USOO9084743B2
(12) UnitedO States Patent (10) Patent No.: US 9,084,743 B2 Teschner et al. (45) Date of Patent: Jul. 21, 2015
(54) STABLE CO-FORMULATION OF 4,396,608 A 8/1983 Tenold ...... 424,177.1 HYALURONIDASE AND ; : A 3: Set al. r. 3:13; IMMUNOGLOBULIN, AND METHODS OF 26736. A 75 FAI. 53. USE THEREOF 4,876,088 A 10/1989 Hirao et al...... 424,101 4,952.496 A 8, 1990 Studier et al. .. 435,9141 (75) Inventors: Wolfgang Teschner, Vienna (AT); Sonja 5,033,252 A 7/1991 Carter ...... 53.425 Svatos, Berg (AT); Leopold 5,052,558 A 10/1991 Carter ...... 206,439 Bruckschwaiger, Vienna (AT); Alfred 5,122,373 A 6, 1992 Eibl et al. .... 424,171.1 5,122,614 A 6/1992 Zalipsky ...... 548,520 Weber, Vienna (AT), Hans-Peter. 5,177,194. A 1/1993 Sarno et al. . 530.390.1 Schwarz, Vienna (AT); Laura Lei, Los 5, 180,810 A 1/1993 Gomi et al. . 530/350 Angeles, CA (US) 5,323,907 A 6/1994 Kalvelage ...... 206,531 5,324,844 A 6/1994 Zalipsky ...... 548,520 (73) Assignees: Baxter International Inc., Deerfield, IL 5,409,990 A 4, 1995 Linnau et al. 525,541 (US);US); Baxter Healthcare S.Asa. Kes 5,608,0385,446,090 A 3,8/1995 1997 EibletHarris al...... 530,387.1525,541 Zurich-Opfikon (CH) 5,612.460 A 3/1997 Zalipsky ...... 530,391.9 5,643,575 A 7/1997 Martinez et al...... 424,194.1 (*) Notice: Subject to any disclaimer, the term of this 5,665,069 A 9/1997 Cumer et al...... 604,116 patent is extended or adjusted under 35 5,672,662 A 9, 1997 Harris et al. ... 525/408 U.S.C. 154(b) by 713 days 5,721,348 A 2f1998 Primakoff et al...... 536, 22.1 M YW- y yS. 5,747,027 A 5/1998 Stern et al...... 424/94.62 5,766,581 A 6/1998 Bartley et al...... 424/85.1 (21) Appl. No.: 12/807,991 5,795,569 A 8/1998 Bartley et al. ... 424/85.1 5,808.096 A 9/1998 Zalipsky ... 548,520 (22) Filed: Sep. 16, 2010 5,827,721. A 10/1998 Stern et al...... 435,201 5,854,046 A 12/1998 Au-Young et al...... 435/201 O O 5,871,736 A 2/1999 Bruegger et al...... 424/177.1 (65) Prior Publication Data 5,900.461. A 5/1999 Harris ...... 525,54.11 US 2011 FOO661 11 A1 Mar 17, 2011 5,919,455 A 7/1999 Greenwald et al...... 424,178.1 s 5,932,462 A 8/1999 Harris et al...... 435.188 Related U.S. Application Data (Continued) (60) Provisional application No. 61/277,045, filed on Sep. FOREIGN PATENT DOCUMENTS 17, 2009. CH 684 164 T 1994 (51) Int. Cl. EP O177836 4f1986 A 6LX39/395 (2006.01) (Continued) A6 IK39/00 (2006.01) A6 IK9/00 (2006.01) OTHER PUBLICATIONS A6 IK 47/42 (2006.01) Bee et al., “Recombinant human PH2O is well tolerated at higher C07K 16/00 (2006.01) intravenous and Subcutaneous doses in cynomolgus monkeys.” C07K 16/06 (2006.01) EUFEPS2008, Munich, Germany, 3 pages. CI2N 9/26 (2006.01) Berger et al., “Immunoglobulin replacement therapy by slow Subcu (52) U.S. Cl. taneous infusion.” Ann Intern Med 93:55-56 (1980). CPC ...... A61K 39/00 (2013.01); A61 K9/002I Berger et al., “Subcutaneous immunoglobulin therapy in primary (2013.01); A61 K39/39591 (2013.01); A61 K immunodeficiencies.” Clin Immuno. 112:1-7 (2004). 47/42 (2013.01); C07K 16/00 (2013.01); C07K Gardulfetal. “Safety of rapic Subcutaneous gammaglobulin by rapid 16/06 (2013.01); C12N 9/2474 (2013.01); infusion in patients with primary antibody deficiency.” CI2Y 302/01035 (2013.01); A61K 2039/505 Immunodeficiency 4:81-84 (1993). (2013.01); C07K 2317/21 (2013.01) (Continued) (58) Field of Classification Search None See application file for complete search history. Primary Examiner — Yunsoo Kim (74) Attorney, Agent, or Firm — McKenna Long & Aldridge (56) References Cited LLP; Stephanie Seidman U.S. PATENT DOCUMENTS (57) ABSTRACT 3,869,436 A 3/1975 FalkSveden et al...... 260f 112 3,966,906 A 6, 1976 Schultze et al...... 424,177.1 Provided herein are stable co-formulations of immunoglobu 4,002,531 A 1/1977 Royer ...... 435.188 lin and hyaluronidase that are stable to storage in liquid form 4,093,606 A 6, 1978 Coval .... 530.390.5 4,124,576 A 11, 1978 Coval ...... 530.390.5 at room temperature for at least 6 months and at Standard 4,126,605 A 11, 1978 Schneider et al. 530.390.5 refrigerator temperatures for 1-2 years. Such co-formulations 4,165,370 A 8, 1979 Coval ...... 424,177.1 can be used in methods of treating IG-treatable diseases or 4,179,337 A 12, 1979 Davis et al...... 435/181 conditions by Subcutaneous administration. 4,186,192 A 1, 1980 Lundbladet al...... 424,177.1 4,362,661 A 12, 1982 Ono et al...... 424,177.1 4,374,763. A 2/1983 Takagi ...... 530.390.5 29 Claims, No Drawings US 9,084,743 B2 Page 2
(56) References Cited 2010.0003238 A1 1/2010 Frost et al...... 424/94.62 2010, 0074885 A1 3/2010 Schiff et al. U.S. PATENT DOCUMENTS 2010.01434.57 A1 6/2010 Wei et al...... 424/450 2010/0172892 A1 7/2010 Uvarkina et al...... 424/94.62 5,945,098 A 8, 1999 Sarno et al...... 424,85.5 2010, 0196423 A1 8/2010 Bookbinder et al...... 424,247.1 5,958,750 A 9, 1999 Au-Young etal 435,201 2010, 0211015 A1 8, 2010 Bookbinder et al...... 604,187 5,985,263. A 11/1999 Lee et al...... 424/85.2 2010/0330.071 A1 12/2010 Teschner et al...... 530/412 5.990,237 A 11, 1999 Bentley et al...... 525,542 2011/O152359 A1 6, 2011 Bookbinder et al. ... 435/200 6,057,110 A 5, 2000 Au-Young et al...... 435/6 2011/0212074 A1 9/2011 Frost et al...... 424,85.1 6,069,236 A 5/2000 Burnouf-Radosevich 2011/0293598 Al 12/2011 Bruckschwaiger et al. .. 424/530 et al...... 530/416 2011/0293638 Al 12/2011 Bruckschwaiger 6,113,906 A 9/2000 Greenwald et al...... 424,194.1 et al...... 424,140.1 6,123,938 A 9, 2000 Stern et al...... 424/94.62 2012/0020951 A1 1/2012 Shepard et al...... 424,130.1 6,193,963 B1 2/2001 Stern et al. ... 424/94.6 2012/0076772 A1 3/2012 Butterweck et al. ... 424/133.1 6.2 14,966 B1 4/2001 Harris ...... 528,322 2012/0076779 A1 3/2012 Butterweck et al...... 424,130.1 6,258,351 B1 7/2001 Harris ...... 424/78.3 2012fOO9377O A1 4/2012 Bookbinder et al...... 424/94.62 6,340,742 B1 1/2002 Burg et al. .. 530,351 2012/O148555 A1 6/2012 Bookbinder et al...... 424/94.3 6,395,880 B1 5/2002 Linnau et al. 530,393 2012/0171153 A1 7/2012 Frost et al...... 424/78.17 6,413.507 B1 7/2002 Bentley et al. 424/78.02 2013/0022588 A 1 1/2013 Yang et al. ... 424/94.3
6.420,339 B1 7/2002 Gegg et al...... 514/12 2013/0251786 A1 9/2013 Li et al. .... 424/94.62 6,437,025 B1 8, 2002 Harris et al. 523,406 2013/0302275 A1 11/2013 Wei et al...... 424/94.62 6,448,369 B1 9, 2002 Bentley et al...... 528,425 2013/0302400 A1 11/2013 Maneval et al. .... 435/195 6,461,802 B1 10/2002 Van Thillo et al. 430,336 2014,0199282 A1 7/2014 Bookbinder et al...... 435/200 6,495,659 B2 12/2002 Bentley et al. ... 528,425 6,552,170 B1 4/2003 Thompson et al. 530,351 FOREIGN PATENT DOCUMENTS 6,737,505 B2 5/2004 Bentley et al...... 528,425 6,828.401 B2 12/2004 Nho et al...... 526,333 EP O246579 5, 1987 6,828,431 B1 12/2004 Frudakis et al...... 536,231 EP O278422 8, 1988 6,858,736 B2 2/2005 Chang-min ETAL ...... 546,290 EP O440483 8, 1991 6,875,848 B2 4/2005 Ristol Debart et al. .... 530/390.1 EP O822199 2, 1998 7,105,330 B2 9, 2006 Stern et al...... 435/200 EP 1064951 6, 2000 7,148,201 B2 12/2006 Stern et al...... 514/44 JP 54O2O124 2, 1979 7.309,810 B2 12/2007 Takai et al...... 8003 JP 57031623 2, 1982 7,368,108 B2 5/2008 DeFrees et al...... 424/94.5 JP 571286.35 8, 1982 7,544.499 B2 6, 2009 Frost et al...... 435/200 JP S63-192724 8, 1988 7,767.429 B2 8, 2010 Bookbinder et al. . 435,201 JP H10-265407 A 10, 1998 7,781,397 B2 8/2010 Stern et al...... 514/2 JP 4346934 12/2002 8, 105,586 B2 1/2012 Bookbinder et al. . 424/94.3 JP 2004-238.392 A 8, 2004 8, 187.855 B2 5/2012 Baker et al...... 435,201 JP 2006-524.507 A 11, 2006 8,202,517 B2 6, 2012 Bookbinder et al...... 424/94.62 JP 2007-511566. A 5/2007 8,431,124 B2 4/2013 Bookbinder et al. 424/94.62 WO WO94,28024 12/1994 8,431,380 B2 4/2013 Bookbinder et al...... 435,201 WO WO94,29334 12/1994 8.450,470 B2 5, 2013 Bookbinder et al...... 536,23.2 WO WO96,07429 3, 1996 8,580,252 B2 11/2013 Bookbinder et al...... 424/85.2 WO WO 98.042376 10, 1998 8,765,685 B2 7, 2014 Bookbinder et al. . 514, 20.9 WO WOOO?O2017 1, 2000 8,772.246 B2 7, 2014 Bookbinder et al. . 435/200 WO WOOOf 176640 10, 2001 2001, 0021763 A1 9, 2001 Harris .. 528,75 WO WOO1,87925 11, 2001 2001/0044526 A1 11/2001 Shen ...... 530/409 WO WO O2/49673 6, 2002 2001/0046481 A1 1 1/2001 Bentley et al. 424/78.18 WO WO 2004/056312 T 2004 2002fOO52430 A1 5, 2002 Harris et al...... 523,406 WO WO 2004/078140 9, 2004 2002fOO72573 A1 6/2002 Bentley et al. . 525/409 WO WO 2005/OOO360 1, 2005 2002/0156047 A1 10, 2002 Zhao ...... 514/58 WO WO 2005/049078 6, 2005 2003.0114647 A1 6, 2003 Harris et al. 530/402 WO WO 2006/09 1871 8, 2006 2003.0143596 A1 7/2003 Bentley et al...... 435/6 WO WO 2008,127 271 10, 2008 2003. O158333 A1 8/2003 Roberts et al...... 525,54.11 WO WO 2009/111066 9, 2009 2003/0170243 A1 9/2003 Stern et al...... 424,146.1 WO WO 2009/117085 9, 2009 2003/0220447 A1 11/2003 Harris ...... 525,54.1 WO WO 2009/128917 10/2009 2004, OO13637 A1 1/2004 Bentley et al. 424/78.17 WO WO 2010/138736 12/2010 2004/0096921 A1 5, 2004 Stern et al. .. 435,792 WO WO 2011/034604 3, 2011 2004/O142867 A1 7/2004 Oi et al...... 514/12 2004/0235.734 A1 11/2004 Bossard et al...... 514/12 OTHER PUBLICATIONS 2004/0268425 A1 12/2004 Bookbinder et al. . ... 800/18 2005.0053598 A1* 3, 2005 Burke et al. 424,130.1 Halozyme Therapeutics, Analyst and Investor Meeting presentations 2005/011.4037 A1 5/2005 Desjarlais et al...... 7O2/19 “Matrix Therapeutics for Life' presentations including Lim, J., 2005/0171328 A1 8/2005 Harris ...... 528,322 “Introduction and strategy overview, Roche program update.” 2005/0209416 A1 9/2005 Harris ...... 525,523 Gustaf K. “Strategic depl t of cash. W. R 2005/0260186 A1 11/2005 Bookbinder et al...... 424/94.61 ustaison, M. Strategic deployment of cash, wasserman, K., 2005/0287134 A1 12/2005 Klein ...... 424/94.61 “HyO treatment of primary immunodeficiency patients.” Muchmore, 2006, O104968 A1 5, 2006 Bookbinder et al. 424/94.61 D., “Ultrafast insulin-clinical results and ongoing trials.” Cefalu, W., 2007/0134228 A1 6, 2007 Stern et al...... 424/94.61 “Unmet needs in diabetes management.” Little, R., Market overview 2007/014.8156 A1 6, 2007 Frost et al. .. 424/94.61 ultrafast insulin and SC immunoglobin and Frost, G., “PEGPH2O 2008.0171014 A1 7/2008 Wu et al...... 530,387.9 s 2009/O123367 A1 5, 2009 Bookbinder et al. ... 424/1.49 and HTI-501 status report.” Presented 10.14.10 in New York, NY. 2009,0181013 A1 7/2009 Bookbinder et al. 424,130.1 (124 pages). 2009, O181032 A1 7/2009 Bookbinder et al. 424,141.1 Hofer, “Human Recombinant Hyaluronidase Increases the Convec 2009/0214505 A1 8, 2009 Bookbinder et al...... 424/94.1 tion of Molecules up to 0.2 um in Athymic Nude Mice.” American 2009/0253175 A1 10, 2009 Bookbinder et al...... 435/69.1 Association for Laboratory Animal Science, 2006, Salt Lake City, 2009/0304665 A1 12/2009 Frost et al...... 424/94.5 UT. Abstract published in J. Am. Assoc. Lab. Animal Sci., 45:120, 2009/0311237 A1 12/2009 Frost ...... 424/94.62 2006, abstract P97.2 pages. US 9,084,743 B2 Page 3
(56) References Cited World Health Organization (WHO) guideline: cf. D21, WHO Tech nical Report Series, No. 814 "Guidelines for assuring the quality of OTHER PUBLICATIONS pharmaceutical and biological products prepared by recombinant DNA technology.” (1991). Horowitzetal. “Viral safety of solvent? detergent-treated blood prod McCoy et al., “Pharmacokinetics of 10% immunoglobulin adminis ucts.” Blood Coagul. Fibrin. 5(3): S21-S28 (1994). tered intravenously or Subcutaneously alone or following recombi Kempfet al., “Virus inactivation during production of intravenous nant human hyaluronidase in subjects with PID,” XIVth Meeting of immunoglobulin.” Transfusion 31(5):423-427 (1991). the European Society for Immunodeficienies (ESID) Istanbul, Tur Mayer, M., “Quantitative C* Fixation Analysis, Complement and key Oct. 6-10, 2010. Poster, 1 page. Complement Fixation.” in Experimental Immunochemistry Eds. Schiff et al., “Tolerability of immunoglobulin subcutaneous 10% Kabat, E. And M. Meyer, Thomas, Springfield, II., pp. 214-216 and administered SC following administration of recombinant human pp. 227-228 (1961). hyaluronidase in subjects with PID,” XIVth Meeting of the European McCoy et al., “Pharmacokinetics of 10% Immunoglobulin Admin Society for Immunodeficienies (ESID) Istanbul, Turkey Oct. 6-10, istered Intravenously or Subcutaneously Alone of Following Recom 2010, Poster, 1 page. binant Human Hyaluronidase in Subjects with PID.” Retrieved from Stein et al., “Tolerability and efficacy of recombinant human the Internet: (56) References Cited hyaluronidase hyaluronidase (IGHy) in a subset of patients with primary immunodeficiency (PI) Presented May 18, 2012 at the Clini OTHER PUBLICATIONS cal Immunology Society Annual Meeting: Primary Immune Defi ciency Disease North American Conference May 17-20, 2012, Chi 2009 Retrieved from:(56) References Cited Csoka et al., “The six hyaluronidase-like genes in the human and mouse genomes.” Matrix Biol. 20:499-508 (2001). OTHER PUBLICATIONS CZitrom et al., “The function of antigen-presenting cells in mice with severe combined immunodeficiency.” J Immunol 134:2276-2280 Bookbinder et al., “A recombinant human enzyme for enhanced (1985). interstitial transport of therapeutics.” JControl Release. 114(2):230 Dalakas, M.. “The use of intravenous immunoglobulin in the treat 241 (2006) Epub 2006 Jun 7. ment of autoimmune neuromuscular diseases: evidence-based indi Bookbinder et al., “Biochemical Characterization of Recombinant cations and safety profile.” Pharmacol Ther 102(3):177-193 (2004). Human PH2O (SPAM1) Hyaluronidase.” Hyaluronan (ISHAS) Dalakas et al., “A controlled trial of high-dose intravenous immune 2007, Charleston, SC, 2 pages. globulin infusions as treatment for dermatomyositis.” N Engl J Med Bookbinder et al., “Enhancing Drug Transport Through Temporary 329(27): 1993-2000 (1993). Matrix Depolymerization.” Keystone Symposia 2005, 13 pages. Dalakas et al., “High-dose intravenous immune globulin for stiff Bookbinder et al., “EnhanzeTM Technology for Antibody Disper person syndrome." N Engl J Med 345(26): 1870-1876 (2001). sion.” Strategic Research Institute Antibody World Summit, 2005, Dalakas et al., “A controlled study of intravenous immunoglobulin Jersey City, NJ. 41 pages. combined with prednisone in the treatment of IBM.” Neurology 56(3):323-327 (2001). Bookbinder et al., “Evaluation of the compatibility and Danilkovitch-Miagkova, et al., “Hyaluronidase 2 negatively regu pharmacokinetics of co-formulated biologics with recombinant lates RON receptor tyrosine kinase and mediates transformation of human hyaluronidase: Dose Response.” American Association of epithelial cells by jaagsiekte sheep retrovirus.” Proc Natl Acad Sci Pharmaceutical Scientists Conference, Nov. 2006, 3 pp. USA. 100(8):4580-4585 (2003). Bordier, C., “Phase separation of integral membrane proteins in Deboeret al., “The tac promoter: a functional hybrid derived from the Triton X-114 solution.” J. Biol. Chem. 256: 1604-1607 (1981). trp and lac promoters.” Proc. Natl. Acad. Sci. USA 80:21-25 (1983). Bouffard et al., “An in vitro assay for hepatitis C virus NS3 serine Delpech et al., “Enzyme-linked hyaluronectin: a unique reagent for proteinase.” Virology 209:52-59 (1995). hyaluronan assay and tissue location and for hyaluronidase activity Brinster et al., “Regulation of metallothionein-thymidine kinase detection.” Anal. Biochem. 229:35-41 (1995). fusion plasmids injected into mouse eggs.” Nature 296:39-42 (1982). Derwent patent abstract citing JP 4346934 published Dec. 2, 2002, Brumeanu et al., “Derivatization with monomethoxypolyethylene for: “Liq. CompSn. For intravenous injection for infectious disease glycol of Igs expressing viral epitopes obviates adjuvant require treatment-comprises chemically unmodified mol. Type gamma ments.” J Immunol. 154:3088-3095 (1995). globulin with low conductivity and contains no sorbitol.” Inventor: Buckley, R. And R. Schiff, “The use of intravenous immune globulin Kamimura et al. Dialog File No. 351. Accession No. 62312172 in immunodeficiency diseases.” n. Engl J Med. 325(2): 110-117 pages. (1991). Derwent patent abstract citing JP 54020124 published Feb. 15, 1979, Byerley et al., "Cutting out the bull. Recombinant human for: “Intraveneously injectable gamma-globulin compSn. Prodn.-by hyaluronidase: Moving to an animal-free system.” Association of addin. Ofamino acids, Sugars and neutral Salts as dissociation agents.” Clinical Embryologists, 2006, Dublin, Ireland. Abstract published in Inventor: Funakoshi et al. Dialog File No. 351. Accession No. Human Fertility 9(2): 110 (2006). 16998.072 pages). Caliceti, P. And F. Veronese, "Pharmacokinetic and biodistribution Derwent patent abstract citing Jp. 57128635 published Aug. 10, 1982, properties of poly(ethylene glycol)-protein conjugates. Adv. Drug for: "Gamma-globulin prepn. For intravenous injection-contains Deliv. Rev. 55(10): 1261-1277 (2003). Sodium chloride and L-arginine or L-lysine.” Inventor: Matsuo et al. Carrillo, H. And D. Lipman, “The multiple-sequence alignment Dialog File No. 351. Accession No. 24967032 pages). problem in biology.” Siam J Applied Math 48(5):1073-1082 (1988). Devereux, J., et al., “A comprehensive set of sequence analysis pro Chapel et al., “Randomised trial of intravenous immunoglobulin as grams for the VAX.” Nucleic Acids Research 12:387-395 (1984). prophylaxis against infection in plateau-phase multiple myeloma. Dodel et al., “Intravenous immunoglobulins containing antibodies The UK Group for Immunoglobulin Replacement Therapy in Mul against beta-amyloid for the treatment of Alzheimer's disease.” J tiple Myeloma.” Lancet 343:1059-1063 (1994). Neurol Neurosurg. Psychiatry 75:1472-1474 (2004). Chapman et al., “Therapeutic antibody fragments with prolonged in D'Souza et al., “In vitro cleavage of hepatitis C virus polyprotein vivo half-lives.” Nature Biotech. 17:780-783 (1999). Substrates by purified recombinant NS3 protease.” J. Gen. Virol. Cheng et al., “PEGylated adenoviruses for gene delivery to the intes 76:1729-1736 (1995). tinal epithelium by the oral route.” Pharm. Res. 20(9): 1444-1451 Ellmeier et al., “Severe B cell deficiency in mice lacking the tec (2003). kinase family members Tec and Btk.” J Exp Med. 192:1611-1624 Cherr et al., “The dual functions of Gpi-anchored Ph-20: (2000). hyaluronidase and intracellular signaling.” Matrix Biol. 20:515-525, Federal Register Sep. 23, 1970 (35 FR 14800); Wydase NDA 6-343, 2001. (40 pages). Cherr et al., “The PH-20 protein in cynomolgus macaque Felix et al., “Pegylated peptides. IV. Enhanced biological activity of spermatozoa: identification of two different forms exhibiting site-directed pegylated GRF analogs.” Int. J. Peptide Res.46:253-264 hyaluronidase activity.” Dev. Biol. 175: 142-153 (1996). (1995). Cho et al., “Construction of hepatitis C-SIN virus recombinants with Fernandes, P. And J. Lundblad, “preparation of a stable intavenous replicative dependency on hepatitis C virus serine protease activity.” gamma-globulin: process design and scale-up. Vox Sang 39:101 J. Virol. Meth. 65:201-207 (1997). 112 (1980). Christadoss et al., “Animal models of myasthenia gravis.” Clin Filocamo et al., “Chimeric Sindbis viruses dependent on the NS3 Immunol. 94:75-87 (2000). protease of hepatitis C virus,” J.Virology 71(2): 1417-1427 (1997). Church et al., “Efficacy, safety and tolerability of a new 10% liquid Form 10-Q for Halozyme Therapeutics dated May 8, 2009, retrieved intravenous immune globulin IGIV 10% in patients with primary from the Internet:. immunodeficiency,” US-PID-IGIV 10% -Study Group 10. J. Clin retrieved on Nov. 25, 20096 pages). Immunol. 26(4):388-395 (2006). Frost et al., “Puntuated Equilibrium: The Evolution of Recombinant Cohn et al., “Preparation and properties of serum and plasma pro Human Hyaluronidase.”Ophthalmic Anesthesia Society, 2006, Chi teins; a system for the separation into fractions of the protein and cago, IL, 36 pages. lipoprotein components of biological tissues and fluids. J. Am. Frost et al., “Purification, cloning, and expression of human plasma Chem. Soc. 68:459-467 (1946). hyaluronidase.” Biochem. Biophys. Res. Commun. 236:10-15 Csoka et al., “Hyaluronidases in tissue invasion.” Invasion Metastasis (1997). 17:297-311 (1997). Frost et al., “Subcutaneous Strategies for Monoclonal Antibody Csoka et al., “Purification and microSequencing of hyaluronidase Delivery.” Drug Delivery 2007: Where Science and Business Meet, isozymes from human urine.” FEBS Lett., 417(3):307-310 (1997). 2007, San Diego, CA, 1 page. US 9,084,743 B2 Page 6
(56) References Cited Hakim et al., “Generation of a novel poliovirus with a requirement of hepatitis C virus protease NS3 activity.” Virology 226:318-326 OTHER PUBLICATIONS (1996). Haller et al., “Escaping the Interstitial Matrix With Enzyme-Medi Frost G., “Subcutaneous Strategies for Monoclonal Antibody Deliv ated Drug Delivery,” Drug Delivery Technology 5(5):1-6 (2005). ery.” IBC Life Sciences Antibodies and Beyond Antibodies: Opti Haller et al., “Recombinant Human Hyaluronidase for the Interstitial mizing Antibody Leads and Exploring Next Generation Scaffolds for Transport of Therapeutics.” American Association of Pharmaceutical Protein Therapeutics, Coronado CA. 2006.20 pages). Scientists Conference, Jun. 2006, San Antonio, TX. 2 pages. Frost, G. And R. Stem, "A microtiter-based assay for hyaluronidase Haller et al., “Recombinant Human Hyaluronidase for the Interstitial activity not requiring specialized reagents.” Anal. Biochem. 251:263 Transport of Therapeutics.” Controlled Release Society Conference, 269 (1997). Vienna, Austria, 2006, 2 pages. Frost, G., “Recombinant human hyaluronidase (rHuPH2O): an Haller et al., “Revolutionizing Drug Dispersion with Enhanze Tech enabling platform for Subcutaneous drug and fluid administration.” nology,” Biotechnology Industry Organization (Bio) Annual Meet Expert Opin. Drug. Deliv. 4:427-440 (2007). ing, 2005, Philadelphia, PA. Jun. 19-22, 4 pages. Gardner et al., “The complete nucleotide sequence of an infectious Haller et al., “Revolutionizing Drug Dispersion with Enhanze Tech nology.” American Association of Pharmaceutical Scientists Annual clone of cauliflower mosaic virus by M13mp7 shotgun sequencing.” Meeting, Nov 6-10, 2005, Nashville, TN, 1 page. Nucleic Acids Res. 9:2871-2888 (1981). Haller et al., “The Effects of Recombinant Human Hyaluronidase on Gardulf et al., “Subcutaneous immunoglobulin replacement in Drug Dispersion.” American Association of Pharmaceutical Scien patients with primary antibody deficiencies: Safety and costs' Lancet tists Annual Meeting, Nashville, TN, abstract in AAPSJournal 7(S2) 345:365-369 (1995). May 5, 2005; 3 pages. Gardulf et al., “Home treatment of hypogammaglobulinaemia with Haller, “Enhanze Technology —An Enzymatic Drug Delivery Sys Subcutaneous gammaglobulin by rapid infusion.” Lancet 338:162 tem (DDS).” Japanese Export Trade Organization, Nov. 2005, Santa 166 (1991). Clara, CA, 2 pages. Gardulf et al., “Lifelong treatment with gammaglobulin for primary Haller, "Halozyme's Enhanze Technology for the Enhanced Disper antibody deficiencies: the patients’ experiences of subcutaneous self sion of Co-Injected Pharmaceuticals.” Japanese Export Trade Orga infusions and home therapy,” J Adv. Nurs. 21:917-927 (1995). nization, Sep. 2004, Chicago, IL, 2 pages. Gardulf et al., “Rapid subcutaneous IgG replacement therapy is Haller, M.. “Converting intravenous dosing to Subcutaneous dosing effective and safe in children and adults with primary with recombinant human hyaluronidase.” Pharmaceut Tech. News immunodeficiencies--a prospective, multi-national study. J Clin. letter, Oct. 2007, 14pgs. Immunol. 26(2):177-185 (2006). Haller, “Enzyme-facilitated Parenteral Drug Transport.” Strategic Gardulfetal. “The life situations of patients with primary antibody Research Institute's 10th Anniversary Drug Delivery Technology and deficiency untreated or treated with Subcutaneous gammaglobulin Deal-making Summit, 2005 New Brunswick, NJ 24 pages. Haller, M., "Focus on Enhanced and Innovative Recombinant Human infusions.” Clin. Exp. Immunol. 92:200-204 (1993). Enzymes.” Japanese Export Trade Organization, Sep. 2004, Chicago, Gardulf, a. And U. Nicolay, "Replacement IgG therapy and self IL, 16 pages. therapy at home improve the health-related quality of life in patients Halozyme Therapeutics, “Halozyme Therapeutic, Inc. Prospectus with primary antibody deficiencies.” Curr. Opin. Allergy Clin. Supplement.” Jun. 23, 2009 84 pages). Immunol. 6:434-442 (2006). Halozyme Therapeutics, “Securities and Exchange Comission Form Gellene, D., "San Diego's Halozyme Injects New Life into Old 10O.” Nov. 6, 2009 44 pages. Drugs' Feb. 28, 2010, Retrieved from the Internet: Halozyme Therapeutics, “Securities and Exchange Comission Form (56) References Cited ment of peripheral neuropathies in children' abstract for the XIth world congress of ICNC Cairo May 2-7, 2010. Retrieved from the OTHER PUBLICATIONS Internet: (56) References Cited from the Internet: , accessed on Nov. 6, 20099 pages). light (accessed Jan. 6, 2009), 2 pages. News Release, Halozyme Therapeutics Inc. Q3 2009 Earnings Call News Release, Halozyme Therapeutics Inc., “Phase III Trial Begins Transcript retrieved from the Internet:, accessed on Nov. 6, 2009 11 pages. from the Internet: (56) References Cited Solomon, B., “Intravenous immunoglobulin and Alzheimer's disease immunotherapy,” (2007) Curr. Opin. Mol. Ther. 9:79-85 (2007). OTHER PUBLICATIONS Sommer et al., “Paraneoplastic stiff-person syndrome: passive trans fer to rats by means of IgG antibodies to amphiphysin.” Lancet Pham et al., "Large-scale transient transfection of serum-free Suspen 365: 1406-1411 (2005). sion-growing HEK293 EBNA1 cells: peptone additives improve cell Steinkuhler et al., “Product inhibition of the hepatitis C virus NS3 growth and transfection efficiency.” Biotechnology and Bioengineer protease.” Biochem. 37:8899-8905 (1998). ing 84:332-342 (2003). Stiehm et al. "Slow subcutaneous human intravenous Pinkert et al., “An albumin enhancer located 10 kb upstream func immunoglobulin in the treatment of antibody immunodeficiency: tions along with its promoter to direct efficient, liver-specific expres Use of an old method with a new product” J Allergy Clin Immunol sion in transgenic mice.”Genes and Devel. 1(3):268-276 (1987). 101:848-849 (1998). Pinkstaffet al., “Evaluation of the Compatibility and Pharmacokinet StrongWater et al., “A murine model of polymyositis induced by ics of Co-formulated Biologics with Recombinant Human coxsackievirus B1 (Tucson strain).” Arthritis Rheum. 