lH samples (2mg/ml in phosphate buffered saline either pH 7.2 or pH 6.0) to give a final concentration of about 2mM with respect to citrate of EDTA.

Copper was added up to 3mM as Oto 6µ1 aliquots of O.lM Cuc1 .2 o per 2 2 200µ1 Campath-lH (2mg/ml) sample. 4µ1 water was added to samples without copper. Samples were incubated at 62°c for 24 hours, centrifuged to remove any precipitated material and 50µ1 aliquots analysed by size exclusion HPLC in the manner described in Example 8. Results, recorded as% "peak B" (whole Campath-lH) are set out in the following Table 11.

IPR Page 2068 of 2482 W093/08837 PCT/GB92/01970

- 20 -

TABLE 11

Added % Peak B

Cu PBS only PBS+2mM EDTA PBS+2Mm CIT

(mM) pH 7 .2 pH 6.0 pH 7 .2 pH 6.0 pH 7 .2 pH 6.0

0 93.54 95.29 91.41 92.91 93.17 89.25

0.5 3.24 38.46 92.86 94.87 64.81 86.63

LO 17.27 12.89 94.47 93.56 66.77 84.96

2.0 6.5 0 95.14 13 18.36 0.74

2.5 25 0 12.92 0 38.41 0.8

3.0 15.44 0 13.2 0 37.5 0.93

2 The above table shows the approximate stoichiometry of binding of Cu +_ by 2mM-EDTA and 2mM-citrate and the contributory effect of pH. 2mM.-EDTA in phosphate buffered saline, pH 7.2, is the most effective in suppressing copper induced cleavage of Campath-lH. An approximate 1:1 stoichiometry of binding is indicated. at pH 7.2. Copper concentrations in excess of 2mM. cause cleavage of Campath-lH in 2mM. EDTA.

IPR Page 2069 of 2482 W093/08837 PCT/GB92/0l 970

CLAIMS:

."!' 1. A stabilised irnmunoglobulin composition comprising at 5 least one immunoglobulin together with a stabilising amount of a chelator of copper ions.

2. A composition as claimed in Claim 1, wherein the irnmunoglobulin is of the class IgG. 10 3. A composition as claimed in Claim 1 or 2, wherein the irnmunoglobulin is a recombinant CDR-grafted antibody.

4. A composition as claimed in Claim 3, wherein the 15 antibody is an antibody against the CO2, CD3, CD4, CDS, CD7, CD8, CD11a,b, CD18, CD19, CD25, CD33, CDw52 or CD54 antigen.

5. A composition as claimed in Claim 3, wherein the 20 antibody is an antibody against the CDw52 antigen.

6. A composition as claimed in Claim 5, wherein the antibody is CAMPATH-lH.

25 7. A composition as claimed in any of Claims 1 to 6 1 wherein the chelator of copper ions is ethy lenediarnine tetraacetic acid.

8. A composition as claimed in any of Claims 1 to 6 30 wherein the chelator of copper ions is citrate ion.

9. A composition as claimed in any of Claims 1 to 8, in the form of a liquid preparation suitable for parenteral adrninist;ration. 35 10. A composition as claimed in any of Claims 1 to 8, in

IPR Page 2070 of 2482 W093/08837 PCT/GB92/01970

22 lyophilised form suitable for reconstitution into a liquid preparation suitable for parenteral administration.

11. Use of a chelator of copper ions to stabilise an 5 immunoglobulin against degradation on storage.

12. Use according to Claim 11, wherein the chelator of copper ions is ethylenediamine tetraacetic acid.

10 13. Use according to Claim 11, wherein the chelator of copper ions is citrate ion.

14. Use according to any of Claims 11 to 13, wherein the antibody is a recombinant CDR-grafted antibody against the 15 CDW52 antigen.

15. Use according to Claim 14, wherein the antibody is CAMPATH-1H.

20 16. A process for enhancing the stability of an immunoglobulin which comprises subjecting the immunoglobulin to a purification procedure capable of removing copper ions therefrom.

25 17 . A process as claimed in Claim 16, wherein the purification procedure is dialysis versus potassium cyanide containing phosphate buffer followed by gel filtration to remove copper as copper cyanide.