27:433-442 Hyaluronidase: Dose Response.” American Association of Pharma (1984). Sudo et al., “Establishment of an in vitro assay system for Screening ceutical Scientists Conference, Jun. 2006, San Antonio, TX 3 hepatitis C virus protease inhibitors using high performance liquid pages. chromatography.” Antiviral Res. 32:9-18 (1996). Pinkstaff et al., “Recombinant Human Hyaluronidase for Drug and SuperSaxo et al., “Effect of molecular weight on the lymphatic Fluid Dispersion.” American Association of Pharmaceutical Scien absorption of water-soluble compounds following Subcutaneous tists Annual Meeting, Nov. 2006, Boston, MA3 pages). administration.” Pharm. Res. 7(2):167-169 (1990). Pinlcstaff et al., “Recombinant Human Hyaluronidase for Use with Swift et al., “Tissue-specific expression of the rat pancreatic elastase Therapeutic Antibodies.” Controlled Release Society Conference, I gene in transgenic mice.” Cell 38:639-646 (1984). Vienna, Austria, 20062 pages. Takahashi et al., "A fluorimetric Morgan-Elson assay method for Poelsler et al., “A new liquid intravenous inmmunoglobulin with hyaluronidase activity.” Anal. Biochem. 322:257-263 (2003). three dedicated virus reduction steps: virus and prion reduction Takeshita et al., “An enzyme-linked immunosorbent assay for detect capacity” Vox Sang 94(3):184-192 (2007). ing proteolytic activity of hepatitis C virus proteinase.” Anal. Polson et al., “The Fractionation of protein mixtures by linear poly Biochem. 247:242-246 (1997). mers of high molecular weight.” Biochim. Biophys. Acta. 82:463 Taliani et al., “A continuous assay of hepatitis C virus protease based 475 (1964). on resonance energy transfer depsipeptide Substrates.” Anal. Readhead et al., “Expression of a myelin basic protein gene in Biochem. 240:60-67 (1996). transgenic shiverer mice: correction of the dysmyelinating pheno Teschner et al., “A new liquid, intravenous immunoglobulin product type.” Cell 48:703-712 (1987). (IGIV 10%) highly purified by state-of-the-art process' Vox Reipert et al., “Fc function of a new intravenous immunoglobulin Sanguinis 92(1):42-55 (2007). product:IGIV 10% triple virally inactivated solution.” Vox Sang Trebstet al., “Expression ofchemokine receptors on peripheral blood 91(3)256-263 (2006). mononuclear cells of patients with immune-mediated neuropathies Relkin et al., "18-Month study of intravenous immunoglobulin for treated with intravenous immunoglobins.” Eur JNeurology 13:1359 treatment of mild Alzheimer disease.” Neurobiol Aging (2008). I9 1363 (2006). pages. Tsubery et al., “Prolonging the action of protein and peptide drugs by Roberts et al., “Chemistry for peptide and protein PEGylation.” Adv. a novel approach of reversible polyethylene glycol modification.” J Drug Deliv. Rev. 54:459-476 (2002). Biol. Chem 279(37):381 18-3.8124 (2004). Sato, H., “Enzymatic procedure for site-specific pegylation of pro van Schaik et al., “Intravenous immunoglobulin for chronic inflam teins.” Adv. Drug Deliv. Rev. 54:487-504 (2002). matory demyelinating polyradicloneuropathy: a systematic review.” Schiffet al., “Alterations in the half-life and clearance of IgG during Lancet Neurol. 1:497-498 (2002). therapy with intravenous gamma-globulin in 16 patients with severe Varga et al., “Efficacy and safety of IGIV. 10% TVR solution, a new primary humoral immunodeficiency.” J. Clin. Immunol. 6:256-264 intravenous immunoglobulin, in adult Subjects with chronic idio (1986). pathic thrombocytopenic purpura' TransfMed Hemother 33:509514 Schiff et al. “Use of a new chemically modified intravenous IgG (2006). preparation in severe primary humoral immunodeficiency: clinical Veronese et al., “Branched and Linear Poly(Ethylene Glycol): Influ efficacy and attempts to individualize dosage.” Clin Immunol ence of the Polymer Structure on Enzymological, Pharmacokinetic, Immunopathol. 31(1): 13-23 (1984). and Immunological Properties of Protein Conjugates.” J. Bioactive Schiff et al., “Multicenter Crossover Comparison of the Safety and Compatible Polymers 12:197-207 (1997). Efficacy of Intraglobin-F with Gamimune-N, Sandoglobulin and Wagner et al., "Nucleotide sequence of the thymidine kinase gene of Gammagard in Patients with Primary Immunodeficiency Diseases” herpes simplex virus type 1.” Proc. Natl. Acad. Sci. USA 78:1441 Journal of Clinical Immunology 17(1):21-28 (1997). 1445 (1981). Schiff, R., “Individualizing the dose of intravenous immune serum Walter et al., “High-dose immunoglobulin therapy in sporadic inclu globulin for therapy of patients with primary humoral sion body myositis: a double-blind, placebo-controlled study.” J immunodeficiency.” Vox Sang. 49 Suppl 1:15-24 (1985). Neurol 247(1):22-28 (2000). Schiff, R. “Half-life and clearance of pH 6.8 and pH 4.25 Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, the immunoglobulin G intravenous preparations in patients with primary Benjamin/Cummings Pub. co., p. 224). disorders of humoral immunity” Rev Infect Dis (4):S449-S456 Wei et al., “Functions of N-linked glycans on human hyaluronidase (1986). PH2O.” poster 83, 1 page (2009). Schwartz and Dayhoff, eds., Atlas of Protein Science and Structure, Wei et al., “Structure function analysis of the human hyaluronidase National Biomedical Research Foundation, pp. 353-358 (1979). enzymes.” American Society for Matrix Biology Biennial Meeting, Shani. M.. “Tissue-specific expression of rat myosin light-chain.” San Diego, CA, Dec. 5, 2008, B4 (2 pages). Nature 314:283-286 (1985). Weksler et al., “Drug-Ranging Study of Intravenous Shapiro et al., “Intravenous gamma globulin inhibits the production Immunoglobulin in Patients with Alzheimer's Disease.” Abstracts: of matrix metalloproteinase-9 in macrophages.” Cancer 95:2032 Pharmacological Treatments 1(Suppl 1): S94-S95 (2005). 2037 (2002). Welch, M. And E. Stiehm, "Slow subcutaneous immunoglobulin Shimizu, Y. And H. Yoshikura, “Multicycle infection of hepatitis C therapy in a patient with reactions to intramuscular virus in cell culture and inhibition by alpha and beta interferons.” J. immunoglobulin.” J. Clin Imrnunol 3(3):285-286 (1983). Virol. 68:8406-8408 (1994). Wilson, M.. “Enhanze Technology —An Enzymatic Drug Delivery Smith, T. And M. Waterman, "Comparison of biosequences.” System (DDS).” Japanese Export Trade Organization, 2005, Santa Advances in Applied Mathematics 2:482-489 (1981). Clara, CA, 22 pages. US 9,084,743 B2 Page 10
(56) References Cited News Release, “Baxterpresents long-term data on HyO during Aaaai annual meeting.” Published.On Mar. 2, 2012 online retrieved on OTHER PUBLICATIONS Nov. 6, 2012 Retrieved from:(56) References Cited Office Action, received Dec. 19, 2013, in connection with corre sponding Eurasian Patent Application No. 201200490 English OTHER PUBLICATIONS translation, 3 pages. Instructions, dated Apr. 15, 2014, for response to Office Action, NewsReleases/News-Release-Details/2013/Baxter-Receives-Mar received Dec. 19, 2013, in connection with corresponding Eurasian keting-Authorization-for-HyOvia-inEuropean-Union/default.aspx Patent Application No. 201200490, 19 pages. 4 pages. Official Action, mailed Feb. 4, 2014, in connection with correspond News Release, "Baxter Submits Amended Bla to U.S. FDA for ing Japanese Patent Application No. 2012-529751 Summary, HyOvia for Primary Immunodeficiency.” Dec. 2, 2013 English translation, and original document in Japanese, 6 pages. online retrieved on Dec. 16, 2013, Retrieved from: Instructions, dated Jul. 23, 2014 for response to Official Action, SYMBOL Gene, 4th Edition, 1987. The Benjamin/Cummings Pub.co. p. 224). Such Substitutions can be made in accordance with 1-Letter 3-Letter AMINO ACID 45 those set forth in TABLE 1A as follows: Y Tyr Tyrosine G Gly Glycine TABLE 1A Phe Phenylalanine M Met Methionine Exemplary A. Ala Alanine Original conservative S Ser Serine 50 residue Substitution Ile Isoleucine Leu Leucine Ala (A) Gly: Ser Thr Threonine Arg (R) Lys V Wall Valine ASn (N) Gln: His C Pro proline Cys (C) Ser K Lys Lysine 55 Gln (Q) ASn H His Histidine Glu (E) Asp Q Gln Glutamine Gly (G) Ala: Pro E Glu glutamic acid His (H) ASn; Glin Z. Glx Glu and for Glin Ile (I) Leu; Val W Trp Tryptophan Leu (L) Ile: Val R Arg Arginine Lys (K) Arg: Gln: Glu D Asp aspartic acid 60 Met (M) Leu: Tyr; Ile N ASn asparagine Phe (F) Met; Leu: Tyr B ASX ASn and/or Asp Ser (S) Thr C Cys Cysteine Thr (T) Ser X Xaa Unknown or other Trp (W) Tyr Tyr (Y) Trp; Phe 65 Val (V) Ile: Leu It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation US 9,084,743 B2 13 14 Other substitutions also are permissible and can be deter ods to measure identity between two polynucleotide or mined empirically or in accord with known conservative Sub polypeptides, the term “identity” is well known to skilled stitutions. artisans (Carillo, H. & Lipton, D., SIAM J Applied Math As used herein, a DNA construct is a single or double 48: 1073 (1988)). Stranded, linear or circular DNA molecule that contains seg As used herein, homologous (with respect to nucleic acid ments of DNA combined and juxtaposed in a manner not and/or amino acid sequences) means about greater than or found in nature. DNA constructs exist as a result of human equal to 25% sequence homology, typically greater than or manipulation, and include clones and other copies of manipu equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% lated molecules. sequence homology; the precise percentage can be specified As used herein, a DNA segment is a portion of a larger 10 DNA molecule having specified attributes. For example, a if necessary. For purposes herein the terms "homology' and DNA segment encoding a specified polypeptide is a portion “identity” are often used interchangeably, unless otherwise of a longer DNA molecule. Such as a plasmid or plasmid indicated. In general, for determination of the percentage fragment, which, when read from the 5' to 3' direction, homology or identity, sequences are aligned so that the high encodes the sequence of amino acids of the specified 15 est order match is obtained (see, e.g.: Computational Molecu polypeptide. lar Biology, Lesk, A. M., ed., Oxford University Press, New As used herein, the term polynucleotide means a single- or York, 1988: Biocomputing: Informatics and Genome double-stranded polymer of deoxyribonucleotides or ribo Projects, Smith, D. W., ed., Academic Press, New York, 1993; nucleotide bases read from the 5' to the 3' end. Polynucle Computer Analysis of Sequence Data, Part I, Griffin, A. M., otides include RNA and DNA, and can be isolated from and Griffin, H. G., eds., Humana Press, New Jersey, 1994: natural Sources, synthesized in vitro, or prepared from a com Sequence Analysis in Molecular Biology, Von Heinje, G., bination of natural and synthetic molecules. The length of a Academic Press, 1987; and Sequence Analysis Primer, Grib polynucleotide molecule is given herein in terms of nucle skov, M. and Devereux, J., eds., MStockton Press, New York, otides (abbreviated “nt') or base pairs (abbreviated “bp'). 1991; Carillo et al. (1988)SIAMJApplied Math 48:1073). By The term nucleotides is used for single- and double-stranded 25 sequence homology, the number of conserved amino acids is molecules where the context permits. When the term is determined by Standard alignment algorithms programs, and applied to double-stranded molecules it is used to denote can be used with default gap penalties established by each overall length and will be understood to be equivalent to the Supplier. Substantially homologous nucleic acid molecules term base pairs. It will be recognized by those skilled in the art would hybridize typically at moderate stringency or at high that the two strands of a double-stranded polynucleotide can 30 stringency all along the length of the nucleic acid of interest. differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded poly Also contemplated are nucleic acid molecules that contain nucleotide molecule can not be paired. Such unpaired ends degenerate codons in place of codons in the hybridizing will, in general, not exceed 20 nucleotides in length. nucleic acid molecule. As used herein, “similarity” between two proteins or 35 Whether any two molecules have nucleotide sequences or nucleic acids refers to the relatedness between the sequence amino acid sequences that are at least 60%, 70%. 80%, 85%, of amino acids of the proteins or the nucleotide sequences of 90%. 95%, 96%, 97%, 98% or 99% “identical or “homolo the nucleic acids. Similarity can be based on the degree of gous' can be determined using known computer algorithms identity and/or homology of sequences of residues and the such as the “FASTA” program, using for example, the default residues contained therein. Methods for assessing the degree 40 parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. of similarity between proteins or nucleic acids are known to USA 85:2444 (other programs include the GCG program those of skill in the art. For example, in one method of assess package (Devereux, J., et al., Nucleic Acids Research 12(I): ing sequence similarity, two amino acid or nucleotide 387 (1984)). BLASTP, BLASTN, FASTA (Atschul, S. F., et sequences are aligned in a manner that yields a maximal level al., J Molec Biol 215:403 (1990)); Guide to Huge Computers, of identity between the sequences. “Identity” refers to the 45 Martin J. Bishop, ed., Academic Press, San Diego, 1994, and extent to which the amino acid or nucleotide sequences are Carillo et al. (1988) SIAM J Applied Math 48: 1073). For invariant. Alignment of amino acid sequences, and to some example, the BLAST function of the National Center for extent nucleotide sequences, also can take into account con Biotechnology Information database can be used to deter servative differences and/or frequent Substitutions in amino mine identity. Other commercially or publicly available pro acids (or nucleotides). Conservative differences are those that 50 grams include, DNAStar “MegAlign' program (Madison, preserve the physico-chemical properties of the residues Wis.) and the University of Wisconsin Genetics Computer involved. Alignments can be global (alignment of the com Group (UWG) “Gap' program (Madison Wis.). Percent pared sequences over the entire length of the sequences and homology or identity of proteins and/or nucleic acid mol including all residues) or local (the alignment of a portion of ecules can be determined, for example, by comparing the sequences that includes only the most similar region or 55 sequence information using a GAP computer program (e.g., regions). Needleman et al. (1970).J. Mol. Biol. 48:443, as revised by “Identity perse has an art-recognized meaning and can be Smith and Waterman ((1981) Adv. Appl. Math. 2:482). calculated using published techniques. (See, e.g.: Computa Briefly, the GAP program defines similarity as the number of tional Molecular Biology, Lesk, A. M., ed., Oxford Univer aligned symbols (i.e., nucleotides or amino acids), which are sity Press, New York, 1988: Biocomputing. Informatics and 60 similar, divided by the total number of symbols in the shorter Genome Projects, Smith, D. W., ed., Academic Press, New of the two sequences. Default parameters for the GAP pro York, 1993: Computer Analysis of Sequence Data, Part I, gram can include: (1) a unary comparison matrix (containing Griffin, A. M., and Griffin, H. G., eds., Humana Press, New a value of 1 for identities and 0 for non-identities) and the Jersey, 1994; Sequence Analysis in Molecular Biology, Von weighted comparison matrix of Gribskov et al. (1986) Nucl. Heinje, G., Academic Press, 1987; and Sequence Analysis 65 Acids Res. 14:6745, as described by Schwartz and Dayhoff, Primer, Gribskov, M. and Devereux, J., eds., M. Stockton eds., ATLAS OF PROTEINSEQUENCE AND STRUCTURE, Press, New York, 1991). While there exists a number of meth National Biomedical Research Foundation, pp. 353-358 US 9,084,743 B2 15 16 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 person can readily adjust these parameters to achieve specific penalty for each symbol in each gap; and (3) no penalty for hybridization of a nucleic acid molecule to a target nucleic end gaps. acid molecule appropriate for a particular application. Therefore, as used herein, the term “identity” or “homol Complementary, when referring to two nucleotide sequences, ogy” represents a comparison between a test and a reference means that the two sequences of nucleotides are capable of polypeptide or polynucleotide. As used herein, the term at hybridizing, typically with less than 25%, 15% or 5% mis least"90% identical to refers to percent identities from 90 to matches between opposed nucleotides. If necessary, the per 99.99 relative to the reference nucleic acid or amino acid centage of complementarity will be specified. Typically the sequence of the polypeptide. Identity at a level of 90% or two molecules are selected such that they will hybridize under more is indicative of the fact that, assuming for exemplifica 10 conditions of high stringency. tion purposes a test and reference polypeptide length of 100 As used herein, Substantially identical to a product means amino acids are compared. No more than 10% (i.e., 10 out of sufficiently similar so that the property of interest is suffi 100) of the amino acids in the test polypeptide differs from ciently unchanged so that the Substantially identical product that of the reference polypeptide. Similar comparisons can be can be used in place of the product. made between test and reference polynucleotides. Such dif 15 As used herein, it also is understood that the terms "sub ferences can be represented as point mutations randomly stantially identical' or “similar varies with the context as distributed over the entire length of a polypeptide or they can understood by those skilled in the relevant art. be clustered in one or more locations of varying length up to As used herein, an allelic variant or allelic variation refer the maximum allowable, e.g. 10/100 amino acid difference ences any of two or more alternative forms of a gene occu (approximately 90% identity). Differences are defined as pying the same chromosomal locus. Allelic variation arises nucleic acid or amino acid substitutions, insertions or dele naturally through mutation, and can result in phenotypic tions. At the level of homologies or identities above about polymorphism within populations. Gene mutations can be 85-90%, the result should be independent of the program and silent (no change in the encoded polypeptide) mean encode gap parameters set; such high levels of identity can be polypeptides having altered amino acid sequence. The term assessed readily, often by manual alignment without relying 25 “allelic variant also is used herein to denote a protein on Software. encoded by an allelic variant of a gene. Typically the refer As used herein, an aligned sequence refers to the use of ence form of the gene encodes a wildtype form and/or pre homology (similarity and/or identity) to align corresponding dominant form of a polypeptide from a population or single positions in a sequence of nucleotides or amino acids. Typi reference member of a species. Typically, allelic variants, cally, two or more sequences that are related by 50% or more 30 which include variants between and among species typically identity are aligned. An aligned set of sequences refers to 2 or have at least 80%, 90% or greater amino acid identity with a more sequences that are aligned at corresponding positions wildtype and/or predominantform from the same species; the and can include aligning sequences derived from RNAS, Such degree of identity depends upon the gene and whether com as ESTs and other cDNAs, aligned with genomic DNA parison is interspecies or intraspecies. Generally, intraspecies Sequence. 35 allelic variants have at least about 80%, 85%, 90% or 95% As used herein, “primer' refers to a nucleic acid molecule identity or greater with a wildtype and/or predominant form, that can act as a point of initiation of template-directed DNA including 96%, 97%, 98%, 99% or greater identity with a synthesis under appropriate conditions (e.g., in the presence wildtype and/or predominant form of a polypeptide. Refer of four different nucleoside triphosphates and a polymeriza ence to an allelic variant herein generally refers to variations tion agent, such as DNA polymerase, RNA polymerase or 40 in proteins among members of the same species. reverse transcriptase) in an appropriate buffer and at a suitable As used herein, “allele,” which is used interchangeably temperature. It will be appreciated that a certain nucleic acid herein with “allelic variant” refers to alternative forms of a molecules can serve as a “probe' and as a “primer.” A primer, gene or portions thereof. Alleles occupy the same locus or however, has a 3' hydroxyl group for extension. A primer can position on homologous chromosomes. When a Subject has be used in a variety of methods, including, for example, 45 two identical alleles of a gene, the subject is said to be polymerase chain reaction (PCR), reverse-transcriptase (RT)- homozygous for that gene or allele. When a Subject has two PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, cap different alleles of a gene, the subject is said to be heterozy ture PCR, expression PCR, 3' and 5' RACE, in situ PCR, gous for the gene. Alleles of a specific gene can differ from ligation-mediated PCR and other amplification protocols. each other in a single nucleotide or several nucleotides, and As used herein, “primer pair refers to a set of primers that 50 can include Substitutions, deletions and insertions of nucle includes a 5' (upstream) primer that hybridizes with the 5' end otides. An allele of a gene also can be a form of a gene of a sequence to be amplified (e.g. by PCR) and a 3’ (down containing a mutation. stream) primer that hybridizes with the complement of the 3' As used herein, species variants refer to variants in end of the sequence to be amplified. polypeptides among different species, including different As used herein, “specifically hybridizes” refers to anneal 55 mammalian species, such as mouse and human. ing, by complementary base-pairing, of a nucleic acid mol As used herein, a splice variant refers to a variant produced ecule (e.g. an oligonucleotide) to a target nucleic acid mol by differential processing of a primary transcript of genomic ecule. Those of skill in the art are familiar with in vitro and in DNA that results in more than one type of mRNA. Vivo parameters that affect specific hybridization, such as As used herein, the term promoter means a portion of a length and composition of the particular molecule. Param 60 gene containing DNA sequences that provide for the binding eters particularly relevant to in vitro hybridization further of RNA polymerase and initiation of transcription. Promoter include annealing and washing temperature, buffer composi sequences are commonly, but not always, found in the 5' tion and salt concentration. Exemplary washing conditions non-coding region of genes. for removing non-specifically bound nucleic acid molecules As used herein, isolated or purified polypeptide or protein at high stringency are 0.1xSSPE, 0.1% SDS, 65° C., and at 65 or biologically-active portion thereof is substantially free of medium stringency are 0.2xSSPE, 0.1% SDS, 50° C. Equiva cellular material or other contaminating proteins from the cell lent stringency conditions are known in the art. The skilled or tissue from which the protein is derived, or substantially US 9,084,743 B2 17 18 free from chemical precursors or other chemicals when tion into an appropriate host cell, results in expression of the chemically synthesized. Preparations can be determined to be cloned DNA. Appropriate expression vectors are well known substantially free if they appear free of readily detectable to those of skill in the art and include those that are replicable impurities as determined by standard methods of analysis, in eukaryotic cells and/or prokaryotic cells and those that Such as thin layer chromatography (TLC), gel electrophoresis remain episomal or those which integrate into the host cell and high performance liquid chromatography (HPLC), used genome. by those of skill in the art to assess such purity, or sufficiently As used herein, vector also includes “virus vectors' or pure such that further purification would not detectably alter “viral vectors.” Viral vectors are engineered viruses that are the physical and chemical properties, such as enzymatic and operatively linked to exogenous genes to transfer (as vehicles biological activities, of the substance. Methods for purifica 10 tion of the compounds to produce Substantially chemically or shuttles) the exogenous genes into cells. pure compounds are known to those of skill in the art. A As used herein, operably or operatively linked when refer Substantially chemically pure compound, however, can be a ring to DNA segments means that the segments are arranged mixture of stereoisomers. In Such instances, further purifica so that they function in concert for their intended purposes, tion might increase the specific activity of the compound. 