30 18. A purified immunoglobulin substantially free from copper ions.

19. A purified immunoglobulin in which no copper can be detected by atomic absorption spectroscopy. 35 20. An immunoglobulin as claimed in Claim 18 or 19, which

IPR Page 2071 of 2482 W093/08837 PCT/GB92/01970

23

is a recombinant CDR-grafted antibody against the CDw52 antigen.

,., 21. An irnrnunoglobulin as claimed in Claim 20, wherein the 5 antibody is CAMPATH-lH.

IPR Page 2072 of 2482 INTERNATIONAL SEARCH REPORT PCT/GB 92/01970 lnlernarional Application No J L CLASSIFlCATION OF SUBJECT MATTER (if several classification symbols apply, iodic:ale all)' Ac:mrding to lntemational Patent Classification (IPq or to both National Classification and IPC Int.Cl. 5 A61K39/395; G01N33/577; C12P21/08; //C07K3/28 G01N21/31

ll. FIELDS SEARCHED Minimum Documentation Searched'7 .. Classification S~tem Classification Symbols

Int.Cl. 5 C07K ; A61K

Documentation Searched other than Minimum Documentation to the Extent that such Documents aze Included in the Fields Searched1

m. DOCUMENTS CONSIDERED TO BE RELEVANT' Category·0 Citation of Document, 11 with indication, where appropriate, of the relev.mt passages ll llelewot to Claim No)l

X EP,A,O 391 526 (BIOPROBE INTERNATIONAL, 1-21 INC.) 10 October 1990 see the whole document --- A BIOCHEMICAL PHARMACOLOGY 1-21 vol. 21, no. 8, 15 Apri 1 1972, OXFORD, GB pages 1097 - 1105 M. CHVAPIL ET AL. 'Effects of selected chelating agents and metals on the stability of 1 iver lysosomes. I see abstract see page 1099, line 12 - page 1101, line 10 --- -/--

0 Special categories of cited documents : 10 •r later document published after the international filing date or priority date and not in mnflict with the :/!llcation but "A" document defining the general state of the art wllicb is not cited to 11Dderstand the principle or theory un etlying the considered to be of particular relevance invention ~· earlier document but published on or after the international "X' document of particular relev:ance; the claimed Invention filing date cannot be considered nowl or cannot be considl!ffil to "L"' document ,wch may tflrow doubts onl,riority claim(s) or involve an inventive step wbicb is cited to establish the publica n date of another "'Y• dOClllllent of particular relev:ance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the "'0' document refening to an or:ll disclosure, use, lldlibition or document is combined with one or more other such doc:u- other means menu. suc:h combination being obvious to a person skilled "P' document published prior to the international filing date but In tbe an. later than the priority date daimed "&" document member of tlle same patent family

IV. CERTIFlCATION 2 Date of the Actual Completion of the International Search Date of Mailing of this International Search Repon 12 JANUARY 1993 tf&,O'l. 93

International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE NOOIJ F .J.M. ~1v~1- 1.,__ - I Fam PCf/lSA/210 (r.ecaad IMdl (J--, 1915)

IPR Page 2073 of 2482 PCT/GB 92/01970 International Application No DL DOCUMENTS CONSIDERED TO BE REI.EVANT (CONTINUED FR.OM THE SECOND SHEE1') Cltegory". a ration of Document, with indication. where appropriate, of the relevant passages

A BIOTECHNOLOGY PROGRESS 1-21 vol. 5, no. 3, September 1989, NEW YORK, USA pages 119 - 125 W. VELANDER ET AL. 1 Process implications for metal-dependent immunoaffinity interactions. 1 see the whole document P,X US,A,5 087 695 (W. MCAULEY) 1-21 11 February 1992 see the whole document

IPR Page 2074 of 2482 ANNEX TO IBE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 9201970 SA 65929

This annex lists the patent family members relating to the patent documents c:ited in the above-mentioned intematiooal llearch report. The members are as contained in the European Patent Office mp file on The European Patent Office is in no way liable for these partic:ulan which are merely given for the purpose of information.12/01/93

Patent document Publication Patent family Publication cited in 5e811:b report I date I manber(ll) I date EP-A-0391526 10-10-90 US-A- 4933435 12-06-90 CA-A- 2010835 05-10-90 JP-A- 2290900 30-11-90 US-A-5087695 11-02-92 None

t•

i ::e ;1------'s l:i For more details about this 111U1ex : see Official JolU'Dal of the European Palmt Office, No. 12/82

IPR Page 2075 of 2482 WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)_

(51) International Patent Classification 6 : (11) International Publication Number: WO 99/37329 A61K 39/395 Al (43) International Publication Date: 29 July 1999 (29.07.99)

(21) International Application Number: PCT/SE99/00049 (81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, (22) International Filing Date: 15 January 1999 (15.01.99) GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, (30) Priority Data: SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, 9800170-4 22 January 1998 (22.01.98) SE ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG, 9800766-9 9 March 1998 (09.03.98) SE ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (71) Applicant (for all designated States except US): ASTRA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, AKTIEBOLAG [SE/SE]; S-151 85 Sodertalje (SE). SN, TD, TG).

(72) Inventors; and (75) Inventors/Applicants (for US only): FLINK, Ola [SE/SE]; Published Astra Arcus AB, S-151 85 Sodertalje (SE). PETREN, Sven With international search report. [SE/SE]; Astra Arcus AB, S-151 85 SMertlllje (SE).

(74) Agent: ASTRA AKTIEBOLAG; Intellectual Property, Patents, S-151 85 SMertalje (SE).

(54) Title: PHARMACEUTICAL FORMULATION COMPRISING AN ANTIBODY AND A CITRATE BUFFER

(57) Abstract

The present invention relates to an isotonic pharmaceutical formulation comprising an antibody and a buffer, wherein the antibody is present at 0.5 mg/ml to 10 mg/ml, the buffer is a citrate buffer present at 5 mmol/1 to 20 mmol/1 and the pH of the formulation is 5.3 to 7.2. The invention also relates to a method for the storage of antibodies by use of such formulations and to the use of such formulations in therapy.

IPR Page 2076 of 2482 FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AL Albania ES Spain LS Lesotho SI Slovenia AM Armenia FI Finland LT Lithuania SK Slovakia AT Austria FR France LU Luxembourg SN Senegal AU Australia GA Gabon LV Latvia sz Swaziland AZ Azerbaijan GB United Kingdom MC Monaco TD Chad BA Bosnia and Herzegovina GE Georgia MD Republic of Moldova TG Togo BB Barbados GH Ghana MG Madagascar TJ Tajikistan BE Belgium GN Guinea MK The former Yugoslav TM Turkmenistan BF Burkina Faso GR Greece Republic of Macedonia TR Turkey BG Bulgaria HU Hungary ML Mali TT Trinidad and Tobago BJ Benin IE Ireland MN Mongolia UA Ukraine BR Brazil IL Israel MR Mauritania UG Uganda BY Belarus IS Iceland MW Malawi us United States of America CA Canada IT Italy MX Mexico uz Uzbekistan CF Central African Republic JP Japan NE Niger VN Viet Nam CG Congo KE Kenya NL Netherlands YU Yugoslavia CH Switzerland KG Kyrgyzstan NO Norway zw Zimbabwe Cl Ci\te d'Ivoire KP Democratic People's NZ New Zealand CM Cameroon Republic of Korea PL Poland CN China KR Republic of Korea PT Portugal cu Cuba KZ Kazakstan RO Romania CZ Czech Republic LC Saint Lucia RU Russian Pederation DE Germany LI Liechtenstein SD Sudan DK Denmark LK Sri Lanka SE Sweden EE Estonia LR Liberia SG Singapore

IPR Page 2077 of 2482 WO 99/37329 PCT/SE99/00049

PHARMACEUTICAL FORMULATION COMPRISING AN ANTIBODY AND A CITRATE BUFFER

The invention relates generally to antibody formulations and particularly to stabilised

5 antibody formulations for storage and for therapeutic administration.