15 e.g., transcription initiates in the promoter and proceeds The term substantially free of cellular material includes through the coding segment to the terminator. preparations of proteins in which the protein is separated As used herein the term assessing is intended to include from cellular components of the cells from which it is isolated quantitative and qualitative determination in the sense of or recombinantly-produced. In one embodiment, the term obtaining an absolute value for the activity of a protein, Such substantially free of cellular material includes preparations of as an enzyme or protease, or a domain thereof, present in the enzyme proteins having less that about 30% (by dry weight) sample, and also of obtaining an index, ratio, percentage, of non-enzyme proteins (also referred to herein as a contami visual or other value indicative of the level of the activity. nating protein), generally less than about 20% of non-enzyme Assessment can be director indirect and the chemical species proteins or 10% of non-enzyme proteins or less that about 5% actually detected need not of course be the endproduct of a of non-enzyme proteins. When the enzyme protein is recom 25 reaction, such as a proteolysis product itself, but can for binantly produced, it also is substantially free of culture example be a derivative thereof or some further substance. medium, i.e., culture medium represents less than about or at For example, assessment can be detection of a cleavage prod 20%, 10% or 5% of the volume of the enzyme protein prepa uct of a protein, such as by SDS-PAGE and protein staining ration. with Coomasie blue. As used herein, the term substantially free of chemical 30 As used herein, biological activity refers to the in vivo precursors or other chemicals includes preparations of activities of a compound or physiological responses that enzyme proteins in which the protein is separated from result upon in vivo administration of a compound, composi chemical precursors or other chemicals that are involved in tion or other mixture. Biological activity, thus, encompasses the synthesis of the protein. The term includes preparations of therapeutic effects and pharmaceutical activity of Such com enzyme proteins having less than about 30% (by dry weight) 35 pounds, compositions and mixtures. Biological activities can 20%, 10%, 5% or less of chemical precursors or non-enzyme be observed in in vitro systems designed to test or use Such chemicals or components. activities. Thus, for purposes herein a biological activity of a As used herein, synthetic, with reference to, for example, a protease is its catalytic activity in which a polypeptide is synthetic nucleic acid molecule or a synthetic gene or a syn hydrolyzed. thetic peptide refers to a nucleic acid molecule or polypeptide 40 As used herein equivalent, when referring to two sequences molecule that is produced by recombinant methods and/or by of nucleic acids, means that the two sequences in question chemical synthesis methods. encode the same sequence of amino acids or equivalent pro As used herein, production by recombinant means by using teins. When equivalent is used in referring to two proteins or recombinant DNA methods means the use of the well known peptides, it means that the two proteins or peptides have methods of molecular biology for expressing proteins 45 Substantially the same amino acid sequence with only amino encoded by cloned DNA. acid substitutions that do not substantially alter the activity or As used herein, vector (or plasmid) refers to discrete ele function of the protein or peptide. When equivalent refers to ments that are used to introduce a heterologous nucleic acid a property, the property does not need to be present to the into cells for either expression or replication thereof. The same extent (e.g., two peptides can exhibit different rates of vectors typically remain episomal, but can be designed to 50 the same type of enzymatic activity), but the activities are effect integration of a gene or portion thereof into a chromo usually substantially the same. Some of the genome. Also contemplated are vectors that are As used herein, "modulate” and “modulation' or “alter' artificial chromosomes. Such as yeast artificial chromosomes refer to a change of an activity of a molecule. Such as a and mammalian artificial chromosomes. Selection and use of protein. Exemplary activities include, but are not limited to, such vehicles are well known to those of skill in the art. 55 biological activities, such as signal transduction. Modulation As used herein, an expression vector includes vectors can include an increase in the activity (i.e., up-regulation or capable of expressing DNA that is operatively linked with agonist activity) a decrease in activity (i.e., down-regulation regulatory sequences, such as promoter regions, that are or inhibition) or any other alteration in an activity (such as a capable of effecting expression of such DNA fragments. Such change in periodicity, frequency, duration, kinetics or other additional segments can include promoter and terminator 60 parameter). Modulation can be context dependent and typi sequences, and optionally can include one or more origins of cally modulation is compared to a designated State, for replication, one or more selectable markers, an enhancer, a example, the wildtype protein, the protein in a constitutive polyadenylation signal, and the like. Expression vectors are state, or the protein as expressed in a designated cell type or generally derived from plasmid or viral DNA, or can contain condition. elements of both. Thus, an expression vector refers to a 65 As used herein, a composition refers to any mixture. It can recombinant DNA or RNA construct, such as a plasmid, a be a solution, Suspension, liquid, powder, paste, aqueous, phage, recombinant virus or other vector that, upon introduc non-aqueous or any combination thereof. US 9,084,743 B2 19 20 As used herein, a combination refers to any association As used throughout this application, the term is intended to between or among two or more items. The combination can encompass IG and hyaluronidase compositions contained in be two or more separate items, such as two compositions or articles of packaging. two collections, can be a mixture thereof. Such as a single As used herein, fluid refers to any composition that can mixture of the two or more items, or any variation thereof. flow. Fluids thus encompass compositions that are in the form The elements of a combination are generally functionally of semi-solids, pastes, solutions, aqueous mixtures, gels, associated or related. lotions, creams and other Such compositions. As used herein, a kit is a packaged combination that option As used herein, a “kit' refers to a combination of compo ally includes other elements, such as additional reagents and sitions provided herein and another item for a purpose includ instructions for use of the combination or elements thereof. 10 ing, but not limited to, activation, administration, diagnosis, As used herein, “disease or disorder” refers to a pathologi and assessment of a biological activity or property. Kits cal condition in an organism resulting from cause or condition optionally include instructions for use. including, but not limited to, infections, acquired conditions, As used herein, a cellular extract or lysate refers to a prepa genetic conditions, and characterized by identifiable symp ration or fraction which is made from alysed or disrupted cell. toms. Diseases and disorders of interest herein are those that 15 As used herein, animal includes any animal. Such as, but are treatable by immune globulin. are not limited to primates including humans, gorillas and As used herein, “treating a subject with a disease or con monkeys; rodents, such as mice and rats; fowl. Such as chick dition means that the Subjects symptoms are partially or ens; ruminants, such as goats, cows, deer, sheep; ovine. Such totally alleviated, or remain static following treatment. Hence as pigs and other animals. Non-human animals exclude treatment encompasses prophylaxis, therapy and/or cure. humans as the contemplated animal. The enzymes provided Prophylaxis refers to prevention of a potential disease and/or herein are from any source, animal, plant, prokaryotic and a prevention of worsening of symptoms or progression of a fungal. Most enzymes are of animal origin, including mam disease. Treatment also encompasses any pharmaceutical use malian origin. of an immune globulin preparation and compositions pro As used herein, a control refers to a sample that is substan vided herein. 25 tially identical to the test sample, except that it is not treated As used herein, a pharmaceutically effective agent, with a test parameter, or, if it is a plasma sample, it can be from includes any therapeutic agent or bioactive agents, including, a normal volunteer not affected with the condition of interest. but not limited to, for example, anesthetics, vasoconstrictors, A control also can be an internal control. dispersing agents, conventional therapeutic drugs, including As used herein, the singular forms “a,” “an and “the Small molecule drugs and therapeutic proteins. 30 include plural referents unless the context clearly dictates As used herein, treatment means any manner in which the otherwise. Thus, for example, reference to a compound, com symptoms of a condition, disorder or disease or other indica prising “an extracellular domain” includes compounds with tion, are ameliorated or otherwise beneficially altered. one or a plurality of extracellular domains. As used herein therapeutic effect means an effect resulting As used herein, ranges and amounts can be expressed as from treatment of a Subject that alters, typically improves or 35 “about a particular value or range. About also includes the ameliorates the symptoms of a disease or condition or that exact amount. Hence “about 5 bases’ means “about 5 bases' cures a disease or condition. A therapeutically effective and also “5 bases.” amount refers to the amount of a composition, molecule or As used herein, “optional' or “optionally’ means that the compound which results in a therapeutic effect following Subsequently described event or circumstance does or does administration to a subject. 40 not occur, and that the description includes instances where As used herein, the term “subject” refers to an animal, said event or circumstance occurs and instances where it does including a mammal. Such as a human being. not. For example, an optionally substituted group means that As used herein, a patient refers to a human Subject. the group is unsubstituted or is Substituted. As used herein, amelioration of the symptoms of a particu As used herein, the abbreviations for any protective groups, lar disease or disorder by a treatment, such as by administra 45 amino acids and other compounds, are, unless indicated oth tion of a pharmaceutical composition or other therapeutic, erwise, in accord with their common usage, recognized refers to any lessening, whether permanent or temporary, abbreviations, or the IUPAC-IUB Commission on Biochemi lasting or transient, of the symptoms that can be attributed to cal Nomenclature (see, (1972) Biochem. 11:1726). or associated with administration of the composition orthera B. STABLE CO-FORMULATIONS OF IMMUNE peutic. 50 GLOBULIN (IG) ANDHYALURONIDASE As used herein, prevention or prophylaxis refers to meth Provided herein are stable co-formulations containing ods in which the risk of developing disease or condition is immune globulin (IG) and hyaluronidase. The co-formula reduced. tions retain IG molecular size distribution and hyaluronidase As used herein, a “therapeutically effective amount’ or a activity after extended storage in liquid form at room tem “therapeutically effective dose” refers to the quantity of an 55 perature (e.g. 28 to 32°C.) for at least six months. Generally, agent, compound, material, or composition containing a com the co-formulations also retain IG molecular size distribution pound that is at least Sufficient to produce atherapeutic effect. and hyaluronidase activity at standard refrigerator tempera Hence, it is the quantity necessary for preventing, curing, tures for at least 1-2 years. The co-formulations can be used ameliorating, arresting or partially arresting a symptom of a for treating IG-treatable diseases and conditions. In particu disease or disorder. 60 lar, the stable co-formulations provided herein are formulated As used herein, unit dose form refers to physically discrete for Subcutaneous administration. units suitable for human and animal Subjects and packaged 1. Immune Globulin Therapy individually as is known in the art. Immune globulin is a therapeutic that is primarily given to As used herein, a single dosage formulation refers to a treat individuals with immune deficiencies. Immunoglobulin formulation for direct administration. 65 deficiency disorders are a Subset of immunodeficiency dis As used herein, an “article of manufacture' is a product eases characterized by missing or reduced levels of serum that is made and sold. immunoglobulins, leading to increased Susceptibility to bac US 9,084,743 B2 21 22 terial infections, especially of the Sinopulmonary tract. preparations cannot be infused intravenously. Such subcuta Immunodeficiency diseases are either primary (genetic) or neous methods of immunoglobulin replacement therapy are secondary (acquired). Primary immunodeficiency diseases considered to be effective, safe and also highly appreciated by are rare and include X-linked agammaglobulinemia, immu patients, as it has a low risk of systemic adverse reactions and noglobulin heavy chain deletion, selective immunoglobulin 5 leads to higher trough serum IgG concentrations compared to G (IgG) Subclass deficiency, common variable immunodefi monthly IV infusions (Gardulf et al. (1995) J. Adv. Nurs., ciency, or X-linked hyperimmunoglobulin M syndrome. 21:917-27; Gardulfetal. (1993) Clin. Exp. Immunol.,92:200 Decreased immunoglobulin levels also are found in individu 4; Gardulfetal. (1991) Lancet, 338:162-6). als having combined immunodeficiencies due to defects in T In addition to the decreased bioavailability associated with and B cells, such as, but not limited to, severe combined 10 Subcutaneous administration of IG, another distinction immunodeficiency or Wiskott Aldrich Syndrome (IUIS Sci between SC and IV administration is that only small volumes entific Committee, 1999). More common are secondary can be infused subcutaneously at each site, necessitating the immunodeficiencies, induced by factors including, but not use of multiple sites on a weekly or biweekly (ever other limited to, malnutrition, viruses, aging and leukemia. Indi week) basis. In general, however, adults can only be infused viduals with these diseases require replacement therapy with 15 with 20-40 mL at a single subcutaneous site, with lower immunoglobulin products to prevent or reduce the severity of volumes per site for children. Currently, the accepted practice infections. for IG administration is 300-600 mg/kg intravenously once Immunoglobulin replacement therapy was first used in every 3-4 weeks or 100-200 mg/kg/wk subcutaneously 1952 and was administered intramuscularly and Subcutane (Berger (2008) Immunol. Allergy Clin. North Am. 28(2):413 ously. However, to effectively treat disease, larger amounts of 438). Thus, up to 15 g of IG is administered per week subcu IG are necessary, which led to the development of intrave taneously. This means that administration of a 16-20% IG nously administrable products with lower IG concentrations preparation at least 3 sites per week is required. Even though (50-100 mg/mL). Since 1981, the majority of immunoglobu weekly or biweekly administration has the added advantage lin products available in the United States are administered of maintaining better trough levels than monthly IV infu intravenously. Generally, IG preparations are sterile, purified 25 sions, the requirement of multiple needle insertions has been products that contain immunoglobulin G (IgG, IgM, IgA or a a deterrent for many patients. combination of those). Typically, IG products contain Nevertheless, subcutaneous methods of immunoglobulin 95-99% IgG and only trace amounts of immunoglobulins A replacement therapy are becoming an increasingly popular (IgA), M (IgM), D (Ig)) and E (IgE). IG preparations for IV alternative to IVIG therapy. Patients having severe reactions administration are generally formulated at 3 to 12% IG. 30 to IVIG infusions can often tolerate subcutaneously admin More recently, immunoglobulin preparations have been istered IG. Subcutaneous administration is considered to be developed for subcutaneous administration (Gardulf et al. effective, safe and also highly appreciated by patients, as it (2006) Curr. Opin. Allergy Clin. Immunol. 6: 434–42; Gardulf has a low risk of systemic adverse reactions and can be et al. (2006).J. Clin. Immunol. 26: 177-85; Ochs et al. (2006) administered at home or in the hospital (Gardulfetal. (1995) J. Clin. Immunol. 26:265-73), and at least one product, Viva 35 J Adv. Nurs. 21: 917-27; Gardulf et al. (1993) Clin. Exp. globin R, is licensed for Subcutaneous administration in the Immunol.92: 200-4: Gardulfetal. (1991) Lancet 338: 162-6). United States. A subcutaneous route of administration of IG 2. Subcutaneous Administration of Immune Globulin and has several advantages compared to the IV route Such as Hyaluronidase Formulations better tolerability and the possibility of home care treatment. The bioavailability of subcutaneously administered IG is The bioavailability of immunoglobulin administered sub 40 increased in combination with hyaluronidase administration, cutaneously generally is less than that infused intravenously. thereby permitting Subcutaneous administration of immune Following IV administration, immunoglobulin is immedi globulin at dosages and frequencies similar to IVIG treatment ately available in the blood, and slowly equilibrates to the (see e.g. U.S. Patent Application No. 2010-0074885 and extra-vascular compartment over 3 to 5 days (Schiff et al. International PCT No. WO 2009-117085, each incorporated (1986) J. Clin. Immunol. 6:256-64). Subcutaneously admin 45 by reference herein). The subcutaneous (SC) space, formed istered immunoglobulin is slowly absorbed from the subcu by a collagen network filled with hyaluronic acid, a gel-like taneous space into the blood and at the same time equilibrates Substance, is largely responsible for the resistance to fluid with the extra-vascular compartment; there is no high IV flow through the tissues. Hyaluronidase is a family of natu spike. The bioavailability has not been extensively studied, rally occurring enzymes that break down hyaluronic acid, but in a recent trial of the ZLB-Behring preparation (i.e., 50 which is a space-filling 'gel-like substance found in the Vivaglobin R), it was determined by measuring the area under extracellular matrix and in tissues throughout the body Such the curve (AUC) that only 67% of the immunoglobulin was as the skin and eye. Hyaluronidase acts by splitting the glu absorbed, and thus, the recommended dose was 137% of the cosaminidic bond in hyaluronic acid between the C of an IV dose (Ochs et al. (2006) J. Clin. Immunol. 26:265-73). N-acetylglucosamine moiety and C of a glucuronic moiety. Despite the technical difficulties of comparing the AUC for 55 This temporarily decreases the viscosity of the cellular two different routes and frequency of administration, studies cement and promotes diffusion of injected fluids, thus facili of intradermally administered immunoglobulin in rabbits tating their absorption. Afterwards, hyaluronic acid is regen Suggests there is decreased bioavailability through the Sub erated naturally within 24 hours. Accordingly, the bioavail cutaneous route. This may be due to the mode of absorption of ability, pharmacokinetics and/or pharmacodynamic large protein molecules, which cannot readily diffuse through 60 characteristics of co-formulations containing hyaluronidase the capillary walls and must be absorbed via the lymphatics are improved. Based on experiments in animals, the increased (Supersaxo et al. (1990) Pharm. Res. 7:167-9). fluid dispersion permits administration of up to 1 L per hour All of the immunoglobulin preparations presently used for via the subcutaneous route, which is an IV-like flow rate. subcutaneous administration are formulated at 16% IG, com In the presence of hyaluronidase, the bioavailability of pared to IVIG preparations formulated at 5 to 12% IG. The 65 Subcutaneously administered IG is increased, typically to higher concentration of IG in Subcutaneous preparations rela more than 90% of the bioavailability of IG following IVIG tive to IV preparations allows smaller infusion volumes; such treatment. Further, co-administration with a soluble hyalu US 9,084,743 B2 23 24 ronidase permits infusion of large Volumes at a single Subcu Generally, the stable co-formulation is a liquid formula taneous site. For example, volumes up to 600 mL or greater of tion. Storage of the co-formulation directly in a liquid form IG can be administered at a single site in a single sitting, for takes advantage of the convenience of having storage stability example 200 mL,300 mL 400 mL, 500 mL, 600 mL or more in the liquid form, ease of administration without reconstitu can be administered at a single site in a single administration. 5 tion, and ability to supply the formulation in prefilled, ready For example, an IG preparation formulated at or between to-use Syringes or as multidose preparations. Hence the liquid 5-12%, for example at 10% protein, which typically are used co-formulations provide a ready-to-use preparation of IG and only for IVIG therapy can be co-administered subcutane hyaluronidase for Subcutaneous administration to a subject ously with a soluble hyaluronidase at dosages equivalent to without having to reconstitute the preparation accurately and 10 aseptically and waiting for a period of time until the Solution once monthly IVIG doses, for example, at or about 100 clarifies before administering the formulation to the subject. mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 It simplifies the procedure of administering the formulation to mg/kg or more. IG preparations at higher concentrations of a Subject for a healthcare professional. In addition, the manu protein, for example, 12-25% IG such as 15%, 16%, 17%, facturing process of the liquid formulations is simplified and 18%, 19%, 20%, 21%, 22% or more also can be administered 1 5 more efficient than the manufacturing process for the lyo Subcutaneously in the presence of hyaluronidase. The dos philized version because all stages of the manufacturing of ages can be administered as a single dose or can be divided the liquid formulations are carried out in an aqueous solution, into multiple doses given daily or weekly, such as once a week involving no drying process. Such as lyophilization and or every two, three or four weeks or combinations thereof. freeze-drying. Accordingly, it is more cost effective as well. Thus, IG, when administered Subcutaneously in the presence 20 The stable co-formulation can be provided as a liquid solution of hyaluronidase, can be administered once monthly at pre in a container or Syringe. Such a co-formulation can be con vailing IVIG doses for the particular indication. Further, Veniently dispensed to humans or other mammalian species because hyaluronidase acts to open flow channels in the skin, as a pharmaceutical without further re-constitution by the it can speed infusion rates. Hence, Subcutaneously adminis physician or patient. tering IG administered with hyaluronidase increases infusion 25 Furthermore, due to its high stability during the storage, the rates and thereby decreases time of delivery of IG therapy. co-formulations can contain high protein concentrations in By administering IG. Subcutaneously in the presence of a the range of about 10% to 22% IG, such as 10% to 20% IG hyaluronidase, one or all of the considerations and problems without causing an adverse effect on the biological activity associated with Subcutaneous administration of IG are (ies) of IG due to protein aggregation and/or fragmentation addressed. Thus, by virtue of the dispersion properties of 30 during a prolonged storage. Such stability not only ensures hyaluronidase, Subcutaneously administering IG in the pres the efficacy of the IG co-formulation, but also reduces pos ence of a soluble hyaluronidase permits administration of sible risks of causing adverse effects on a subject. Hence, the IVIG doses at once monthly IVIG frequencies, while main stable co-formulations provided herein retain hyaluronidase taining IVIG bioavailability. enzymatic activity and IG activity while minimizing IG self 3. Stable Co-Formulations 35 association and aggregation. Generally, the activity is Since Subcutaneously administrable immune globulin retained at a temperature that is up to 32°C., for example at or preparations have the advantages of home-care treatment, a about 0°C. to 32° C., generally at or about 28°C. to 32° C. stable, ready-for-use preparation of IG and hyaluronidase is The stability of the co-formulation is maintained over pro contemplated. Proteins used for therapy are typically sub longed periods of time, for example, daily, weekly, monthly, jected to a range of conditions during processing and storage, 40 yearly or more. The co-formulations have the advantage that including low pH, fluctuations in temperature, various buffer they are stable in liquid form during storage for prolonged components and ionic strengths, and, often, high protein con periods of time of at least 6 months. In one example, the stable centration in the final preparation. To be effective, however, co-formulations are stable in liquid for at least 1 year or the co-formulation should retain sufficient activity of the IG longer, for example, 1 year to 2 years, such as 1 year, 2 years, and hyaluronidase. Thus, a co-formulation of IG and hyalu 45 or more at Standard refrigerator temperatures (approximately ronidase must be provided as a stable solution for storage as 4+2°C., or about 2-8°C., or, more generally, ranging from an aqueous Solution without deteriorating for prolonged peri about 0-10°C.). In another example, the co-formulations are ods of time. Hence, provided herein is a stable liquid co stable in liquid form during storage at room temperature (in formulation of IG and hyaluronidase. The co-formulation is the range of 18-32° C., for example, 28°C. to 32° C.) for at Such that it is provided as a dosage form that can be used for 50 least six months. For example, the stable co-formulations direct injection, i.e. not diluted before use. generally have a shelf-life of at least or about 6 months to 18 It was found herein that a co-formulated product prepared months, for example 6 months, 12 months, 18 months, or by the addition of a hyaluronidase designated rHuPH20 to a more when stored at room temperature. preparation of IG before administration was not stable at The following sections describe the formulations provided room temperature. The addition of salt improves the stability 55 herein, including exemplary immunoglobulins and hyalu of the formulation, in particular, by maintaining the activity of ronidases in the formulations, methods of making them, and the hyaluronidase in the formulation. Thus, in addition to methods of using the stable co-formulations to treat IG-treat containing an effective amount of IG and hyaluronidase, the able diseases and conditions. stable co-formulations provided herein also contain at least C. IMMUNE GLOBULIN AND PREPARATION OF 50 mM of an alkali metal chloride salt, for example, NaCl or 60 IMMUNE GLOBULIN KCl. Typically, the stable co-formulations also contain an Provided herein are immune globulins (IG, also referred to amino acid, for example glycine, as a stabilizer and are pro as immunoglobulin, gamma globulin or IgG) that can be vided at a pH of about or at 4 to 5. In general, the ratio of formulated in stable compositions with hyaluronidase. The hyaluronidase to IG in a co-formulated product is greater than stable co-formulations can be used for use in treating IG the ratio when the same products (IG and hyaluronidase) and 65 treatable diseases and conditions. the same amount of IG are Subcutaneously administered Immunoglobulins are gamma globulin proteins produced separately, for example, in a leading edge administration. by the humoral immune system and found in the plasma of US 9,084,743 B2 25 26 higher animals. IG acts to strengthen the immune system by a. Cohn-Oncley Method modulating the activity of complement, Suppressing autoan Conventional industrial methods of immune globulin puri tibody production, Saturating or blocking Fc receptors on fication from blood plasma are based on cold ethanol frac macrophages and B lymphocytes, and Suppressing the pro tionation, which co-precipitates groups of proteins based on duction of inflammatory mediators such as cytokines, their isoelectric points at given alcohol concentrations at Sub chemokines and metalloproteinases. IG is composed of five Zero temperatures, originally employed by Cohn and modi classes, or isotypes, of antibodies (IgG, IgA, IgM, Ig|D and fied by Oncley (see, e.g., Cohn et al. (1946).J. Am. Chem. Soc. IgE) and various Subclasses, each with varying specificities. 68:459-75: Oncley et al. (1949).J. Am. Chem. Soc. 71:541 IgG is the most predominate class of IG found in the blood 50). The use of alcohol in the purification process can inac 10 tivate potentially contaminating viruses, however, with and is important in secondary immune responses and protect increasing temperature and alcohol concentration, the Cohn ing tissues against infection. Table 2 illustrates typical Oncley method can result in denatured and aggregated pro amounts of immunoglobulins found in the serum, although teins. These high molecular weight forms can act as antibody preparations of IG for treatment can employ purification steps antigen complexes having the capacity to freely fix to alter ratios of a particular immunoglobulin class or classes. 15 complement. For example, protein A, protein G or protein H Sepharose b. Modified Cohn-Oncley Procedures chromatography can be used to enrich a mixture of immuno To prevent the unwanted effects of the Cohn-Oncley globulins for IgG, or for specific IgG subtypes (see, e.g., method, modified Cohn-Oncley methods have been devel Harlow and Lane (1999) Using Antibodies, Cold Spring Har oped for the preparation and purification of IG. Various such bor Laboratory Press; Harlow and Lane (1988) Antibodies, A procedures are known and can be adapted and modified for Laboratory Manual, Cold Spring Harbor Laboratory Press; producing the IG preparations herein. It is within the skill of U.S. Pat. No. 5,180,810). the art to prepare IG preparations in view of the detailed methods known and available in the art. TABLE 2 Typically, IG is manufactured using a primary cold ethanol 25 fractionation and a secondary fractionation that can include, Serum Immunoglobulin for example, any one or more of the following steps to obtain Serum Level a product having a low anti-complementary activity (ACA): Ig Class mg/mL (%) Function separation of IG aggregates by conventional techniques, such as ultra-centrifuging or exclusion chromatography; chemical IgG 1200 (77) Major IG class in humans; secondary immune response; protects against infection 30 modification of the IG molecules by alcoholization, alkyla IgA 200 (13) Protects mucosa tion, Sulfonation and treatment with reducing agents (see e.g., IgM 150 (9) Major IG for primary immune responses U.S. Pat. No. 6,875,848); incubation at a moderately acidic IgD 2 (<1) Regulates B cells pH (pH 4.0) with or without pepsin, plasmin and immobilized IgE <1 (trace) Major IG in allergic response trypsin, fractionating human plasma by means of ethyleneg 35 lycol polymers (Polson et al. (1964) Biochim. Biophys. Acta. 1. Preparation and Purification 82: 463-475), incorporation of polyethyleneglycol (PEG) as a The immunoglobulin preparations provided herein can be purification agent for material separated from the Cohn frac prepared from any suitable starting materials. For example, tionation (fraction II or II+III, see e.g., U.S. Pat. Nos. 4,093, immune globulins can be isolated from human or animal 606 and 4,165.370), fractionation methods which use poly blood, for example, from human donor serum, or produced by 40 ethylene glycol as a precipitating agent, and other techniques other means, for example, by recombinant DNA technology described in U.S. Pat. Nos. 4,093,606, 4,126,605, 3,966,906, or hybridoma technology. Hence, immunoglobulin prepara and 4,124,576, and other similar methods of purification pro tions can include monoclonal or recombinant immunoglobu cesses with polyethyleneglycol (EP 0246.579); B-propiolac lins. For example, immune globulin can be obtained from tone treatment; ion exchange chromatography to eliminate tissues, lymphocyte hybridoma cultures, blood plasma or 45 undesirable contaminants from the starting materials used to serum, or recombinant cell cultures using any Suitable proce obtain the IG preparations (see e.g., U.S. Pat. No. 3.869,436, dure. Such as, for example, precipitation (Cohn alcohol frac EP 91300790 and WO 94/29334). EP 0440483 describes a tionation or polyethylene glycol fractionation); chromato combination of techniques useful for facilitating the intrave graphic methods (ion exchange chromatography, affinity nous preparation of the product based on ion exchange chro chromatography, immunoaffinity chromatography); ultra 50 matography and diafiltration at a weakly acidic pH; enzy centrifugation; or electrophoretic preparation (see, e.g., Cohn matic cleavage; Solvent/detergent treatment; and diafiltration et al. (1946) J. Am. Chem. Soc. 68:459-75: Oncley et al. and ultrafiltration. Other methods also are described in the art (1949).J. Am. Chem. Soc., 71:541-50; Barandernet al. (1962) and are known to one of skill in the art (see e.g., U.S. Pat. Nos. Vox Sang..., 7:157-74; Koblet et al. (1967) Vox Sang, 13:93 5,177,194 and 6,875,848). 102; U.S. Pat. Nos. 5,122,373 and 5,177,194). Typically, 55 Purified Cohn Fraction II is commonly used. The starting immunoglobulin is prepared from gamma globulin-contain Cohn Fraction II paste is typically about 95 percent IgG and ing products produced by alcohol fractionation and/or ion also contains the four IG subtypes. The different subtypes are exchange and affinity chromatography methods well known present in Fraction II in approximately the same ratio as they to those of skill in the art. are found in the pooled human plasma from which they are Preparative steps can be used to enrich a particular isotype 60 obtained. The Fraction II is further purified before formula or subtype of immunoglobulin. For example, protein A, pro tion into an administrable product. For example, the Fraction tein G or protein H Sepharose chromatography can be used to II can be dissolved in cold purified aqueous alcohol Solution enrich a mixture of immunoglobulins for IgG, or for specific and impurities removed via precipitation and filtration. Fol IgG subtypes. (See generally Harlow and Lane. Using Anti lowing the final filtration, the immunoglobulin Suspension bodies, Cold Spring Harbor Laboratory Press (1999); Harlow 65 can be dialyzed or diafiltered (e.g. using ultrafiltration mem and Lane, Antibodies, A Laboratory Manual, Cold Spring branes having a nominal molecular weight limit of less than Harbor Laboratory Press (1988): U.S. Pat. No. 5,180,810). or equal to 100,000 daltons) to remove alcohol. The solution US 9,084,743 B2 27 28 can be concentrated or diluted to obtain the desired protein e. Exemplary IG Preparations concentration and can be further purified by techniques well i. 10%. IG known to those skilled in the art. Exemplary of an IG preparation is Immune Globulin Intra c. Viral Processing venous (Human), 10% (IVIG, 10%, marketed as Gamma The IG preparations should be treated to remove viral load. 5 gard R. liquid, Baxter Healthcare Corporation), which is a There are two methods of viral processing: viral inactivation liquid unmodified IgG preparation, with a distribution of IgG and viral partitioning or removal. Viral inactivation renders Subclasses similar to that of normal plasma. The preparation viruses inactive by, for example, chemically altering the lipid contains intact fragment crystallizable (Fc) and fragment or protein coat, or by completely denaturing the virus. Exem antigen binding (Fab) regions. The preparations contain 100 10 mg/mL protein, with at least 98% being IgG, IgA is present at plary of viral inactivation methods include, but are not limited a concentration of 37 ug/mL, and IgM is present only in trace to, heating (pasteurization), solvent/detergent (S/D) treat amounts. It has an osmolality that is similar to physiologic ment and exposure to an acidic environment (low pH). The osmolality, and contains no added Sugars, sodium or preser S/D process is the most widely used viral inactivation method vatives. It is formulated with glycine for stabilization at a pH in the blood plasma industry, used to inactivate viruses con 15 of 4.6 to 5.1. The manufacturing process employs a modified taining a lipid coat. For example, the S/D process has been Cohn-Oncley cold alcohol fractionation procedure and fur demonstrated to have virucidal action against VSV (vesicular ther purifications by a continuous process through the use of stomatitits virus), Sindbis virus, HIV, HBV (hepatitis B virus) weak cation exchange chromatography and weak anion and HCV (hepatitis C virus). exchange chromatography. The manufacturing process also Viral removal is a method that completely removes all 20 includes 3 independent viral inactivation or removal steps: viruses from the sample. Exemplary of viral partitioning or solvent/detergent (S/D) treatment, nanofiltration and incuba removal include, but are not limited to, cold ethanol fraction tion at a low pH and elevated temperature. Preparation of a ation, phase partitioning or PEG precipitation, affinity chro 10% IVIG preparation is described in Example 1. matography, ion exchange or gel exclusion chromatography ii. High Concentration IG Preparations (e.g. 20%. IG) and nanofiltration. 25 The generation of high concentration immunoglobulin d. Protein concentration preparations are described in U.S. Provisional Application Immunoglobulins can be prepared at varying concentra No. 61/181,606. Exemplary of preparations containing tions. For example, IG can be prepared at protein concentra 18-22% IG are highly purified, isotonic liquid formulations of tions ranging from at or about 3-25%. IG, typically at or about immunoglobulin (at least 95% IgG) formulated in 0.25 mM 10% to 22%, such as 10% - 20% w/v. For example, IG 30 glycine at pH 4.4 to 4.9, represented in the Examples below. preparations can be at or about 18% to 22% IG w/v. The IG The high concentration IgG products described herein are preparations provided herein generally are prepared at IG produced by a process having many of the same or similar concentrations of at or about 10%, 11%, 12%, 13%, 14%, steps as in the process of producing traditional IVIG prepa 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22% or more. The rations (e.g. 10% IG). The additional steps, ultrafiltration/ final protein concentration depends largely on the method of 35 diafiltration using open channel membranes with a specifi generation and purification. It is contemplated herein that any cally designed post-wash and formulation near the end of the immune globulin preparation can be used herein for stable production process, render the resulting IG compositions co-formulations with hyaluronidase. It is within the level of about twice as high in protein concentration (200 mg/mL) one of skill in the art to empirically determine the appropriate compared to state of the art IVIGs (e.g., Gammagard Liquid), concentration of IG for inclusion in the stable co-formula- 40 without affecting yield and storage stability. With most com tions herein. The choice of IG preparation will depend on a mercially available ultrafiltration membranes, a concentra variety of factors such as the administration route, the patient tion of 200 mg/mL IgG cannot be reached without major to be treated and the type of condition to be treated. protein losses. These membranes become blocked early, con For example, any known or existing preparation of IG can sequently adequate post-wash is difficult to achieve. There be used. These include preparations of IG typically used for 45 fore, open channel membrane configurations have to be used. IV administration (IVIG). In general, final IG preparations Further, a specifically designed post-wash procedure is for intravenous administration have a protein concentration employed to obtain the required IG concentration without of about 3 to 12% w/v., or typically 10% w/v. For example, significant protein loss (less than 2% loss); the higher protein WIG is commercially available as CarimuneR NF, Fleboga concentration of 200 mg/mL does not affect the virus inacti mma(R) 5%, Gammagard R. Liquid, Gammagard(R) S/D, 50 Vation capacity of the low pH storage step. GamunexR), Iveegam.R EN, Octagam.R) and Polygam.R S/D. The general process of producing the high concentration Typically, Such preparations use a method of cold alcohol IG composition includes the following steps which are fractionation, but differ in the methods used to isolate and described in further detail in Example 2. First, the cryopre purify the immune globulin and methods to reduce potential cipitates are separated from previously frozen plasma to yield virus contamination. 55 a liquid "cryo-poor plasma, which is processed in the next Further, other preparations presently formulated for intra step to obtain the Supernatant (or Fractionation I). Adjustment muscular or Subcutaneous administration can be used in the of pH and ethanol concentration, typically to 7 and 20 to 25% compositions and methods provided herein. For example, IG V/V, respectively, followed by Subsequent centrifugation preparations for intramuscular administration and Subcutane while decreasing temperature, separates the liquid and Solid. ous administration are commercially available as Gam- 60 The precipitate from this step is then extracted, mixed with aSTANR) S/D and Vivaglobin R, respectively. Typically, such fumed silica, and filtered, all steps performed at low tempera preparations use cold ethanol fractionation from human tures, typically 2 to 8° C. The filtrate is then mixed with plasma and have an IgG concentration of about 15 to 18% or polysorbate-80 and sodium citrate dehydrate while stirring at 10 to 22%, respectively. U.S. Provisional Application No. 2 to 8° C. Precipitate G is then obtained, in a manner similar 61/181,606 describes the generation of a highly purified and 65 to the precipitation step of Cohn II, in which the pH and concentrated immunoglobulin composition from pooled alcohol concentration is adjusted. Precipitate G is dissolved plasma for Subcutaneous administration. and filtered with a depth filter of a nominal pore size of 0.2 um US 9,084,743 B2 29 30 (e.g., Cuno VR06 filter or equivalent) to obtain a clear filtrate. romolecule, a polyol and another protein to stabilize against Subsequent solvent/detergent treatment, typically using 1.0% anti-complement generation; U.S. Pat. No. 5,945,098 dis (v/v) Triton X-100, 0.3% (v/v) Tween-80, and 0.3% (v/v) closes the stabilization of isotonic solutions by the addition of TNBP, at 18 to 25° C. for at least 60 minutes, followed by amino acids (0.1 to 0.3 M glycine) and non-ionic detergents cation exchange chromatography, anion exchange chroma (polysorbate and PEG); U.S. Pat. No. 4,186,192 discloses tography and nanofiltration using, e.g., an Asahi Planova 35N various additives, including amino acids: WO 2005/049078 filter or equivalent. Subsequent to nanofiltration, the filtrate is discloses the stabilization with maltose, and additionally, gly concentrated to a protein concentration of 5+1% w/v by ultra cine to 0.1 M: U.S. Pat. No. 4,362,661 discloses the use of filtration. In some examples, the ultrafiltration is carried out in neutral and basic amino acids to impart stability on a 5% IG a cassette with an open channel screen and the ultrafiltration 10 preparation. Stable liquid formulations can also be prepared membrane has a nominal molecular weight cut off (NM WCO) of 50 kDa or less. Upon completion of the ultrafiltra using carbohydrates in an aqueous medium with very low tion step, the concentrate is diafiltered against a 0.25 M gly ionic strength and a pH of 4.25 (U.S. Pat. No. 4.396,608) or a cine solution with a low pH. Typically, the minimum weakly acidic pH of 5-6 (EP 0278422). exchange Volume is 6 times the original concentrate Volume, 15 Dimer formation of IG preparations also can be controlled. and the Solution is concentrated to a protein concentration of For example, U.S. Pat. No. 5,871,736 discloses IG prepara more than 20% w/v. At the end of the diafiltration and con tions, particularly liquid preparations, containing one or more centration process, the pH of the solution is typically between amphiphilic stabilizers against dimer formation. The 4.4 to 4.9. For formulation, the protein concentration of the amphiphilic stabilizers include nicotinic acid and its deriva solution is then adjusted to just over 20% w/v. e.g., 20.4+04% tives, in particular nicotinamide, and mainly in conjunction w/v, with the diafiltration buffer. The formulated bulk solu with amino acids having uncharged lipophilic side chains, tion is further sterilized by first filtering through a membrane e.g., phenylalanine, methionine, leucine, isoleucine, proline filter with an absolute pore size of 0.2 micron or less. Then the and valine. Solution is aseptically dispensed into final containers for b. pH proper sealing, with samples taken for testing. The final step 25 The IG preparations can be prepared by methods known in is storing the sealed containers at 30 to 32°C. for an extended the art, such as any described herein. Generally, however, the time period, e.g., 21 to 22 days. pH of the final preparation is adjusted to a relatively high pH, Incorporating ultrafiltration and formulations steps in the namely in the range of about pH 4.0 to 7.4. It has been found manufacturing process is an improvement over previously that the pH of the immune globulin preparation is an impor used IG purification and concentration methods, resulting in 30 tant factor relative to the IgG monomer content of the final preparations with higher IG concentrations without signifi product. Generally, a 5 percent immune globulin preparation cant IG activity loss while maintaining a low pH in the final has a pH of 4.2+0.5. Ten percent preparations are most stable formulation. Typically, the products have a protein concen at a pH of 5.2+0.2. Optimal pH is obtained by formulation tration of at least 18% weight/volume (w/v), of which the vast techniques well known to those skilled in the art. For majority (typically no less than 95%) is IgG, and a pH in the 35 example, optimal pH can be determined from size exclusion range of pH 3-6, which facilitates inactivation of pathogens chromatography determinations as well as heat stability data Such as viruses that may be present in the plasma. Due to the and anticomplement titers of the various preparations under high IG concentration and therefore reduced volume in differing pH conditions. administration, the high concentration preparations are Suit D. Hyaluronidase able for Subcutaneous administration. In some embodiments, 40 Provided herein are stable co-formulations containing the IG products have a viscosity no greater than 18 immunoglobulin and a hyaluronidase, typically a soluble mPascal second and may therefore be suitable for intrave hyaluronidase. Hyaluronidases are members of a large family nous administration as well. Simple dilution can also permit of enzymes that degrade hyaluronic acid, which is an essen intravenous administration. tial component of the extracellular matrix and a major con 2. Storage Stability 45 stituent of the interstitial barrier. By catalyzing the hydrolysis Final, purified IG formulations must be prepared to retain of hyaluronic acid, a major constituent of the interstitial bar activity of the IG and avoid excessive aggregation. Upon rier, hyaluronidase lowers the viscosity of hyaluronic acid, storage of the IG preparations, aggregation can be minimized thereby increasing tissue permeability. As such, hyalu and stability improved by, for example, the addition of pro ronidases have been used, for example, as a spreading or tein-Stabilizing excipients or adjusting the pH of the solution. 50 dispersing agent in conjunction with other agents, drugs and a. Protein-Stabilizing Excipients proteins to enhance their dispersion and delivery. Exemplary A way to increase the stability of IG preparations that is of hyaluronidases in the co-formulations provided herein are well known in the art is to add protein-Stabilizing excipients soluble hyaluronidases. to the IG preparation. Known excipients include, but are not There are three general classes of hyaluronidases; mam limited to, Sugars, polyols, amino acids, amines, salts, poly 55 malian hyaluronidase, bacterial hyaluronidase and hyalu mers and surfactants. For example, U.S. Pat. No. 4,499,073 ronidase from leeches, other parasites and crustaceans. describes stabilization as a result of ionic strength and pH of Mammalian-type hyaluronidases (EC 3.2.1.35) are endo the storage solution; JP Patent 54020124 discloses the addi B-N-acetyl-hexosaminidases that hydrolyze the B1-4 glyco tion of an amino acid to an intramuscular preparation to sidic bond of hyaluronan into various oligosaccharide lengths render the preparation stable and safe for storage; JP 60 Such as tetrasaccharides and hexasaccharides. They have both 57031623 and JP 57128635 disclose the use of arginine and/ hydrolytic and transglycosidase activities, and can degrade or lysine with NaCl in 5 to 15% IG preparations to achieve hyaluronan and chondroitin sulfates (CS), generally C4-S and long-term stability in an intramuscular preparation; JP C6-S. Hyaluronidases of this type include, but are not limited 4346934 discloses the use of low conductivity (less than 1 to, hyaluronidases from cows (bovine) (SEQID NOS:10 and mmho), pH 5.3 to 5.7 and optionally one or more stabilizers, 65 11), mouse (SEQID NOS:17-19, 32), pig (SEQID NOS:20 including PEG, human serum albumin and mannitol; U.S. 21), rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), Pat. No. 4,439.421 teaches the addition of a hydrophilic mac sheep (ovine) (SEQID NOS:26 and 27), orangutan (SEQID US 9,084,743 B2 31 32 NO:28), cynomolgus monkey (SEQ ID NO:29), guinea pig termini due to the absence of a GPI anchor in the bovine (SEQ ID NO:30), and human hyaluronidases. polypeptide (see e.g., Frost GI (2007) Expert Opin. Drug. Mammalian hyaluronidases can be further subdivided into Deliv. 4:427-440). In fact, clear GPI anchors are not predicted those that are neutral active, predominantly found in testes in many other PH20 species besides humans. Thus, PH20 extracts, and acid active, predominantly found in organs Such polypeptides produced from Ovine and bovine naturally exist as the liver. Exemplary neutral active hyaluronidases include as soluble forms. Though bovine PH20 exists very loosely PH20, including but not limited to, PH20 derived from dif attached to the plasma membrane, it is not anchored via a ferent species such as ovine (SEQ ID NO:27), bovine (SEQ phospholipase sensitive anchor (Lalancette et al. (2001) Biol ID NO:11) and human (SEQID NO:1). Human PH20 (also Reprod. 65(2):628–36). This unique feature of bovine hyalu known as SPAM1 or sperm surface protein PH2O), is gener 10 ronidase has permitted the use of the soluble bovine testes ally attached to the plasma membrane via a glycosylphos hyaluronidase enzyme as an extract for clinical use (Wy phatidyl inositol (GPI) anchor. It is naturally involved in dase(R), Hyalase(R). sperm-egg adhesion and aids penetration by sperm of the The human PH20 mRNA transcript is normally translated layer of cumulus cells by digesting hyaluronic acid. to generate a 509 amino acid precursor polypeptide (SEQID Besides human PH20 (also termed SPAM1), five hyalu 15 NO:1) containing a 35 amino acid signal sequence at the ronidase-like genes have been identified in the human N-terminus (amino acid residue positions 1-35) and a 19 genome, HYAL1, HYAL2, HYAL3, HYAL4 and HYALP1. amino acid glycosylphosphatidylinositol (GPI) anchor HYALP1 is a pseudogene, and HYAL3 (SEQID NO:38) has attachment signal sequence at the C-terminus (amino acid not been shown to possess enzyme activity toward any known residue positions 491-509). The mature PH20 is, therefore, a substrates. HYAL4 (precursor polypeptide set forth in SEQ 474 amino acid polypeptide set forth in SEQID NO:2. Fol ID NO:39) is a chondroitinase and exhibits little activity lowing transport of the precursor polypeptide to the ER and towards hyaluronan. HYAL1 (precursor polypeptide set forth removal of the signal peptide, the C-terminal GPI-attachment in SEQID NO:36) is the prototypical acid-active enzyme and signal peptide is cleaved to facilitate covalent attachment of a PH20 (precursor polypeptide set forthin SEQID NO:1) is the GPI anchor to the newly-formed C-terminal amino acid at the prototypical neutral-active enzyme. Acid-active hyalu 25 amino acid position corresponding to position 490 of the ronidases, such as HYAL1 and HYAL2 (precursor polypep precursor polypeptide set forth in SEQID NO:1. Thus, a 474 tide set forth in SEQ ID NO:37) generally lack catalytic amino acid GPI-anchored mature polypeptide with an amino activity at neutral pH (i.e. pH 7). For example, HYAL1 has acid sequence set forth in SEQID NO:2 is produced. little catalytic activity in vitro over pH 4.5 (Frost et al. (1997) Compared to other hyaluronidases, including bee and Anal. Biochemistry, 251:263-269). HYAL2 is an acid-active 30 honey Venom hyaluronidase and mouse, monkey and guinea enzyme with a very low specific activity in vitro. The hyalu pig PH20, human PH20 contains a common region of 340 ronidase-like enzymes can also be characterized by those amino acids with 57 conserved amino acids (see e.g. Arming which are generally attached to the plasma membrane via a et al. (1997) Eur: I Biochem., 247:810-814). The conserved glycosylphosphatidylinositol anchor Such as human HYAL2 amino acids include four cysteine residues that form disulfide and human PH20 (Danilkovitch-Miagkova et al. (2003) Proc 35 bridges at amino acid residues 25, 189, 203 and 316 in the Natl Acad Sci USA. 100(8):4580-5), and those which are sequence of amino acids set forth in SEQ ID NO:2 (corre generally soluble such as human HYAL1 (Frost et al., (1997) sponding to residues 60,224, 238 and 351 in the sequence of Biochem Biophy's Res Commun. 