Purified proteins, particularly those produced using recombinant DNA technology are now well established as pharmaceutical agents. Such proteins do however present a range of problems associated with their stable formulation. Many protein preparations are

10 particularly unstable in dilute solutions and must be formulated in such a way as to prevent significant levels of denaturation, agglomeration or degradation. These problems are particularly acute in the formulation of large proteins such as immunoglobulins. Immunoglobulins or antibodies are known to be prone to form aggregates and particulates in solution and this has long provided special problems in generating suitable formulations

15 for the storage and administration of therapeutic antibodies. Existing antibody formulations frequently require to be filtered before injection to remove aggregates or particulate matter which is inconvenient and tends to reduce the accuracy of the injected dose.

Various attempts have been made to overcome the problems of antibody formulation:

20 EP O 073 371 describes intravenously administrable immunoglobulin compositions which have their pH adjusted to 3.5 to 5.0 as proteins are known to be more stable at low pH. Such low pHs however tend to result in undesirable reactions at the site of injection .

25 US 4650772 describes a method for stabilising thermally unstable monoclonal antibodies which requires the presence of 0.25% to 5% hydrolysed ovalbumin. The use of ovalbumin in pharmaceutical formulations results in the induction of an allergic response which prevents its effective use for repeated administrations.

30 WO 90/11091 describes the us~ of maltose and buffers in a lyophilised formulation of monoclonal antibodies. Lyophilisation is however an expensive process and the need to

IPR Page 2078 of 2482 WO 99/37329 PCT/SE99/00049 2

resuspend the formulation prior to administration adds to the complexity of the treatment regimen. It was suggested that citrate buffer may be used to buffer the pH at between 3 .0 and 6.0. s The present invention provides a more simple antibody formulation than those presently known, providing a formulation which is both suitable for administration and has improved storage properties. Existing antibody formulations require the use both of a stabiliser and of a buffer. However antibodies in formulations of the present invention are stabilised only by citrate buffer in a saline solution at a physiologically preferable pH.

10 There is therefore provided according to the present invention, an isotonic pharmaceutical formulation comprising an antibody and a buffer, wherein the antibody is present at 0.5mg/ml to 1Omg/ml, the buffer is a citrate buffer present at Smmol/1 to 20mmol/l and the pH of the formulation is 5.3 to 7.2.

15 A citrate buffer for use in the present invention may be generated by dissolution of free citric acid or preferably a pharmaceutically acceptable salt of citrate, preferably a sodium salt.

20 A formulation of the present invention may be generated by solubilising the relevant antibody, preferably in saline and adding an amount of citrate buffer necessary to obtain a pH of the solution in the range 5.3 to 7.2. The citrate buffer is preferably present at Smmol/1 to 20mmol/l.

2s A formulation of the present invention may additionally contain other substances desirable for therapeutic efficacy of the antibody e.g. chelators, or other therapeutic compounds desirable to be coformulated with the antibody but it is preferably substantially free of any additional compound known for use in antibody stabilising e.g. Tween, mannitol or maltose. By 'substantially free' it is meant that such additional compounds known for use in

30 antibody stabilising formulations may not be present in formulations of the present

IPR Page 2079 of 2482 W099/37329 PCT/SE99/00049 3

invention in an amount capable alone or in combination with one or more other stabilisers, of having a stabilising effect upon an antibody in a formulation.

In preferred embodiments of the present invention citrate buffer is present in the s formulation at 7.Smmol/1 to 15mmol/l and most preferably at lOmmol/1. Any pharmaceutically acceptable citrate buffer may be used in the present invention but the citrate buffer is preferably sodium citrate. It is more preferable that sodium citrate dihydrate is used and most preferable that the citrate buffer be generated from a mixture of sodium citrate dihydrate and citric acid monohydrate. In the preferred embodiments of the

10 present invention, the formulation contains about 2.4 mg/ml sodium citrate dihydrate and about 0.387 mg/ml citric acid monohydrate.

The present invention is suitable for the formulation of any antibody or antibody fragment. Any reference to an antibody herein will be taken to include a :fragment of such antibody.