236(1):10-5). amino acids set forth in SEQID NO:1). Disulfide bonds form 1. PH2O between the cysteine residues C60 and C351 and between PH20, like other mammalian hyaluronidases, is an endo 40 C224 and C238 to form the core hyaluronidase domain. How B-N-acetyl-hexosaminidase that hydrolyzes the B1->4 gly ever, additional cysteines are required in the carboxy termi cosidic bond of hyaluronic acid into various oligosaccharide nus for neutral enzyme catalytic activity Such that amino lengths such as tetrasaccharides and hexasaccharides. They acids 36 to 464 of SEQIDNO:1 contains the minimally active have both hydrolytic and transglycosidase activities and can human PH20 hyaluronidase domain. A further four disulfide degrade hyaluronic acid and chondroitin Sulfates, such as 45 bonds are formed between the cysteine residues C376 and C4-S and C6-S. PH20 is naturally involved in sperm-egg C387; between C381 and C435; between C437 and C443; adhesion and aids penetration by sperm of the layer of cumu and between C458 and C464 of the polypeptide exemplified lus cells by digesting hyaluronic acid. PH20 is located on the in SEQID NO: 1 (corresponding to residues C341 and C352: sperm surface, and in the lysosome-derived acrosome, where between C346 and C400; between C402 and C408; and it is bound to the inner acrosomal membrane. Plasma mem 50 between C423 and C429 of the mature polypeptide set forth in brane PH20 has hyaluronidase activity only at neutral pH, SEQ ID NO:2, respectively). while inner acrosomal membrane PH20 has activity at both In addition, other conserved residues are likely involved in neutral and acid pH. In addition to being a hyaluronidase, Substrate binding and catalysis. Amino acid residues atamino PH20 also appears to be a receptor for HA-induced cell sig acid positions 111, 113, 176,249 and 252 corresponding to naling, and a receptor for the Zona pellucida Surrounding the 55 residues in SEQID NO:2 appear to be involved in the activity oocyte. of PH20, since mutation at these position renders the enzyme Exemplary PH20 proteins include, but are not limited to, devoid of enzymatic activity or leave only residual activity human (precursor polypeptide set forth in SEQ ID NO:1, compared to wild-type PH20 not containing the mutations mature polypeptide set forth in SEQID NO: 2), bovine (SEQ (see e.g. Arming et al. (1997) Eur: J. Biochem., 247:810-814). ID NOS: 11), rabbit (SEQID NO:25), ovine PH20 (SEQID 60 There are seven potential N-linked glycosylation sites at NOS: 27), Cynomolgus monkey (SEQ ID NO: 29), guinea N82, N166, N235, N254, N368, N393, N490 of human PH20 pig (SEQID NO:30), rat (SEQID NO:31) and mouse (SEQ exemplified in SEQID NO: 1. Disulfide bonds form between ID NO:32) PH20 polypeptides. the cysteine residues C60 and C351 and between C224 and Bovine PH20 is a 553 amino acid precursor polypeptide C238 to form the core hyaluronidase domain. Since amino (SEQID NO:11). Alignment of bovine PH20 with the human 65 acids 36 to 464 of SEQID NO:1 contain the minimally active PH20 shows only weak homology, with multiple gaps exist human PH20 hyaluronidase domain, N-linked glycosylation ing from amino acid 470 through to the respective carboxy site N-490 is not required for proper hyaluronidase activity. US 9,084,743 B2 33 34 2. Soluble Hyaluronidase example, Soluble forms include, but are not limited to, any Generally, the hyaluronidase in the stable co-formulations having C-terminal truncations to generate polypeptides con provided herein are soluble hyaluronidases. Soluble hyalu taining amino acid 1 to amino acid 467 to 483, for example, ronidases, when expressed in cells, are secreted into the 467,477,478,479,480,481,482 and 483. When expressed in media. Solubility can be demonstrated by partitioning of the mammalian cells, the 35 amino acid N-terminal signal protein into the aqueous phase of Triton X-114 solution. sequence is cleaved during processing, and the mature form Accordingly, it is understood that a soluble hyaluronidase of the protein is secreted. Thus, the mature soluble polypep does not include any hyaluronidase that contains a GPI tides contain at least amino acids 36 to 464 of SEQID NO:1. anchor, rendering the polypeptide attached to the cell mem For example, mature soluble polypeptides contain amino brane. For example, full-length human PH20 (set forth in its 10 acids 36 to 467 to 36 to 483 of SEQID NO:1, for example 36 mature form as SEQID NO:2) contains a GPI anchor and is to 467,477,478,479,480,481,482 and 483 of SEQIDNO:1. not soluble. In contrast, bovine and ovine PH20 polypeptides Deletion mutants ending at amino acid position 477 to 483 do not containa GPI anchor that is sufficient for attachment to (corresponding to the precursor polypeptide set forth in SEQ the GPI anchor, and thus are considered to be soluble pro ID NO: 1) exhibit higher secreted hyaluronidase activity than teins. Further, the soluble hyaluronidase that are included in 15 the full length GPI-anchored form. Hence, exemplary of the co-formulations provided herein generally are Substan soluble hyaluronidases are those that are 442, 443, 444, 445, tially purified proteins. Also, soluble hyaluronidases retain 446 or 447 amino acids in length, Such as set forth in any of hyaluronidase activity. For example, soluble human PH20 SEQID NOS:4-9, or allelic or species variants or other vari retains neutral activity. ants thereof. Soluble hyaluronidases include hyaluronidases that do not b. Recombinant Soluble Human PH20 (rHuPH20) naturally include a GPI anchor or an anchor sufficient for Recombinant soluble forms of human PH20 designated as attachment to the membrane, including, but not limited to, rHuPH20 have been generated and can be produced and puri Hyal 1, bovine PH20 and ovine PH20, allelic variants thereof fied using the methods described herein. The generation of and other variants. Also included among soluble hyalu Such soluble forms of ruPH20 are described in U.S. Patent ronidase are any hyaluronidase that has been modified to be 25 Application Ser. Nos. 11/065,716 and 1 1/238,171 (published soluble. For example, human PH20, which is normally mem as U.S. published patent application Nos. US20050260186 brane anchored via a GPI anchor, can be made soluble by and US 20060104968), and in Examples 3 below. Exemplary truncation of and removal of all or a portion of the GPI anchor of Such polypeptides are those generated from a nucleic acid at the C-terminus. Soluble hyaluronidases also include neu molecule encoding amino acids 1-482 set forth in SEQ ID tral active and acid active hyaluronidases, however, neutral 30 NO:3. Post translational processing removes the 35 amino active hyaluronidases are contemplated for use herein for acid signal sequence, resulting in the secretion of a 447 amino purposes of subcutaneous administration. acid soluble rHuPH20 (SEQ ID NO:4). Resulting purified Thus, exemplary of a soluble hyaluronidase is PH20 from rHuPH20 can be heterogenous due to peptidases present in any species, such as any set forth in any of SEQID NOS: 1, 2, the culture medium upon production and purification. Typi 11, 25, 27, 30, 31 and 32, or truncated forms thereof lacking 35 cally, rHuPH20 is produced in cells that facilitate correct all or a portion of the C-terminal GPI anchor, so long as the N-glycosylation to retain activity, such as CHO cells (e.g. hyaluronidase is soluble and retains hyaluronidase activity. DG44 CHO cells). Also included among soluble hyaluronidases are allelic Vari 3. Glycosylation ants or other variants of soluble forms of any of SEQID NOS: Glycosylation, including N- and O-linked glycosylation, 1, 2, 11, 25, 27.30,31 and 32, such as truncated forms thereof. 40 of some hyaluronidases can be very important for their cata Allelic variants and other variants are known to one of skill in lytic activity and stability. While altering the type of glycan the art, and include polypeptides having 60%, 70%. 80%, modifying a glycoprotein can have dramatic affects on a 90%, 91%, 92%, 93%, 94%, 95% or more sequence identify protein's antigenicity, structural folding, Solubility, and sta to any of SEQ ID NOS: 1, 2, 11, 25, 27, 30 and 31, or bility, most enzymes are not thought to require glycosylation truncated forms thereof. 45 for optimal enzyme activity. Such hyaluronidases are unique Typically, co-formulations herein contain a soluble human in this regard, in that removal of N-linked glycosylation can PH20. Although PH20 from other animals can be utilized, result in near complete inactivation of the hyaluronidase Such preparations are potentially immunogenic, since they activity. For such hyaluronidases, the presence of N-linked are animal proteins. For example, a significant proportion of glycans is critical for generating an active enzyme. patients demonstrate prior sensitization secondary to ingested 50 N-linked oligosaccharides fall into several major types foods, and since these are animal proteins, all patients have a (oligomannose, complex, hybrid, Sulfated), all of which have risk of Subsequent sensitization. Thus, non-human prepara (Man) 3-GlcNAc-GlcNAc-cores attached via the amide tions may not be Suitable for chronic use. If non-human nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser preparations are desired, it is contemplated herein that Such sequences (where Xaa is not Pro). Glycosylation at an-Asn polypeptides can be prepared to have reduced immunogenic 55 Xaa-Cys-site has been reported for coagulation protein C. In ity. Such modifications are within the level of one of skill in Some instances, the hyaluronidase can contain both N-glyco the art. sidic and O-glycosidic linkages. For example, PH20 has a. Soluble Human PH2O O-linked oligosaccharides as well as N-linked oligosaccha Exemplary of a soluble hyaluronidase is soluble human rides. There are seven potential N-linked glycosylation sites PH20, Soluble forms of recombinant human PH20 have been 60 at N82, N166, N235, N254, N368, N393, N490 of human produced and can be included in the co-formulations PH20 exemplified in SEQ ID NO: 1. As noted above, described herein. The production of such soluble forms of N-linked glycosylation at N490 is not required for hyalu PH20 is described in U.S. Patent Application Nos. 2005 ronidase activity. 0260186 and 2006-0104968. Soluble forms include, but are 4. Modifications of Hyaluronidases to Improve their Phar not limited to, any having C-terminal truncations to generate 65 macokinetic Properties polypeptides containing amino acid 1 to amino acid 464 or of Hyaluronidases provided in the co-formulations can be the sequence of amino acids set forth in SEQID NOS 1. For modified to improve their pharmacokinetic properties, such US 9,084,743 B2 35 36 as increasing their half-life in vivo and/or activities. The rHuPH20, has a molecular weight of 5, 10, 15, 20, 25, 30, 35, modification of hyaluronidases for use in co-formulations 40, 45, 50, 55, 60 or more than 60 kDa. provided herein can include attaching, directly or indirectly Various methods of modifying polypeptides by covalently via a linker, such as covalently or by other stable linkage, a attaching (conjugating) a PEG or PEG derivative (i.e. "PEGy polymer, such as dextran, a polyethylene glycol (PEGylation lation') are known in the art (see e.g., U.S. 2006/0104968; (PEG)) or sialyl moiety, or other such polymers, such as U.S. Pat. No. 5,672,662; U.S. Pat. No. 6,737,505; and U.S. natural or Sugar polymers. 2004/0235734). Techniques for PEGylation include, but are PEGylation of therapeutics is known to increase resistance not limited to, specialized linkers and coupling chemistries to proteolysis, increase plasma half-life, and decrease antige (see e.g., Harris, Adv. Drug Deliv Rev. 54:459-476, 2002), nicity and immunogenicity. Covalent or other stable attach 10 attachment of multiple PEG moieties to a single conjugation ment (conjugation) of polymeric molecules, such as polyeth site (such as via use of branched PEGs; see e.g., Veronese et ylene glycol moiety (PEG), to the hyaluronidase thus can al., Bioorg. Med. Chem. Lett. 12:177-180, 2002), site-specific impart beneficial properties to the resulting enzyme-polymer PEGylation and/or mono-PEGylation (see e.g., Chapman et composition. Such properties include improved biocompat al., Nature Biotech. 17:780-783, 1999), and site-directed ibility, extension of protein (and enzymatic activity) half-life 15 enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv Rev., in the blood, cells and/or in other tissues within a subject, 54:487-504, 2002) (see, also, for example, Lu and Felix effective shielding of the protein from proteases and hydroly (1994) Int. J. Peptide Protein Res. 43:127-138; Lu and Felix sis, improved biodistribution, enhanced pharmacokinetics (1993) Peptide Res. 6: 142-6, 1993: Felix et al. (1995) Int. J. and/or pharmacodynamics, and increased water Solubility. Peptide Res. 46:253-64; Benhar et al. (1994).J. Biol. Chem. Exemplary polymers that can be conjugated to the hyalu 269:13398-404; Brumeanu et al. (1995) J Immunol. 154: ronidase, include natural and synthetic homopolymers. Such 3088-95; see also, Caliceti et al. (2003) Adv. Drug Deliv: Rev. as polyols (i.e. poly-OH), polyamines (i.e. poly-NH2) and 55(10): 1261-77 and Molineux (2003) Pharmacotherapy 23 polycarboxyl acids (i.e. poly-COOH), and further het (8 Pt 2):3S-8S). Methods and techniques described in the art eropolymers i.e. polymers containing one or more different can produce proteins having 1,2,3,4,5,6,7,8,9, 10 or more coupling groups e.g. a hydroxyl group and amine groups. 25 than 10 PEG or PEG derivatives attached to a single protein Examples of suitable polymeric molecules include polymeric molecule (see e.g., U.S. 2006/0104968). molecules selected from among polyalkylene oxides (PAO), Numerous reagents for PEGylation have been described in Such as polyalkylene glycols (PAG), including polypropylene the art. Such reagents include, but are not limited to, N-hy glycols (PEG), methoxypolyethylene glycols (mPEG) and droxysuccinimidyl (NHS) activated PEG, succinimidyl polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), 30 mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinim PEG-oxycarbonylimidazole (CDI-PEG) branched polyethyl idyl alpha-methylbutanoate, mPEG succinimidyl propionate, ene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxy mPEG succinimidylbutanoate, mPEG carboxymethyl 3-hy lates, polyvinylpyrrolidone, poly-D.L-amino acids, polyeth droxybutanoic acid Succinimidyl ester, homobifunctional ylene-co-maleic acid anhydride, polystyrene-co-maleic acid PEG-succinimidyl propionate, homobifunctional PEG propi anhydride, dextrans including carboxymethyl-dextrans, hep 35 onaldehyde, homobifunctional PEG butyraldehyde, PEG arin, homologous albumin, celluloses, including methylcel maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, lulose, carboxymethylcellulose, ethylcellulose, hydroxyeth mPEG-benzotriazole carbonate, propionaldehyde PEG, ylcellulose carboxyethylcellulose and mPEG butyraldehyde, branched mPEG2 butyraldehyde, hydroxypropylcellulose, hydrolysates of chitosan, starches mPEG acetyl, mPEG piperidone, mPEG methylketone, Such as hydroxyethyl-Starches and hydroxypropyl-Starches, 40 mPEG “linkerless” maleimide, mPEG vinyl sulfone, mPEG glycogen, agaroses and derivatives thereof, guar gum, pullu thiol, mPEG orthopyridylthioester, mPEG orthopyridyl dis lan, inulin, Xanthan gum, carrageenan, pectin, alginic acid ulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG hydrolysates and bio-polymers. NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin Typically, the polymers are polyalkylene oxides (PAO), PEG-NHS (see e.g., Monfardini et al., Bioconjugate Chem. such as polyethylene oxides, such as PEG, typically mPEG, 45 6:62-69, 1995; Veronese et al., J. Bioactive Compatible Poly which, in comparison to polysaccharides Such as dextran, mers 12:197-207, 1997: U.S. Pat. Nos. 5,672,662; 5,932,462: pullulan and the like, have few reactive groups capable of 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614; cross-linking. Typically, the polymers are non-toxic poly 5,183,550; 5,324,844; 5,446,090; 5,612,460; 5,643,575: meric molecules such as (m)polyethylene glycol (mPEG) 5,766,581; 5,795,569; 5,808,096; 5,900.461; 5,919,455; which can be covalently conjugated to the hyaluronan 50 5,985,263; 5,990,237; 6,113,906; 6,214,966; 6,258,351: degrading enzyme (e.g., to attachment groups on the protein 6,340,742; 6,413,507; 6,420,339; 6,437,025; 6,448,369; Surface) using a relatively simple chemistry. 6,461,802; 6,828,401; 6,858,736: U.S. 2001/0021763: U.S. Suitable polymeric molecules for attachment to the hyalu 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; roman degrading enzyme include, but are not limited to, poly U.S. 2002/0072573: U.S. 2002/0156047; U.S. 2003/ ethylene glycol (PEG) and PEG derivatives such as methoxy 55 0114647; U.S. 2003/0143596: U.S. 2003/0158333; U.S. polyethylene glycols (mPEG), PEG-glycidyl ethers (Epox 2003/0220447; U.S. 2004/0013637; US 2004/0235734; U.S. PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched 2005/000360; U.S. 2005/0114037; U.S. 2005/0171328; U.S. PEGs, and polyethylene oxide (PEO) (see e.g. Roberts et al., 2005/0209416: EP 01064951; EP 0822199; WO 00176640; Advanced Drug Delivery Review 2002, 54: 459-476; Harris WO 0002017; WO 0249673; WO 9428024; and WO and Zalipsky, S (eds.)“Poly(ethylene glycol), Chemistry and 60 0187925). Biological Applications' ACS Symposium Series 680, 1997: E. Methods of producing nucleic acids encoding a soluble Mehvar et al., J. Pharm. Pharmaceut. Sci., 3(1):125-136, Hyaluronidase and polypeptides thereof 2000; Harris, Nature Reviews 2:215 et seq. (2003); and Tsub Polypeptides of a soluble hyaluronidase set forth herein, ery, J Biol. Chem. 279(37):38118-24, 2004). The polymeric can be obtained by methods well known in the art for protein molecule can be of a molecular weight typically ranging from 65 purification and recombinant protein expression. Any method about 3 kDa to about 60 kDa. In some embodiments the known to those of skill in the art for identification of nucleic polymeric molecule that is conjugated to a protein, such as acids that encode desired genes can be used. Any method US 9,084,743 B2 37 38 available in the art can be used to obtain a full length (i.e., vectors include, but are not limited to, plasmids or modified encompassing the entire coding region) cDNA or genomic viruses, but the vector system must be compatible with the DNA clone encoding a hyaluronidase, Such as from a cell or host cell used. Such vectors include, but are not limited to, tissue source. Modified or variant soluble hyaluronidases, can bacteriophages such as lambda derivatives, or plasmids Such be engineered from a wildtype polypeptide. Such as by site as pCMV4, pBR322 or puC plasmid derivatives or the Blue directed mutagenesis. Typically, hyaluronidases, including script vector (Stratagene, La Jolla, Calif.). Other expression soluble hyaluronidases such as rHuPH20, used in the co vectors include the HZ24 expression vector exemplified formulations provided herein can be recombinantly produced herein. The insertion into a cloning vector can, for example, or can be purified or partially-purified from natural Sources, be accomplished by ligating the DNA fragment into a cloning Such as, for example, from testes extracts. 10 vector which has complementary cohesive termini. Insertion Polypeptides can be cloned or isolated using any available can be effected using TOPO cloning vectors (INVITROGEN, methods known in the art for cloning and isolating nucleic Carlsbad, Calif.). If the complementary restriction sites used acid molecules. Such methods include PCR amplification of to fragment the DNA are not present in the cloning vector, the nucleic acids and screening of libraries, including nucleic ends of the DNA molecules can be enzymatically modified. acid hybridization screening, antibody-based screening and 15 Alternatively, any site desired can be produced by ligating activity-based screening. nucleotide sequences (linkers) onto the DNA termini; these Methods for amplification of nucleic acids can be used to ligated linkers can contain specific chemically synthesized isolate nucleic acid molecules encoding a desired polypep oligonucleotides encoding restriction endonuclease recogni tide, including for example, polymerase chain reaction (PCR) tion sequences. In an alternative method, the cleaved vector methods. A nucleic acid containing material can be used as a and protein gene can be modified by homopolymeric tailing. starting material from which a desired polypeptide-encoding Recombinant molecules can be introduced into host cells via, nucleic acid molecule can be isolated. For example, DNA and for example, transformation, transfection, infection, elec mRNA preparations, cell extracts, tissue extracts, fluid troporation and Sonoporation, so that many copies of the gene samples (e.g. blood, serum, saliva), samples from healthy sequence are generated. and/or diseased Subjects can be used in amplification meth 25 In specific embodiments, transformation of host cells with ods. Nucleic acid libraries also can be used as a source of recombinant DNA molecules that incorporate the isolated starting material. Primers can be designed to amplify a protein gene, cDNA, or synthesized DNA sequence enables desired polypeptide. For example, primers can be designed generation of multiple copies of the gene. Thus, the gene can based on expressed sequences from which a desired polypep be obtained in large quantities by growing transformants, tide is generated. Primers can be designed based on back 30 isolating the recombinant DNA molecules from the transfor translation of a polypeptide amino acid sequence. Nucleic mants and, when necessary, retrieving the inserted gene from acid molecules generated by amplification can be sequenced the isolated recombinant DNA. Generally, hyaluronidases, and confirmed to encode a desired polypeptide. including soluble forms of PH20, are produced using protein Additional nucleotide sequences can be joined to a expression systems that facilitate correct N-glycosylation to polypeptide-encoding nucleic acid molecule, including 35 ensure the polypeptide retains activity, since glycosylation is linker sequences containing restriction endonuclease sites for important for the catalytic activity and stability of hyalu the purpose of cloning the synthetic gene into a vector, for ronidases. Such cells include, for example Chinese Hamster example, a protein expression vector or a vector designed for Ovary (CHO) cells (e.g. DG44 CHO cells). the amplification of the core protein coding DNA sequences. 1. Vectors and Cells Furthermore, additional nucleotide sequences specifying 40 For recombinant expression of one or more of the desired functional DNA elements can be operatively linked to a proteins, such as any described herein, the nucleic acid con polypeptide-encoding nucleic acid molecule. Examples of taining all or a portion of the nucleotide sequence encoding Such sequences include, but are not limited to, promoter the protein can be inserted into an appropriate expression sequences designed to facilitate intracellular protein expres vector, i.e., a vector that contains the necessary elements for Sion, and secretion sequences, for example heterologous sig 45 the transcription and translation of the inserted protein coding nal sequences, designed to facilitate protein secretion. Such sequence. The necessary transcriptional and translational sig sequences are known to those of skill in the art. Additional nals also can be supplied by the native promoter for enzyme nucleotide residues sequences such as sequences of bases genes, and/or their flanking regions. specifying protein binding regions also can be linked to Also provided are vectors that contain a nucleic acid enzyme-encoding nucleic acid molecules. Such regions 50 encoding the enzyme. Cells containing the vectors also are include, but are not limited to, sequences of residues that provided. The cells include eukaryotic and prokaryotic cells, facilitate or encode proteins that facilitate uptake of an and the vectors are any suitable for use therein. enzyme into specific target cells, or otherwise alter pharma Prokaryotic and eukaryotic cells, including endothelial cokinetics of a product of a synthetic gene. For example, cells, containing the vectors are provided. Such cells include enzymes can be linked to PEG moieties. 55 bacterial cells, yeast cells, fungal cells, Archea, plant cells, In addition, tags or other moieties can be added, for insect cells and animal cells. The cells are used to produce a example, to aid in detection or affinity purification of the protein thereof by growing the above-described cells under polypeptide. For example, additional nucleotide residues conditions whereby the encoded protein is expressed by the sequences such as sequences of bases specifying an epitope cell, and recovering the expressed protein. For purposes tag or other detectable marker also can be linked to enzyme 60 herein, for example, the enzyme can be secreted into the encoding nucleic acid molecules. Exemplary of Such medium. sequences include nucleic acid sequences encoding a His tag Provided are vectors that contain a sequence of nucleotides (e.g., 6xHis, HHHHHH: SEQ ID NO:54) or Flag Tag that encodes the Soluble hyaluronidase polypeptide coupled (DYKDDDDK; SEQID NO:55). to the native or heterologous signal sequence, as well as The identified and isolated nucleic acids can then be 65 multiple copies thereof. The vectors can be selected for inserted into an appropriate cloning vector. A large number of expression of the enzyme protein in the cell or such that the vector-host systems known in the art can be used. Possible enzyme protein is expressed as a secreted protein. US 9,084,743 B2 39 40 A variety of host-vector Systems can be used to express the active in liver (Kelsey et al., Genes and Devel. 1:161-171 protein coding sequence. These include but are not limited to (1987)), beta globin gene control region which is active in mammalian cell systems infected with virus (e.g. Vaccinia myeloid cells (Magram et al., Nature 315:338-340 (1985); virus, adenovirus and other viruses); insect cell systems Kollias et al., Cell 46:89-94 (1986)), myelin basic protein infected with virus (e.g. baculovirus); microorganisms such 5 gene control region which is active in oligodendrocyte cells of as yeast containing yeast vectors; or bacteria transformed the brain (Readhead et al., Cell 48:703-712 (1987)), myosin with bacteriophage, DNA, plasmid DNA, or cosmid DNA. light chain-2 gene control region which is active in skeletal The expression elements of vectors vary in their strengths and muscle (Shani, Nature 314:283-286 (1985)), and gona specificities. Depending on the host-vector System used, any dotrophic releasing hormone gene control region which is one of a number of Suitable transcription and translation 10 active in gonadotrophs of the hypothalamus (Mason et al., elements can be used. Science 234:1372-1378 (1986)). Any methods known to those of skill in the art for the In a specific embodiment, a vector is used that contains a insertion of DNA fragments into a vector can be used to promoter operably linked to nucleic acids encoding a desired construct expression vectors containing a chimeric gene con protein, or a domain, fragment, derivative or homolog, taining appropriate transcriptional/translational control sig 15 thereof, one or more origins of replication, and optionally, nals and protein coding sequences. These methods can one or more selectable markers (e.g., an antibiotic resistance include in vitro recombinant DNA and synthetic techniques gene). Exemplary plasmid vectors for transformation of E. and in vivo recombinants (genetic recombination). Expres coli cells, include, for example, the pOE expression vectors sion of nucleic acid sequences encoding protein, or domains, (available from Qiagen, Valencia, Calif.; see also literature derivatives, fragments or homologs thereof, can be regulated published by Qiagen describing the system). pCE vectors by a second nucleic acid sequence so that the genes or frag have a phage T5 promoter (recognized by E. coli RNA poly ments thereof are expressed in a host transformed with the merase) and a double lac operator repression module to pro recombinant DNA molecule(s). For example, expression of vide tightly regulated, high-level expression of recombinant the proteins can be controlled by any promoter/enhancer proteins in E. coli, a synthetic ribosomal binding site (RBS II) known in the art. In a specific embodiment, the promoter is 25 for efficient translation, a 6xHis tag coding sequence, to and not native to the genes for a desired protein. Promoters which T1 transcriptional terminators, Col. 1 origin of replication, can be used include but are not limited to the SV40 early and a beta-lactamase gene for conferring amplicillin resis promoter (Bernoist and Chambon, Nature 290:304-310 tance. The pGE vectors enable placement of a 6xHis tag at (1981)), the promoter contained in the 3' long terminal repeat either the N- or C-terminus of the recombinant protein. Such of Rous sarcoma virus (Yamamoto et al. Cell 22:787-797 30 plasmids include pGE32, pGE30, and pGE31 which provide (1980)), the herpes thymidine kinase promoter (Wagner et al., multiple cloning sites for all three reading frames and provide Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regu for the expression of N-terminally 6xHis-tagged proteins. latory sequences of the metallothionein gene (Brinster et al., Other exemplary plasmid vectors for transformation of E. coli Nature 296:39-42 (1982); prokaryotic expression vectors cells, include, for example, the pET expression vectors (see, such as the B-lactamase promoter (Jay et al., (1981) Proc. 35 U.S. Pat. No. 4,952,496; available from NOVAGEN, Madi Natl. Acad. Sci. USA 78:5543) or the tac promoter (DeBoeret son, Wis.; see, also literature published by Novagen describ al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also ing the system). Such plasmids include pET 11a, which con “Useful Proteins from Recombinant Bacteria': in Scientific tains the T71ac promoter, T7 terminator, the inducible E. coli American 242:79-94 (1980)); plant expression vectors con lac operator, and the lac repressor gene; pET 12a-c, which taining the nopaline synthetase promoter (Herrara-Estrella et 40 contains the T7 promoter. T7 terminator, and the E. coliompT al., Nature 303:209-213 (1984)) or the cauliflower mosaic secretion signal; and pET. 15b and pET19b (NOVAGEN, virus 35S RNA promoter (Garder et al., Nucleic Acids Res. Madison, Wis.), which contain a His-Tag TM leader sequence 9:2871 (1981)), and the promoter of the photosynthetic for use in purification with a His column and a thrombin enzyme ribulose bisphosphate carboxylase (Herrera-Estrella cleavage site that permits cleavage following purification et al., Nature 310:115-120 (1984); promoter elements from 45 over the column, yeast and other fungi such as the GalA promoter, the alcohol the T7-lac promoter region and the T7 terminator. Exem dehydrogenase promoter, the phosphoglycerol kinase pro plary of a vector for mammalian cell expression is the HZ24 moter, the alkaline phosphatase promoter, and the following expression vector. The HZ24 expression vector was derived animal transcriptional control regions that exhibit tissue from the pCI vector backbone (Promega). It contains DNA specificity and have been used in transgenic animals: elastase 50 encoding the Beta-lactamase resistance gene (AmpR), an F1 I gene control region which is active in pancreatic acinar cells origin of replication, a Cytomegalovirus immediate-early (Swift et al., Cell 38:639-646 (1984); Ornitz et al., Cold enhancer/promoter region (CMV), and an SV40 late polyade Spring Harbor Symp. Ouant. Biol. 50:399-409 (1986); Mac nylation signal (SV40). The expression vector also has an Donald, Hepatology 7:425-515 (1987)); insulin gene control internal ribosome entry site (IRES) from the ECMV virus region which is active in pancreatic beta cells (Hanahan et al., 55 (Clontech) and the mouse dihydrofolate reductase (DHFR) Nature 315: 115-122 (1985)), immunoglobulin gene control gene. region which is active in lymphoid cells (Grosschedl et al., 2. Expression Cell 38:647-658 (1984); Adams et al., Nature 318:533-538 Soluble hyaluronidase polypeptides can be produced by (1985); Alexander et al., Mol. Cell. Biol. 7:1436-1444 any method known to those of skill in the art including in vivo (1987)), mouse mammary tumor virus control region which is 60 and in vitro methods. Desired proteins can be expressed in active in testicular, breast, lymphoid and mast cells (Leder et any organism Suitable to produce the required amounts and al., Cell 45:485-495 (1986)), albumin gene control region forms of the proteins, such as for example, needed for admin which is active in liver (Pinckert et al., Genes and Devel. istration and treatment. Expression hosts include prokaryotic 1:268-276 (1987)), alpha-fetoprotein gene control region and eukaryotic organisms such as E. coli, yeast, plants, insect which is active in liver (Krumlauf et al., Mol. Cell. Biol. 65 cells, mammalian cells, including human cell lines and trans 5:1639-1648 (1985); Hammer et al., Science 235:53-58 genic animals. Expression hosts can differ in their protein 1987)), alpha-1 antitrypsin gene control region which is production levels as well as the types of post-translational US 9,084,743 B2 41 42 modifications that are present on the expressed proteins. The can be used for production of proteins, such as any described choice of expression host can be made based on these and herein. Yeast can be transformed with episomal replicating other factors, such as regulatory and safety considerations, vectors or by stable chromosomal integration by homologous production costs and the need and methods for purification. recombination. Typically, inducible promoters are used to Many expression vectors are available and known to those regulate gene expression. Examples of Such promoters of skill in the art and can be used for expression of proteins. include GAL1, GAL7 and GAL5 and metallothionein pro The choice of expression vector will be influenced by the moters, such as CUP1, AOX1 or other Pichia or other yeast choice of host expression system. In general, expression vec promoter. Expression vectors often include a selectable tors can include transcriptional promoters and optionally marker such as LEU2, TRP1, HIS3 and URA3 for selection enhancers, translational signals, and transcriptional and 10 and maintenance of the transformed DNA. Proteins translational termination signals. Expression vectors that are expressed in yeast are often soluble. Co-expression with used for stable transformation typically have a selectable chaperonins such as Bip and protein disulfide isomerase can marker which allows selection and maintenance of the trans improve expression levels and Solubility. Additionally, pro formed cells. In some cases, an origin of replication can be teins expressed in yeast can be directed for secretion using used to amplify the copy number of the vector. 15 secretion signal peptide fusions such as the yeast mating type Soluble hyaluronidase polypeptides also can be utilized or alpha-factor secretion signal from Saccharomyces cerevisae expressed as protein fusions. For example, an enzyme fusion and fusions with yeast cell Surface proteins such as the Aga2p can be generated to add additional functionality to an enzyme. mating adhesion receptor or the Arcula adeninivorans glu Examples of enzyme fusion proteins include, but are not coamylase. A protease cleavage site such as for the Kex-2 limited to, fusions of a signal sequence, a tag such as for protease, can be engineered to remove the fused sequences localization, e.g. a his tag or a myc tag, or a tag for purifica from the expressed polypeptides as they exit the Secretion tion, for example, a GST fusion, and a sequence for directing pathway. Yeast also is capable of glycosylation at ASn-X-Ser/ protein secretion and/or membrane association. Thr motifs. a. Prokaryotic Cells c. Insect Cells Prokaryotes, especially E. coli, provide a system for pro 25 Insect cells, particularly using baculovirus expression, are ducing large amounts of proteins. Transformation of E. coli is useful for expressing polypeptides such as hyaluronidase simple and rapid technique well known to those of skill in the polypeptides. Insect cells express high levels of protein and art. Expression vectors for E. coli can contain inducible pro are capable of most of the post-translational modifications moters, such promoters are useful for inducing high levels of used by higher eukaryotes. Baculovirus have a restrictive host protein expression and for expressing proteins that exhibit 30 range which improves the safety and reduces regulatory con some toxicity to the host cells. Examples of inducible pro cerns of eukaryotic expression. Typical expression vectors moters include the lac promoter, the trp promoter, the hybrid use a promoter for high level expression such as the polyhe tac promoter, the T7 and SP6 RNA promoters and the tem drin promoter of baculovirus. Commonly used baculovirus perature regulated WPL promoter. systems include the baculoviruses such as Autographa Cali Proteins, such as any provided herein, can be expressed in 35 fornica nuclear polyhedrosis virus (AcNPV), and the Bombyx the cytoplasmic environment of E. coli. The cytoplasm is a mori nuclear polyhedrosis virus (BmNPV) and an insect cell reducing environment and for Some molecules, this can result line such as Sf9 derived from Spodoptera frugiperda, Pseu in the formation of insoluble inclusion bodies. Reducing daletia unipuncta (A7S) and Danaus plexippus (DpN1). For agents such as dithiothreitol and B-mercaptoethanol and high-level expression, the nucleotide sequence of the mol denaturants, such as guanidine-HCl and urea can be used to 40 ecule to be expressed is fused immediately downstream of the resolubilize the proteins. An alternative approach is the polyhedrin initiation codon of the virus. Mammalian secre expression of proteins in the periplasmic space of bacteria tion signals are accurately processed in insect cells and can be which provides an oxidizing environment and chaperonin used to secrete the expressed protein into the culture medium. like and disulfide isomerases and can lead to the production of In addition, the cell lines Pseudaletia unipuncta (A7S) and soluble protein. Typically, a leader sequence is fused to the 45 Danaus plexippus (DpN1) produce proteins with glycosyla protein to be expressed which directs the protein to the peri tion patterns similar to mammalian cell systems. plasm. The leader is then removed by signal peptidases inside An alternative expression system in insect cells is the use of the periplasm. Examples of periplasmic-targeting leader stably transformed cells. Cell lines such as the Schneider 2 sequences include the pelB leader from the pectate lyase gene (S2) and Kc cells (Drosophila melanogaster) and C7 cells and the leader derived from the alkaline phosphatase gene. In 50 (Aedes albopictus) can be used for expression. The Droso Some cases, periplasmic expression allows leakage of the phila metallothionein promoter can be used to induce high expressed protein into the culture medium. The secretion of levels of expression in the presence of heavy metal induction proteins allows quick and simple purification from the culture with cadmium or copper. Expression vectors are typically supernatant. Proteins that are not secreted can be obtained maintained by the use of selectable markers such as neomycin from the periplasm by osmotic lysis. Similar to cytoplasmic 55 and hygromycin. expression, in Some cases proteins can become insoluble and d. Mammalian Cells denaturants and reducing agents can be used to facilitate Mammalian expression systems can be used to express solubilization and refolding. Temperature of induction and proteins including Soluble hyaluronidase polypeptides. growth also can influence expression levels and solubility, Expression constructs can be transferred to mammalian cells typically temperatures between 25° C. and 37° C. are used. 60 by viral infection such as adenovirus or by direct DNA trans Typically, bacteria produce aglycosylated proteins. Thus, if fer Such as liposomes, calcium phosphate, DEAE-dextran proteins require glycosylation for function, glycosylation can and by physical means such as electroporation and microin be added in vitro after purification from host cells. jection. Expression vectors for mammalian cells typically b. Yeast Cells include an mRNA cap site, a TATA box, a translational ini Yeasts such as Saccharomyces cerevisae, Schizosaccharo 65 tiation sequence (Kozak consensus sequence) and polyade myces pombe, Yarrowia lipolytica, Kluyveromyces lactis and nylation elements. IRES elements also can be added to permit Pichia pastoris are well known yeast expression hosts that bicistronic expression with another gene. Such as a selectable US 9,084,743 B2 43 44 marker. Such vectors often include transcriptional promoter For secreted molecules, proteins are generally purified from enhancers for high-level expression, for example the SV40 the culture media after removing the cells. For intracellular promoter-enhancer, the human cytomegalovirus (CMV) pro expression, cells can be lysed and the proteins purified from moter and the long terminal repeat of Rous sarcoma virus the extract. When transgenic organisms such as transgenic (RSV). These promoter-enhancers are active in many cell plants and animals are used for expression, tissues or organs types. Tissue and cell-type promoters and enhancer regions can be used as starting material to make a lysed cell extract. also can be used for expression. Exemplary promoter/en Additionally, transgenic animal production can include the hancer regions include, but are not limited to, those from production of polypeptides in milk or eggs, which can be genes Such as elastase I, insulin, immunoglobulin, mouse collected, and if necessary, the proteins can be extracted and mammary tumor virus, albumin, alpha fetoprotein, alpha 1 10 antitrypsin, beta globin, myelin basic protein, myosin light further purified using standard methods in the art. chain 2, and gonadotropic releasing hormone gene control. Proteins, such as soluble hyaluronidase polypeptides, can Selectable markers can be used to select for and maintain cells be purified using standard protein purification techniques with the expression construct. Examples of selectable marker known in the art including but not limited to, SDS-PAGE, size genes include, but are not limited to, hygromycin B phospho 15 fraction and size exclusion chromatography, ammonium Sul transferase, adenosine deaminase, Xanthine-guanine phos fate precipitation and ionic exchange chromatography, Such phoribosyltransferase, aminoglycoside phosphotransferase, as anion exchange. Affinity purification techniques also can dihydrofolate reductase (DHFR) and thymidine kinase. For be utilized to improve the efficiency and purity of the prepa example, expression can be performed in the presence of rations. For example, antibodies, receptors and other mol methotrexate to select for only those cells expressing the ecules that bind hyaluronidase enzymes can be used in affin DHFR gene. Fusion with cell surface signaling molecules ity purification. Expression constructs also can be engineered such as TCR- and FcRI-Y can direct expression of the pro to add an affinity tag to a protein Such as a myc epitope, GST teins in an active state on the cell Surface. fusion or His and affinity purified with myc antibody, glu Many cell lines are available for mammalian expression tathione resin and Ni-resin, respectively. Purity can be including mouse, rat human, monkey, chicken and hamster 25 assessed by any method known in the art including gel elec cells. Exemplary cell lines include but are not limited to CHO, trophoresis and staining and spectrophotometric techniques. Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other F. Preparation, Formulation and Administration of Immune myeloma cell lines, hybridoma and heterohybridoma cell Globulins and Soluble Hyaluronidase Polypeptides lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, Provided herein are co-formulations of IG and hyalu HEK293, 293S, 2B8, and HKB cells. Cell lines also are 30 ronidase that are stable as a liquid formulation for prolonged available adapted to serum-free media which facilitates puri periods of time of at least 6 months attemperatures up to 32 fication of secreted proteins from the cell culture media. C., for example, ranging from at or about 0°C. to 32°C. The Examples include CHO S cells (Invitrogen, Carlsbad, increased stability is characterized by improved storage time, Calif., cat #11619-012) and the serum free EBNA-1 cell line decreased fragmentation, decreased aggregate formation, (Pham et al., (2003) Biotechnol. Bioeng 84:332-42.). Cell 35 decreased dimer formation or/and decreased discoloring, lines also are available that are adapted to grow in special while retaining activity of the IG and hyaluronidase. Such mediums optimized for maximal expression. For example, co-formulations can be provided as “ready-to-use liquid DG44 CHO cells are adapted to grow in suspension culture in formulation without further reconstitution and/or without any a chemically defined, animal product-free medium. requirement for further dilution. The resulting stable co-for e. Plants 40 mulations can be conveniently dispensed to physicians or Transgenic plant cells and plants can be used to express patients in dosage forms for directinjection or administration. proteins such as any described herein. Expression constructs For example, the co-formulations can be infused or injected at are typically transferred to plants using direct DNA transfer home or anywhere. such as microprojectile bombardment and PEG-mediated Soluble hyaluronidases that are co-formulated with transfer into protoplasts, and with agrobacterium-mediated 45 immune globulin permit enhanced delivery of immune globu transformation. Expression vectors can include promoter and linto desired sites within the body by increasing the bioavail enhancer sequences, transcriptional termination elements ability of the immune globulin. Thus, the co-formulations and translational control elements. Expression vectors and achieve elevated and/or more rapidly achieved concentrations transformation techniques are usually divided between dicot of the immune globulin following Subcutaneous administra hosts, such as Arabidopsis and tobacco, and monocot hosts, 50 tion compared to conventional methods of Subcutaneous Such as corn and rice. Examples of plant promoters used for administration, to provide, for example, a more potent and/or expression include the cauliflower mosaic virus promoter, the more rapid response for a given dose. In addition, co-formu nopaline syntase promoter, the ribose bisphosphate carboxy lations of IG containing Soluble hyaluronidases also permit lase promoter and the ubiquitin and UBQ3 promoters. Select lower doses of IG to be administered achieving a given able markers such as hygromycin, phosphomannose 55 response with a lower dose of administered IG. Finally, the isomerase and neomycin phosphotransferase are often used ability of a soluble hyaluronidase to enhance bulk fluid flow at to facilitate selection and maintenance of transformed cells. and near a site of injection or infusion also can improve other Transformed plant cells can be maintained in culture as cells, aspects of associated pharmacologic delivery. For example, aggregates (callus tissue) or regenerated into whole plants. the increase in bulk fluid flow can help to allow the volume of Transgenic plant cells also can include algae engineered to 60 fluid injected to be more readily dispersed from the site of produce hyaluronidase polypeptides. Because plants have injection (reducing potentially painful or other adverse con different glycosylation patterns than mammalian cells, this sequences of injection). This is particularly important for can influence the choice of protein produced in these hosts. Subcutaneous infusions to permit higher doses to be admin 3. Purification Techniques istered. In addition to increased bioavailability, co-formula Method for purification of polypeptides, including soluble 65 tion of IG with hyaluronidase provides for a safer or more hyaluronidase polypeptides or other proteins, from host cells convenient route of administration compared to conventional will depend on the chosen host cells and expression systems. intravenous routes of administration. US 9,084,743 B2 45 46 The co-formulations provided herein are stable for pro require dilution for use. Such liquid preparations can be pre longed periods of time, including at varied temperatures. For pared by conventional means with pharmaceutically accept example, the co-formulations are provided herein are stable able additives Such as Suspending agents (e.g., Sorbitol syrup, and retain activity of the IG and hyaluronidase temperatures cellulose derivatives or hydrogenated edible fats); emulsify up to 32° C. for at least 6 months. For example, the co ing agents (e.g., lecithin or acacia); non-aqueous vehicles formulations are stable at “refrigerator temperatures, for (e.g., almond oil, oily esters, or fractionated vegetable oils); example at 2°C. to 8°C., such as at or about 4°C., for at least and preservatives (e.g., methyl or propyl-p-hydroxyben 6 months to 4 years, such as 1 year to 2 years, for example 6 Zoates or Sorbic acid). In another example, pharmaceutical months, at least 1 year, at least 2 years, at least 3 years or at preparations can be presented in lyophilized form for recon least 4 years or more. In another example, the co-formulations 10 stitution with water or other suitable vehicle before use. are stable and retainactivity at room temperature, for example The pH of the stable co-formulations provided herein is at 18°C. to 32° C., generally 20° C. to 32°C., such as 28°C. Such that the IG in the co-formulation does not aggregate to 32° C., for at least 6 months to 1 year, for example 6 and/or the IG and hyaluronidase retain activity as described in months, at least 7 months, at least 8 months, at least 9 months, Section G. Optimal pH can be obtained by formulation tech or at least 1 year or more. 15 niques known to those skilled in the art. For example, optimal In particular, the stable co-formulations exhibit low to pH can be determined by assessing aggregation and activity undetectable levels of aggregation and/or fragmentation of IG under differing pH conditions using various methods known after storage for defined periods of time. Methods to assess to one of skill in the art, for example, as described in Section aggregation and fragmentation are known to one of skill in the G. Such assays or assessment include, but are not limited to, art, and are exemplified in Section G below. Generally, no size exclusion chromatography, HSPEC determinations, heat more than 0.5% to 5% of IG, for example, no more than 5%, stability data, anticomplement titers of the various prepara no more than 4%, no more than 3%, no more than 2%, no tions and/or hyaluronidase activity assays. Typically, in the more than 1% and generally no more than 0.5% of IG in the co-formulations provided herein the pH can range from 4.0 to co-formulation forms an aggregate, as measured by HPSEC 8.0 as measured in the concentrated solution of the co-formu or other methods, after storage for the defined periods of time 25 lation. Generally, within this range, a lower pH is desired, as set forth above. however, to ensure maximum monomer content. Accord In addition, the IG and hyaluronidase in the stable co ingly, the co-formulations provided herein typically have a formulations provided herein retain one or more activities of pH that is at least or about 4.0 to 7.4, generally at least or about the initial activity of the IG and hyaluronidase prior to stor 4.0 to 6.0, and typically 4.4 to 4.9. As noted, the indicated pH age. One of skill in the art is familiar with activities of IG and 30 is measured in the concentrated Solution of the formulation. hyaluronidase and can assess Such activities. Section G pro pH can be adjusted using acidifying agents to lower the pH or vides exemplary activities and assays to assess activity. Typi alkalizing agents to increase the pH. Exemplary acidifying cally, the stable liquid co-formulations provided herein retain agents include, but are not limited to, acetic acid, citric acid, after storage at least 50%, 60%, 70%, 80%, 90%, 100%, or Sulfuric acid, hydrochloric acid, monobasic sodium phos more of the initial activity of the protein prior to storage, 35 phate solution, and phosphoric acid. Exemplary alkalizing generally at least 70% to 95% of the initial activity. For agents include, but are not limited to, dibasic sodium phos example the stable liquid co-formulations retain after storage phate solution, Sodium carbonate, or Sodium hydroxide. more than 70%, more than 80%, more than 85%, more than Any buffer can be used in the preparation of the liquid 90%, more than 95%, more than 98%, more than 99%, or formulation provided herein so long as it does not adversely more than 99.5% of the initial activity of the respective pro 40 affect the stability of the co-formulation, and supports the tein prior to storage. requisite pH range required. Examples of particularly Suit 1. Formulations and Dosages able buffers include succinate, acetate, phosphate buffers, The co-formulations provided herein are formulated as citrate, aconitate, malate and carbonate. Those of skill in the liquids. The co-formulations contain immune globulin, art, however, will recognize that formulations provided hyaluronidase, at least 0.05 Mofan alkali metal chloride salt, 45 herein are not limited to a particular buffer, so long as the for example, at least 0.05M sodium chloride (NaCl or salt) or buffer provides an acceptable degree of pH stability, or 0.05 M potassium chloride (KCl). The co-formulations also “buffer capacity' in the range indicated. Generally, a buffer are adjusted in pH to limit aggregation and retain activity of has an adequate buffer capacity within about 1 pH unit of its the IG and hyaluronidase. In some examples, the co-formu pK (Lachman et al. 1986). Buffer suitability can be estimated lations do not contain other ingredients except water or Suit 50 based on published pK tabulations or can be determined able solvents. In other examples, the co-formulations further empirically by methods well known in the art. The pH of the contain diluents, carriers or other excipients. solution can be adjusted to the desired endpoint within the Typically, the compounds are formulated into pharmaceu range as described above, for example, using any acceptable tical compositions using techniques and procedures well acid or base. known in the art (see e.g., Ansel Introduction to Pharmaceu 55 a. Immune Globulin tical Dosage Forms, Fourth Edition, 1985, 126). Pharmaceu The IG in the co-formulations is provided at a concentra tically acceptable compositions are prepared in view of tion that is or is about 5% to 22% w/v. for example, that is or approvals for a regulatory agency or other agency prepared in is about 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 accordance with generally recognized pharmacopeia for use mg/mL, 100 mg/mL, 120 mg/mL, 150 mg/mL, 180 mg/mL, in animals and in humans. The formulation should suit the 60 200 mg/mL, 220 mg/mL, 250 mg/mL or more. Generally, the mode of administration. IG in the co-formulation is provided in an amount that is at The co-formulations can be provided as a pharmaceutical least 10% (100 mg/mL) to 20% (200 mg/mL), for example, preparation in liquid form as Solutions, syrups or Suspensions. 