1s The antibody for use in a formulation of the present invention may be natural or recombinant and may be generated according to any known technique. Natural antibodies may be those isolated either by purification from body fluids or from cell lines and may be polyclonal or monoclonal antibodies. Particularly preferred antibodies for use in formulations of the present invention are recombinant antibodies produced from

20 engineered cell lines. Such cell lines will have been engineered to express the relevant antibody gene. The antibody gene may either be a human gene or a gene from another species which has been humanised by modification of the native sequence to prevent rejection when administered to a human, e.g. a humanised recombinant antibody. The antibody is preferably an antibody directed against the human T cell surface receptor TCR

25 V() 5.2/5.3 (the method for constructing such an antibody is described in WO 95/16038 and the description of such methods is hereby incorporated by reference) , and is more preferably an IgG, IgGl or IgG/K. The antibody is most preferably the antibody produced by the cell line deposited on June 22, 1995 under the Budapest treaty as ATCC (CRL 11949) [herein this antibody is referred to as 'TM27'] or is one comprising the following

30 TM27 VK sequence:

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1 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQTPGKAPKLLIYY 50

51 TSSLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYSKLPRTFGQ 100

5 101 GTKLQIT 107,

and further comprises an amino acid sequence selected from the group consisting of the TM27 VH amino acid sequence:

10 1 QVQLQESGPGLVRPSQTLSLTCTVSGFSLTAYGVNWVRQPPGRGLEWLGM 50

51 IWGDGNTDYNSALKSRVTMLKDTSKNQFSLRLSSVTAADTAVYYCARDRV 100

1s 101 TATLYAMDYWGQGSLVTVSS 120,

the TM27 VH sequence wherein amino acid residue 48 is replaced with isoleucine (I),

the TM27 VH sequence wherein amino acid residues 78 and 79 are valine (V) and

20 phenylalanine (F),

theTM27 VH sequence above wherein amino acid residues 67 to 70 VTML are replaced with LSIS respectively and amino acid 73 is an aspargine (N),

2s the TM27 VH sequence wherein amino acid residue 92 is an arginine (R).

The present invention also provides for the use of formulations of the present invention in medical therapy and particularly for the treatment of autoimmune disease and further particularly in the therapy of multiple sclerosis.

30

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In preferred embodiments of the present invention the formulation has a pH in the range 5.5 to 6.5 and is most preferably pH 5.5. The pH may be altered using any pharmaceutically acceptable acid or alkali. s The formulation of the present invention may be prepared under aseptic conditions, leading to a sterile formulation.

The invention will now be illustrated by reference to the following examples which are in no way intended to be limiting of the scope of the invention described herein.

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EXAMPLES

The antibody assayed in the following examples is TM27 which is a humanised antibody s (IgGl) produced in Chinese hamster ovary (CHO) cells by recombinant technology.

The bulk batches of TM27 used were purified by protein-A affinity chromatography. The TM27 preparations were prepared from two different TM27 bulk solutions. One was 10.8 mg TM27 /ml in 10 mmol/1 citrate buffer pH 5.5 and the other was 11.0 mg TM27 /ml

10 in 10 mmol/1 phosphate buffer pH 6.5. The bulk solutions had sodium chloride added to make them isotonic.

The TM27 preparations used in this study were prepared under aseptic conditions. The batches were protected from air with nitrogen during the manufacturing and filling

1s processes. Eight batches with different compositions were prepared using the buffers described.

For manufacturing purposes, TM27 bulk solution was diluted with the appropriate buffer to a concentration of 1 mg/ml TM27 by gentle mixing, while avoiding foaming. The

20 manufactured solutions were filtered through a sterile 0.22 µm MILLEX-GV filter directly into 10 ml sterile glass vials. Filling was performed from the bottom of the vials under nitrogen protection.

The solutions were filled into 10 ml injection vials of neutral Type I glass (Ph Eur), 1

2s ml/vial. Bromobutyl rubber stoppers (FM 257) were used and the vials were sealed with aluminium capsules.

The batches were stored under the following conditions: +5 °C/ambient humidity and +25 °C/30 % relative humidity. All the vials were stored upright. All examples contain

30 antibody at 1 mg/ml.

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