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, In liquid form, the pharmaceutical preparations can be pro 20% or more. vided as a concentrated preparation to be diluted to a thera 65 The immune globulin preparations provided herein can be peutically effective concentration before use. Generally, the formulated as pharmaceutical compositions for single or mul preparations are provided in a dosage form that does not tiple dosage use. Typically, as noted elsewhere herein, the IG US 9,084,743 B2 47 48 in the co-formulation is formulated in an amount Such that it monthly IV dose for the particular indication being treated. In is ready to use and that no further dilution is necessary. Such an example, immune globulin preparations can be for Depending on whether the co-formulation is provided as a mulated for single dose administration in an amount Sufficient single or multiple dosage formulation, one of skill in the art to provide a once monthly dose, but can be provided in lesser can empirically determine the exact amount of IG in the amounts for multiple dosage administrations. For example, co-formulation. once monthly doses of IG preparations can be administered Generally, the immune globulin is provided in a therapeu daily, weekly, biweekly or once a month. Dosage regimes can tically effective amount for the particular dosage regime. be continued for months or years. The particular once Therapeutically effective concentration can be determined monthly IV dose is a function of the disease to be treated, and empirically by testing the compounds in known in vitro and in 10 thus can vary. Vivo systems, such as the assays provided herein. The con Exemplary single dosages ranges, in particular for Subcu centration of a selected immune globulin in the composition taneous administration of IG, are from at or about 1 gram (g) depends on absorption, inactivation and excretion rates of the to 200 g, for example, 1 gram (g), 5 g, 10g, 20g, 30 g, 40 g, complex, the physicochemical characteristics of the complex, 50 g. 60 g, 70 g, 80 g, 90 g, 100 g or 200 g. The particular the dosage schedule, and amount administered as well as 15 dosage and formulation thereof depends upon the indication other factors known to those of skill in the art. For example, it and individual. For example, dosages can be administered at is understood that the precise dosage and duration of treat 50 mg/kg body weight (BW) to 600 mg/kg, BW, for example ment is a function of the tissue being treated and may be 50 mg/kg body weight (BW), 100 mg/kg BW, 200 mg/kg BW. determined empirically using known testing protocols or by 300 mg/kg BW, 400 mg/kg BW, 500 mg/kg BW, 600 mg/kg extrapolation from in vivo or in vitro test data. It is to be noted BW, or more. If necessary dosage can be empirically deter that concentrations and dosage values may also vary with the mined. To achieve Such dosages, Volumes of IG-containing age of the individual treated. It is to be further understood that co-formulations administered Subcutaneously can be at or for any particular subject, specific dosage regimens should be about 10 mL to 700 mL, for example, 100 mL to 500 mL, such adjusted over time according to the individual need and the as 200 mL to 400 mL. For example, volumes of IG-containing professional judgment of the person administering or Super 25 co-formulations administered Subcutaneously can be at or vising the administration of the formulations, and that the about 10 mL, 20 mL, 30 mL, 40 mL, 50 ml, 100 ml, 200 ml, concentration ranges set forth herein are exemplary only and 300 ml, 400 ml, 500 ml, 600 ml, 700 ml or more for single are not intended to limit the scope thereof. The amount of a dosage administration. For example, a 10% liquid IG co selected immune globulin preparation to be administered for formulation (100 mg/ml) for indications described hereincan the treatment of a disease or condition, for example an IG 30 be administered in a volume of 200 ml to 700 ml to achieve a treatable disease or condition, can be determined by standard single dosage of 20 g to 70g of IG. In another example, a 20% clinical techniques. In addition, in vitro assays and animal liquid IG co-formulation (200 mg/mL) for indications models can be employed to help identify optimal dosage described herein can be administered in a volume of 100 mL ranges. Hence, the precise dosage, which can be determined to 350 mL to achieve a similar single dosage of 20 g to 70g of empirically, can depend on the particular immune globulin 35 IG. As noted, IG can be provided in lesser amounts in the preparation, the regime and dosing schedule with the soluble co-formulation for multiple dosage administrations. hyaluronidase, the route of administration, the type of disease b. Hyaluronidase to be treated and the seriousness of the disease. The selected hyaluronidase, in particular a soluble hyalu For example, IG preparations can be formulated in phar ronidase, for example, rHuPH20, is included in the co-for maceutical compositions to achieve dosage regimes (doses 40 mulationata concentration that is at or about 50 U/mL to 300 and frequencies) for which current intravenous (IVIG) prepa U/mL, for example 50 U/ml, 75 U/mL, 100 U/ml, 150 U/ml, rations are prepared and administered for particular IG-treat 200U/ml, 300U/mL,400U/ml or 500U/ml, typically at least able diseases or conditions. One of skill in the art is familiar 100U/mL to 300U/mL, generally at a concentration that is 75 with dosage regimes for IVIG administration of particular U/mL to 350 U/mL. If desired, the hyaluronidase can be diseases or conditions. For example, Section H below pro 45 provided in a more concentrated form, for example at or about vides exemplary dosage regimes (doses and frequencies) of 1000 U/mL to 5000 U/mL, such as 1000 U/ml, 1500 Units/ IG for particular diseases and conditions. Other dosage ml, 2000 U/ml, 4000 U/ml or 5000 U/ml. regimes are well known to those of skill in the art. If neces The hyaluronidase in the co-formulation can beformulated sary, a particular dosage and duration and treatment protocol as a pharmaceutical compositions for single or multiple dos can be empirically determined or extrapolated. 50 age administration. As noted above for IG, the hyaluronidase For example, exemplary doses of intravenously adminis in the co-formulation typically is formulated in an amount tered immune globulin can be used as a starting point to that is ready to use Such that no further dilution is necessary. determine appropriate dosages. Dosage levels can be deter Depending on whether the formulation is provided as a single mined based on a variety of factors, such as body weight of ormultiple dosage form, one of skill in the art can empirically the individual, general health, age, the activity of the specific 55 determine the exact amount of hyaluronidase to include in the compound employed, sex, diet, time of administration, rate of co-formulation. excretion, drug combination, the severity and course of the Generally, the selected hyaluronidase, in particular a disease, and the patient’s disposition to the disease and the soluble hyaluronidase, for example, rhuPH20, is included in judgment of the treating physician. Generally, dosages of the co-formulation in an amount Sufficient to exert a thera immune globulin are from or about 100 mg per kg body 60 peutically useful effect of the IG in the absence of undesirable weight (i.e. 100 mg/kg BW) to 2 g/kg BW. It is understood side effects on the patient treated. The therapeutically effec that the amount to administer will be a function of the indi tive concentration can be determined empirically by testing cation treated, and possibly side effects that will be tolerated. the polypeptides in known in vitro and in Vivo systems such as Dosages can be empirically determined using recognized by using the assays provided herein or known in the art (see models for each disorder. 65 e.g., Taliani et al. (1996) Anal. Biochem., 240: 60-67: Filo In one example, IG is provided in an amount that permits camo et al. (1997) J Virology, 71: 1417-1427; Sudo et al. Subcutaneous administration of a dose equivalent to a once (1996) Antiviral Res. 32:9-18; Buffardet al. (1995) Virology, US 9,084,743 B2 49 50 209:52-59: Bianchi et al. (1996) Anal. Biochem., 237: 239 d. Amino Acid Stabilizer 244; Hamatake et al. (1996) Intervirology 39:249-258: The co-formulation provided herein contains an amino Steinkuhler et al. (1998) Biochem., 37:8899-8905; D'Souza acid stabilizer, which contributes to the stability of the prepa et al. (1995) J. Gen. Virol.., 76:1729-1736: Takeshita et al. ration. The stabilizer can be a non-polar and basic amino (1997) Anal. Biochem. 247:242-246; see also e.g., Shimizu et 5 acids. Exemplary non-polar and basic amino acids include, al. (1994) J Virol. 68:8406-8408; Mizutani et al. (1996) J. but are not limited to, alanine, histidine, arginine, lysine, Virol. 70:7219-7223; Mizutani et al. (1996) Biochem. Bio ornithine, isoleucine, Valine, methionine, glycine and proline. phys. Res. Commun., 227:822-826; Lu et al. (1996) Proc. For example, the amino acid stabilizer is glycine or proline, Natl. Acad. Sci. (USA), 93:1412-1417; Hahm et al., (1996) typically glycine. The stabilizer can be a singleamino acid or Virology, 226:318-326: Ito et al. (1996) J Gen. Virol., 10 it can be a combination of 2 or more Such amino acids. The 77:1043-1054: Mizutani et al. (1995) Biochem. Biophys. Res. amino acid stabilizers can be natural amino acids, amino acid Commun., 212:906-911; Cho et al. (1997) J. Virol. Meth. analogues, modified amino acids or amino acid equivalents. 65:201-207 and then extrapolated therefrom for dosages for Generally, the amino acid is an L-amino acid. For example, humans. when proline is used as the stabilizer, it is generally L-proline. For example, a therapeutically effective dose of hyalu 15 It is also possible to use amino acid equivalents, for example, ronidase for single dosage administration is at or about 500 proline analogues. Units to 500,000 Units, for example, 1000 Units to 100,000 Generally, an amount of one or more amino acids effective Units of hyaluronidase. For example, hyaluronidase can be to maintain the immune globulin in monomeric form is added administered, in particular for Subcutaneous administration, to the solution. The concentration of amino acid stabilizer, for at or about 500 Units, 1000 Units, 2000 Units, 5000 Units, example glycine, included in the liquid co-formulation ranges 10,000 Units, 30,000 Units, 40,000 Units, 50,000 Units, from 0.1 M to 1 Mamino acid, typically 0.1 M to 0.75M, 60,000 Units, 70,000 Units, 80,000 Units, 90,000 Units, 100, generally 0.2M to 0.5M, for example, at least at or about 0.1 000 Units or more. As noted, hyaluronidase can be provided M, 0.15 M, 0.2 M, 0.25M, 0.3 M, 0.35M, 0.4M, 0.45 M, 0.5 in lesser amounts in the co-formulation for multiple dosage M, 0.6 M, 0.7 M, 0.75 M or more. The amino acid, for administrations. 25 example glycine, can be used in a form of a pharmaceutically In some examples, dosages can be provided as a ratio IG acceptable salt, Such as hydrochloride, hydrobromide, Sul administered. For example, hyaluronidase can be adminis fate, acetate, etc. The purity of the amino acid, for example tered at 10U/gram (g) to 2000U/g or more of IG, for example, glycine, should be at least 98%, at least 99%, or at least 99.5% at or about 10U/g, 20U/g,30U/g, 40U/g,50U/g, 60U/g, 70 O. O. U/g,80U/g,90U/g, 100U/g, 150U/g, 200U/g, 250U/g,300 30 e. Other Agents U/g, 400 U/g, 500 U/g, 1000 U/g, 1500 U/g, 2000 U/g, 3000 Optionally, the co-formulations can include carriers such U/g IG or more. In general, the ratio of hyaluronidase to IG in as a diluent, adjuvant, excipient, or vehicle with which a a co-formulated product is greater than the ratio when the hyaluronidase or IG is administered. Examples of suitable same products (IG and hyaluronidase) and the same amount pharmaceutical carriers are described in “Remington's Phar of IG are subcutaneously administered separately, for 35 maceutical Sciences” by E. W. Martin. Such compositions example, in a leading edge administration. Thus, generally will contain a therapeutically effective amount of the com the ratio is at least 100U/g, and generally 250U/g or more, for pound, generally in purified form or partially purified form, example 100 U/g to 3000 U/g IG, such as 250 U/g to 1000 together with a suitable amount of carrier so as to provide the U/g, and in particular 250U/g to 750U/g, such as 500U/g IG. form for proper administration to the patient. Such pharma For example, a co-formulation containing 100 U/mL hyalu 40 ceutical carriers can be sterile liquids, such as water and oils, ronidase, when co-formulated with a 20%. IG (200 mg/mL), is including those of petroleum, animal, vegetable or synthetic provided at a ratio that is or is about 500U/g of IG. Typically, origin, Such as peanut oil, soybean oil, mineral oil, and volumes administered subcutaneously can be at or about 10 sesame oil. Wateris a typical carrier when the pharmaceutical mL to 700 mL, such as 50 mL to 500 mL, for example 100 mL composition is administered intravenously. Saline solutions to 400 mL for a single dosage administration. For example, 45 and aqueous dextrose and glycerol solutions also can be volumes administered subcutaneously can be at or about 10 employed as liquid carriers, particularly for injectable solu mL, 20 mL, 30 mL, 40 mL, 50 ml, 100 ml, 200 ml, 300 ml, tions. 400 ml, 500 ml, 600 ml, 700 ml or more for single dosage For example, pharmaceutically acceptable carriers used in administration. parenteral preparations include aqueous vehicles, nonaque c. Alkali metal Chloride Salt 50 ous vehicles, antimicrobial agents, isotonic agents, buffers, The co-formulation provided herein contain analkali metal antioxidants, local anesthetics, Suspending and dispersing chloride salt that is at least 0.05 M. The alkali metal chloride agents, emulsifying agents, sequestering or chelating agents salt includes, but is not limited to, sodium chloride (NaCl) or and other pharmaceutically acceptable Substances. Examples potassium chloride (KCl). Typically, the alkali metal chloride of aqueous vehicles include Sodium Chloride Injection, salt, for example NaCl or KC1, is provided to retain the 55 Ringers Injection, Isotonic Dextrose Injection, Sterile Water stability and activity of the hyaluronidase. The exact amount Injection, Dextrose and Lactated Ringers Injection. Non of salt can be empirically determined by one of skill in the art. aqueous parenteral vehicles include fixed oils of vegetable For example, the amount of salt in the formulations can be origin, cottonseed oil, corn oil, Sesame oil and peanut oil. determined by assessing aggregation and activity under dif Antimicrobial agents in bacteriostatic or fungistatic concen fering salt conditions using various methods known to one of 60 trations can be added to parenteral preparations packaged in skill in the art, for example, as described in Section G. multiple-dose containers, which include phenols or cresols, Typically, in the co-formulations provided herein, Sodium mercurials, benzyl alcohol, chlorobutanol, methyl and propyl chloride is provided in an amount that is or is about 0.05 M to p-hydroxybenzoic acid esters, thimerosal, benzalkonium 0.3 M, for example, at or about 0.05M, 0.06 M, 0.07 M, 0.08 chloride and benzethonium chloride. Isotonic agents include M, 0.09 M, 0.1 M, 0.15 M, 0.2M, 0.25 Mormore. Typically, 65 sodium chloride and dextrose. Buffers include phosphate and the amount of salt is between 0.05 M to 0.25M, for example citrate. Antioxidants include sodium bisulfate. Local anes O.15 M. thetics include procaine hydrochloride. Suspending and dis US 9,084,743 B2 51 52 persing agents include Sodium carboxymethylcellulose, known and practiced in the art. When provided as a multidose hydroxypropyl methylcellulose and polyvinylpyrrolidone. preparation, the formulation can contain a bacteriostatic Emulsifying agents include Polysorbate 80 (TWEENs 80). A agent. sequestering or chelating agent of metal ions include EDTA. 3. Administration Pharmaceutical carriers also include ethyl alcohol, polyeth Co-formulated compositions provided herein typically are ylene glycol and propylene glycol for water miscible vehicles formulated for parenteral administration, for example, by and sodium hydroxide, hydrochloric acid, citric acid or lactic subcutaneous route. Due to the increased bioavailability of IG acid for pH adjustment. in co-formulations with hyaluronidase, immune globulins Compositions can contain along with an active ingredient: can be administered Subcutaneously at dosages and frequen 10 cies for which current intravenous (IVIG) preparations are a diluent such as lactose, Sucrose, dicalcium phosphate, or prepared and administered. The advantages over current Sub carboxymethylcellulose; a lubricant, such as magnesium cutaneous formulations of IG is that co-formulated hyalu Stearate, calcium Stearate and talc, and a binder Such as starch, ronidase/IG can result in more favorable dosing regimens, for natural gums, such as gum acaciagelatin, glucose, molasses, example, less frequent dosing. By less frequent or lower polyvinylpyrrolidone, celluloses and derivatives thereof, 15 dosing, side effects associated with toxicity can be reduced. poVidone, crospovidones and other Such binders known to Generally, the pharmacokinetic and/or pharmacodynamics of those of skill in the art. Subcutaneous IG therapy is improved. In addition, Subcuta For example, an excipient protein can be added to the neous administrations of IG also has advantages over current co-formulation that can be any of a number of pharmaceuti intravenous infusions. For example, Subcutaneous infusion cally acceptable proteins or peptides. Generally, the excipient permits infusion by the patient or family as opposed to a protein is selected for its ability to be administered to a mam skilled nurse; infusion can be achieved at higher rates Such malian Subject without provoking an immune response. For that IG is infused in 1-3 hours compared to 5-10 hours for example, human serum albumin is well-suited for use in conventional IVIG therapies; there is no requirement for pharmaceutical formulations. Other known pharmaceutical functional veins; there is no infusion related side effects such protein excipients include, but are not limited to, starch, glu 25 as thrombosis, headache, thrombophlebitis, and nausea and cose, lactose. Sucrose, gelatin, malt, rice, flour, chalk, silica less probability of adverse events; and infusion can be per gel, Sodium Stearate, glycerol monostearate, talc, sodium formed at home or anywhere. chloride, dried skim milk, glycerol, propylene, glycol, water, Subcutaneous administration also is desired to ensure that and ethanol. The excipient is included in the formulation at a hyaluronidases are administered so that they reach the inter 30 Stitium of skin or tissues, thereby degrading the interstitial Sufficient concentration to prevent adsorption of the protein to space for Subsequent delivery of immunoglobulin. Thus, the holding vessel or vial. The concentration of the excipient direct administration under the skin, such as by subcutaneous will vary according to the nature of the excipient and the administration methods, is contemplated. concentration of the protein in the co-formulation. Administration can be local, topical or systemic depending A composition, if desired, also can contain minor amounts 35 upon the locus of treatment. Local administration to an area in of wetting or emulsifying agents, or pH buffering agents, for need of treatment can be achieved by, for example, but not example, acetate, sodium citrate, cyclodextrine derivatives, limited to, local infusion, topical application, e.g., in conjunc Sorbitan monolaurate, triethanolamine sodium acetate, tri tion with a wound dressing after Surgery, by injection, by ethanolamine oleate, and other Such agents. means of a catheter, by means of a Suppository, or by means 2. Dosage Forms 40 of an implant. Generally, local administration is achieved by The co-formulations provided herein can be formulated as injection, Such as from a Syringe or other article of manufac single or multiple dosage forms. For example, since the co ture containing a injection device Such as a needle. In another formulation provided herein is stable over prolonged periods example, local administration can be achieved by infusion, of time, the co-formulation can be provided in multiple dos which can be facilitated by the use of a pump or other similar age form for administration over an interval of days, weeks, 45 device. months or years. Thus, the liquid co-formulation can be pre Other modes of administration also are contemplated. pared as unit dosage forms. The concentration of the pharma Pharmaceutical composition can be formulated in dosage ceutically active compound is adjusted so that an injection forms appropriate for each route of administration. The most provides an effective amount to produce the desired pharma Suitable route in any given case depends on a variety of cological effect. For example, each unit dose contains a pre 50 factors, such as the nature of the disease, the progress of the determined quantity of therapeutically active compound Suf disease, the severity of the disease the particular composition ficient to produce the desired therapeutic effect, in association which is used. Other routes of administration, Such as any with the required pharmaceutical carrier, vehicle or diluent. route known to those of skill in the art, include but are not The exact dose depends on the age, weight and condition of limited to intramuscular, intravenous, intradermal, intrale the patient or animal as is known in the art. 55 sional, intraperitoneal injection, epidural, nasal, oral, vaginal, Unit dose forms can be administered in fractions or mul rectal, topical, local, otic, inhalational, buccal (e.g., Sublin tiples thereof. A multiple dose form is a plurality of identical gual), and transdermal administration or any route. Formula unit dosage forms packaged in a single container to be admin tions suited for such routes are knownto one of skill in the art. istered in segregated unit dose form. Hence, multiple dose Compositions also can be administered with other biologi form is a multiple of unit doses that are not segregated in 60 cally active agents, either sequentially, intermittently or in the packaging. same composition. Administration also can include con The unit-dose parenteral preparations are packaged in an trolled release systems including controlled release formula ampoule, a vial or a syringe with a needle. The Volume of tions and device controlled release, such as by means of a liquid solution containing the pharmaceutically active com pump. pound is a function of the disease to be treated and the par 65 Subcutaneous administration, generally characterized by ticular article of manufacture chosen for package. All prepa injection or infusion, is contemplated herein. Injectables can rations for parenteral administration must be sterile, as is be prepared in conventional forms, either as liquid solutions US 9,084,743 B2 53 54 or Suspensions, Solid forms Suitable for Solution or Suspen Techniques for infusion are known to one of skill in the art, sion in liquid prior to injection, or as emulsions. Generally, and are within the skill of a treating physician. Generally, the the co-formulations provided herein are prepared as liquids. appropriate dose of IG/hyaluronidase co-formulation can be Injectables are designed for local and systemic administra pooled into a standard IV bag. For example, a non-vented tion. For purposes herein, local administration is desired for 5 infusion set can be used that has a Y-port near its terminus. A direct administration to the affected interstitium. Prepara 24-gauge subcutaneous infusion needle can be inserted at a tions for parenteral administration include sterile solutions site of the subject’s preferences, but the abdomen and sec ready for injection, sterile dry soluble products, such as lyo ondarily the thighs are recommended because of the volume philized powders, ready to be combined with a solvent just of solution to be infused. The hyaluronidase and IG can be prior to use, including hypodermic tablets, sterile Suspensions 10 ready for injection, sterile dry insoluble products ready to be provided in the sameYport apparatus. Other articles of manu combined with a vehicle just prior to use and sterile emul facture also can be used herein for purposes of infusion by sions. The Solutions may be either aqueous or nonaqueous. If gravity or a pump, and include, but are not limited to tubes, administered intravenously, Suitable carriers include physi bottles, Syringes or other containers. ological saline or phosphate buffered saline (PBS), and solu 15 In the event that an infusion is not tolerated (e.g., it causes tions containing thickening and solubilizing agents. Such as moderate to severe local reactions), a second infusion site can glucose, polyethylene glycol, and polypropylene glycol and be started so that the subject receives the full dosage. mixtures thereof. Further, it is understood that the stable co-formulations Administration methods can be employed to decrease the provided herein are amenable to dosage regimes involving a exposure of selected compounds to degradative processes, periodic frequency of administration. For example, the dos Such as proteolytic degradation and immunological interven age frequency can be daily over an interval of time given over tion via antigenic and immunogenic responses. Examples of consecutive or alternate days, for example, 1, 2, 3, 4, 5, 6, 7, Such methods include local administration at the site of treat 8, 9, 10 or more days. In other examples, the dosage regime is ment. PEGylation of therapeutics has been reported to weekly, for example, once every week, every two weeks, increase resistance to proteolysis, increase plasma half-life, 25 every three weeks, every four weeks, every five weeks, every and decrease antigenicity and immunogenicity. Examples of six weeks or more. Thus, an IG/hyaluronidase preparation PEGylation methodologies are known in the art (see for can be administered at once, or can be divided into a number example, Lu and Felix, Int. J. Peptide Protein Res., 43: 127 of smaller doses to be administered at intervals of time. 138, 1994; Luand Felix, Peptide Res., 6: 142-6, 1993: Felix et Selected IG/hyaluronidase preparations can be adminis al., Int. J. Peptide Res., 46: 253-64, 1995; Benhar et al., J. 30 tered in one or more doses over the course of a treatment time Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., JImmu for example over several hours, days, weeks, or months. In mol., 154:3088-95, 1995; see also, Caliceti et al. (2003) Adv. some cases, continuous administration is useful. It is under Drug Deliv Rev. 55(10): 1261-77 and Molineux (2003) Phar stood that the precise dosage and course of administration macotherapy 23 (8 Pt 2):3S-8S). Pegylation also can be used depends on the indication and patients tolerability. in the delivery of nucleic acid molecules in vivo. For example, 35 Also, it is understood that the precise dosage and duration pegylation of adenovirus can increase stability and gene of treatment is a function of the disease being treated and can transfer (see, e.g., Cheng et al. (2003) Pharm. Res. 2009): be determined empirically using known testing protocols or 1444–51). by extrapolation from in vivo or in vitro test data. It is to be Where large Volumes are administered, administration is noted that concentrations and dosage values also can vary typically by infusion. Subjects can be dosed at rates of infu 40 with the severity of the condition to be alleviated. It is to be sion at or about 0.5 ml/kg/BW/h to 5 ml/kg/BW/h, for further understood that for any particular subject, specific example at or about 0.5 ml/kg/BW/h, 1 ml/kg/BW/h, 2 ml/kg/ dosage regimens should be adjusted over time according to BW/h, 3 ml/kg/BW/h, 4 ml/kg/BW/h, or 5 ml/kg/BW/h. The the individual need and the professional judgment of the infusion rate can be empirically determined, and typically is person administering or Supervising the administration of the a function of the tolerability of the subject. If an adverse 45 compositions, and that the concentration ranges set forth reaction occurs during the infusion, the rate of infusion can be herein are exemplary only and are not intended to limit the slowed to the rate immediately below that at which the Scope or use of compositions and combinations containing adverse event occurred. If the adverse event resolves in them. The compositions can be administered hourly, daily, response to the reduction in rate, the infusion rate can be weekly, monthly, yearly or once. Generally, dosage regimens slowly increased at the discretion of the physician. Subcuta 50 are chosen to limit toxicity. It should be noted that the attend neous infusion of IG co-formulations can be facilitated by ing physician would know how to and when to terminate, gravity, pump infusion or injection of a desired dose, for interrupt or adjust therapy to lower dosage due to toxicity, or example, a full 20-30 gram dose. Generally, for infusions bone marrow, liver or kidney or other tissue dysfunctions. intravenous infusion pumps can be employed. IG/hyalu Conversely, the attending physician would also know how to ronidase co-formulations can be infused at rates at or about 5 55 and when to adjust treatment to higher levels if the clinical ml/h, 10 ml/h, 30 ml/h, 60 ml/h, 120 ml/h, 240 ml/h or 300 response is not adequate (precluding toxic side effects). ml/h. Infusion rates can be increased during the course of G. Methods of Assessing Stability, Activity, Bioavailability treatment so long as the infusion is tolerated by the patient. and Pharmacokinetics Generally, time of administration of infusion is at or about 0.5 The stability and activity of IG and hyaluronidase in the h, 1 h, 1.5 h, 2 h, 2.5h, 3 h, 4 h or more. Due to the high rate 60 formulations can be assessed using various in vitro and in of infusion achieved by subcutaneous administration of IG vivo assays that are known to one of skill in the art. Various co-formulated with hyaluronidase, the time of infusion is analytical techniques for measuring protein stability are significantly less than for conventional IVIG therapies. available in the art and are reviewed in Peptide and Protein Where infusion time exceeds the desired limit, a second infu Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, sion site can be started at the physician and Subjects discre 65 Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug tion. The second site typically is started at least 10 cm from Delivery Rev. 10: 29-90 (1993). Stability can be measured at the initial site. a selected temperature for a selected time period. US 9,084,743 B2 55 56 Assays to assess molecular size (e.g. caused by aggrega ACA titer is not generally a determinative indicator for co tion, denaturation and/or fragmentation) of the IG is an formulations that are formulated for Subcutaneous adminis important consideration for assessing stability of the co-for tration. mulation. In addition, the stability of the liquid formulations Generally, the ACA assay measures the amount of comple also can be assessed by any assays which measure the bio ment that is bound by the mixture of standardized amounts of logical activity of IG and hyaluronidase in the formulation. complement and protein (see e.g., Palmer, D. F. and Whaley, Such assays are well known in the art. In addition to assessing S.D., Complement Fixation Test, in Manual of Clinical Labo the stability of the co-formulation, such assays can be used, ratory Immunology (Ed. N. R. Rose, et al., American Society for example, to determine appropriate dosages of immune for Microbiology, Washington, D.C., 1986) pp. 57-66: 10 Mayer, M. M., Quantitative C' Fixation Analysis, Comple globulin and hyaluronidase, and the frequency of dosing, for ment and Complement Fixation, in Experimental Immu treatment. Further, assays known to one of skill in the art also nochemistry (Ed. E. A. Kabat and M. M. Meyer, Thomas, can be performed to assess the pharmacokinetic properties of Springfield, Ill., 1961), pp. 214-216, 227-228.) Briefly, red Subcutaneously-administered immune globulin, including blood cells that have been sensitized by preincubation with bioavailability, and tolerability. 15 red blood cellantibodies are added to the complement/protein 1. Molecular Size mixture. In the presence of free complement (not already The main stability indicating parameter is molecular size, bound by the protein) these sensitized cells will lyse, releas and a change in size may be the result of degradation by ing hemoglobin which can be quantitated as a measure of the denaturation, aggregation or fragmentation. Aggregation of degree of lysis. In parallel, sensitized red blood cells are also IG is a common problem during storage of IG products. The added to a buffer control-complement mixture, whose degree aggregates are problematic because they can combine with of lysis is defined as 100%. The difference between the actual complement in the patient’s blood and produce an anti amount of complement needed to give 100% lysis and the complement reaction. The ability of IG to bind complement is amount of complement remaining unbound in the presence of greatly increased as a result of denaturation, in particular by protein equals the amount of complement actually bound by aggregation to high molecular weight species. The comple 25 the protein, or anticomplement activity. One unit of ACA ment binding mechanism of these aggregates appears to be activity (one CHso unit) is the amount of protein capable of identical to that of antigen-antibody complexes. Marcus, D. activating 50% of the complement in an optimally titered M., (1960) J. Immunol. 84:273-284. In the case of IgG, it is complement and red blood cell/hemolysin System. Generally, known that the complement binding site requires two mol an acceptable ACA titer is less than 50% CH50 units con ecules close together. It is therefore possible that critical 30 Sumed per mg protein. packing of the molecules is required, rather than any neces In another example, molecular size distribution, for sary conformational change. example due to aggregate formation, during storage of a Methods for monitoring stability of IG are available in the liquid co-formulation can be readily determined by measur art, including those methods described herein and in the ing the change in Soluble protein in Solution over time. examples disclosed herein. There are various methods avail 35 Amount of soluble polypeptide in solution can be quantified able for assessing the stability of protein formulations, by a number of analytical assays. Such assays include, for including antibody or immunoglobulin formulations, based example, reverse phase (RP)-HPLC and UV absorption spec on the physical and chemical structures of the proteins as well troscopy. Determination of both soluble and insoluble aggre as on their biological activities. For example, to study aggre gates during storage in liquid formulations can be achieved, gation, fragmentation and denaturation of proteins, methods 40 for example, using analytical ultracentrifugation to distin Such as charge-transfer absorption, thermal analysis, fluores guish between that portion of the soluble polypeptide that is cence spectroscopy, circular dichroism, NMR, reduced cap present as Soluble aggregates and that portion that is present illary gel electrophoresis (rCGE), and high performance size in the nonaggregate, biologically active molecular form. exclusion chromatography (HPSEC), are available. See, for In a further example, the stability of co-formulations can be example, Wang et al., 1988, J. of Parenteral Science & Tech 45 assessed by heating the finished product to a temperature of nology 42(supp):S4-S26. TherCGE, and HPSEC are the most 57°C. and holding it at that temperature for four hours while common and simplest methods to assess the molecular size examining the product for visual precipitates. (See e.g., Code due to formation of protein aggregates, protein degradation of Federal Regulations 21, Food and Drugs, 640. 101 a (re and protein fragmentation. Further, the anticomplement vised Apr. 1978)). In a modification of the method (see e.g., activity (ACA) can be directly determined. 50 Fernandes and Lundblad, Vox Sang 39:101-112 (1980)), For example, the stability of the liquid formulations can be approximately 2 milliliters of the test product is heated at 57 evaluated by HPSEC or rCGE, where the percentage area of C. for four hours and then the percent change in degree of the peaks represents the non-degraded protein. In one opalescence as measured by recording the transmittance at example, protein is injected onto a TosoH Biosep TSKG3000 580 nm with a laboratory spectrophotometer is evaluated (see SW 600x7.5 mm column. The protein is eluted. Eluted pro 55 also U.S. Pat. No. 4,597,966). tein is detected using UV absorbance at 280 nm. A reference SDS-PAGE also can be used to assess aggregation and/or standard is run in the assay as a control, and the results are fragmentation. The density or the radioactivity of each band reported as the area percent of the product monomer peak stained or labeled with radioisotope can be measured and the compared to all other peaks excluding the included Volume % density or % radioactivity of the band representing non peak. Peaks eluting earlier than the monomer peak are 60 degraded protein can be obtained. recorded as percent aggregate. Generally, the co-formulations exhibit low to undetectable ACA titer also can be determined as described in the Euro levels of aggregation as measured by any of the above assays, pean Pharmacopoeia (European Pharmacopeia, 1997, 2" ed. for example HPSEC or rCGE. For example, the aggregation Part II. Maisonneuve, S.A., Saint Ruffine, France). Gener is, no more than 5%, no more than 4%, no more than 3%, no ally, ACA titer is a specification indicator for intravenous (IV) 65 more than 2%, no more than 1%, and generally no more than administration and is not relevant for Subcutaneous adminis 0.5% aggregate by weight protein, and low to undetectable tration of the co-formulations. Thus, for purposes herein, levels of fragmentation, that is, 80% or higher, 85% or higher, US 9,084,743 B2 57 58 90% or higher, 95% or higher, 98% or higher, or 99% or which residual biotinylated hyaluronic acid is measured fol higher, or 99.5% or higher of the total peak area in the peak(s) lowing incubation with hyaluronidase (see e.g. Frost and representing intact antibodies or fragments thereof. For Stern (1997) Anal. Biochem. 251:263-269, U.S. Patent Pub example, typically, an acceptable aggregation includes >90% lication No. 20050260186). The free carboxyl groups on the monomers and oligo-fcdimers; <5% aggregates, and <5% 5 glucuronic acid residues of hyaluronic acid are biotinylated, fragments. and the biotinylated hyaluronic acid substrate is covalently 2. Biological Activity couple to a microtiterplate. Following incubation with hyalu a. Immune Globulin ronidase, the residual biotinylated hyaluronic acid substrate is The ability of immune globulinto act as a therapeutic agent detected using an avidin-peroxidase reaction, and compared can be assessed in vitro or in vivo. For example, in vitro assays 10 to that obtained following reaction with hyaluronidase stan can be performed to assess the ability of immune globulin to dards of known activity. Other assays to measure hyalu neutralize viral or bacterial infectivity (Hiemstra et al., (1994) ronidase activity also are known in the art and can be used in J Lab Clin Med 123:241-6). Other in vitro assays can be the methods herein (see e.g. Delpech et al., (1995) Anal. utilized to assess other biological activities of immune globu Biochem. 229:35-41: Takahashi et al., (2003) Anal. Biochem. lin. For example, the ability of immune globulin preparations 15 322:257-263). to interact with and modulate complement activation prod The ability of hyaluronidase to act as a spreading or dif ucts, bind idiotypic antibody, bind Fc receptors on macroph fusing agent also can be assessed. For example, trypan blue ages, and Suppress various inflammatory mediators including dye can be injected subcutaneously with or without hyalu cytokines, chemokines, and metalloproteinases, can be ronidase into the lateral skin on each side of nude mice. The assessed using any method known in the art, including, but dye area is then measured, Such as with a microcaliper, to not limited to, ELISA, Western blot, Northern blot, and flow determine the ability of hyaluronidase to act as a spreading cytometry to assess marker expression. For example, the agent (U.S. Patent No. 20060104968). effect of immune globulin on the expression of chemokine 3. Pharmacokinetics and Tolerability receptors on peripheral blood mononuclear cells can be Pharmacokinetic and tolerability studies can be performed assessed using flow cytomtery (Trebst et al., (2006) Eur J 25 using animal models or can be performed during clinical Neurology 13(12): 1359–63). In another example, the effect of studies with patients. Animal models include, but are not immune globulin on metalloproteinase expression in mac limited to, mice, rats, rabbits, dogs, guinea pigs and non rophages can be assessed using Northern blot analysis (Sha human primate models, such as cynomolgus monkeys or piro et al., (2002) Cancer 95:2032-2037). rhesus macaques. In some instances, pharmacokinetic and In vivo studies using animal models also can be performed 30 tolerability studies are performed using healthy animals. In to assess the therapeutic activity of immune globulin. other examples, the studies are performed using animal mod Immune globulin can be administered to animal models els of a disease for which therapy with immune globulin is infected with one or more microorganisms and the effect on considered, such as animal models of any of the diseases and progression of infection can be assessed, such as by measur conditions described below. ing the number of microorganisms or measuring weight as a 35 The pharmacokinetics of Subcutaneously administered marker of morbidity. The therapeutic effect of immune globu immune globulin can be assessed by measuring Such param lin also can be assessed using animal models of the diseases eters as the maximum (peak) plasma immune globulin con and conditions for which therapy using immune globulin is centration (C), the peak time (i.e. when maximum plasma considered. Such animal models are known in the art, and immune globulin concentration occurs; T), the minimum include, but are not limited to, small animal models for 40 plasma immune globulin concentration (i.e. the minimum X-linked agammaglobulinemia (XLA), SCID, Wiskott-Ald plasma concentration between doses of immune globulin; rich syndrome, Kawasaki disease, Guillain-Barré syndrome, C), the elimination half-life (T) and area under the curve ITP polymyositis, Lambert-Eaton myasthenic syndrome, (i.e. the area under the curve generated by plotting time versus Myasthenia gravis and Moersch-Woltmann syndrome (CZ plasma immune globulin concentration; AUC), following itrometal. (1985).J Immunol 134:2276-2280, Ellmeier et al., 45 administration. The absolute bioavailability of subcutane (2000).J Exp Med. 192: 1611-1624. Ohno (2006) Drug Dis ously administered immune globulin is determined by com covery Today. Disease Models 3:83-89, Oyaizu et al. (1988) paring the area under the curve of immune globulin following J Exp Med2017-2022, Hansenet al., (2002) Blood 100:2087 subcutaneous delivery (AUC) with the AUC of immune 2093, Strongwater et al., (1984) Arthritis Rheum. 27:433-42, globulin following intravenous delivery (AUC). Absolute Kim et al. (1998) Annals NYAcadSci 841:670-676, Christa 50 bioavailability (F), can be calculated using the formula: doss et al. (2000) Clin. Immunol. 94:75-87. Sommer et al., F=(AUC.xdose)/(LAUCIxdose). The concentration of (2005) Lancet 365:1406-1411 and U.S. Pat. No. 7,309,810) immune globulin in the plasma following Subcutaneous b. Hyaluronidase administration can be measured using any method known in Hyaluronidase activity can be assessed using methods well the art Suitable for assessing concentrations of immune known in the art. In one example, activity is measured using 55 globulin in samples of blood. Exemplary methods include, a microturbidity assay. This is based on the formation of an but are not limited to, ELISA and nephelometry. insoluble precipitate when hyaluronic acid binds with serum A range of doses and different dosing frequency of dosing albumin. The activity is measured by incubating hyalu can be administered in the pharmacokinetic studies to assess ronidase with sodium hyaluronate (hyaluronic acid) for a set the effect of increasing or decreasing concentrations of period of time (e.g. 10 minutes) and then precipitating the 60 immune globulin and/or hyaluronidase in the dose. Pharma undigested sodium hyaluronate with the addition of acidified cokinetic properties of subcutaneously administered immune serum albumin. The turbidity of the resulting sample is mea globulin, such as bioavailability, also can be assessed with or sured at 640 nm after an additional development period. The without co-administration of hyaluronidase. For example, decrease in turbidity resulting from hyaluronidase activity on dogs, such as beagles, can be administered immune globulin the Sodium hyaluronate Substrate is a measure of hyalu 65 Subcutaneously in combination with hyaluronidase, or alone. ronidase enzymatic activity. In another example, hyalu Intravenous doses of immune globulin also are given to ronidase activity is measured using a microtiter assay in another group of beagles. Blood samples can then be taken at US 9,084,743 B2 59 60 various time points and the amount of immune globulin in the flammatories, non-steroidal anti-inflammatories, and other plasma determine. Such as by nephelometry. The AUC can immunomodulatory agents such as cytokines, chemokines then be measured and the bioavailability of subcutaneously and growth factors. administered immune globulin administered with or without If necessary, aparticular dosage and duration and treatment hyaluronidase can be determined. Such studies can be per protocol can be empirically determined or extrapolated. For formed to assess the effect of co-administration with hyalu example, exemplary doses of intravenously administered ronidase on pharmacokinetic properties, such as bioavailabil immune globulin can be used as a starting point to determine ity, of Subcutaneously administered immune globulin. appropriate dosages. Dosage levels can be determined based Studies to assess safety and tolerability also are known in on a variety of factors, such as body weight of the individual, the art and can be used herein. Following Subcutaneous 10 general health, age, the activity of the specific compound administration of immune globulin, with or without co-ad ministration of hyaluronidase, the development of any employed, sex, diet, time of administration, rate of excretion, adverse reactions can be monitored. Adverse reactions can drug combination, the severity and course of the disease, and include, but are not limited to, injection site reactions, such as the patient’s disposition to the disease and the judgment of the edema or Swelling, headache, fever, fatigue, chills, flushing, 15 treating physician. Exemplary dosages of immune globulin dizziness, urticaria, wheezing or chest tightness, nausea, and hyaluronidase are provided elsewhere herein. It is under Vomiting, rigors, back pain, chest pain, muscle cramps, sei stood that the amount to administer will be a function of the Zures or convulsions, changes in blood pressure and anaphy indication treated, and possibly side effects that will be tol lactic or severe hypersensitivity responses. Typically, a range erated. Dosages can be empirically determined using recog of doses and different dosing frequencies are be administered nized models for each disorder. in the safety and tolerability studies to assess the effect of Upon improvement of a patient's condition, a maintenance increasing or decreasing concentrations of immune globulin dose of immune globulin can be administered subcutaneously and/or hyaluronidase in the dose. in combination with hyaluronidase, if necessary, and the dos H. Methods of Treatment and Therapeutic Uses age, the dosage form, or frequency of administration, or a The IG/hyaluronidase co-formulations described herein 25 combination thereof can be modified. In some cases, a subject can be used for treatment of any condition for which immune can require intermittent treatment on a long-term basis upon globulin is employed. Immune globulin (IG) can be admin any recurrence of disease symptoms. istered subcutaneously in co-formulations with hyalu 1. Primary and Secondary Immune Deficiency ronidase, to treat any condition that is amendable to treatment a. Primary Immune Deficiency with immune globulin. This section provides exemplary 30 More than 80 primary immune deficiency diseases are therapeutic uses of IG/hyaluronidase co-formulations. It is recognized by the World Health Organization and occur in understood that the IG/hyaluronidase co-formulations pro about 1 out of 10,000 individuals. These diseases are charac vided herein can be used in methods, processes or uses to treat terized by an intrinsic defect in the immune system in which, any of the diseases and conditions described below and other in some cases, the body is unable to produce any or enough diseases and conditions known to one of skill in the art that are 35 antibodies against infection. In other cases, cellular defenses treatable by IG. In particular, subcutaneous administration of to fight infection fail to work properly. Immune globulin can the co-formulations is contemplated. Dosages of IG admin be used to treat primary immune deficiency with antibody istered is the same or similar to the dosage administered deficiency. Thus, immune globulin can be administered as intravenously and known to one of skill in the art. The dosage immunoglobulin replacement therapy to patients presenting regime and frequency can vary from intravenous regimes as 40 with Such diseases. described elsewhere herein. The therapeutic uses described Typically, primary immune deficiencies are inherited dis below are exemplary and do not limit the applications of the orders. Exemplary of primary immune deficiencies include, methods described herein. but are not limited to, common variable immune deficiency For example, co-formulations provided herein can be used (CVID), selective IgA deficiency, IgG subclass deficiency, to treat immune deficiencies such as primary immune defi 45 X-linked agammaglobulinemia (XLA), severe combined ciencies, such as X-linked agammaglobulinemia, hypogam immune deficiency (SCID), complement disorders, ataxia maglobulinemia, and acquired compromised immunity con telangiectasia, hyper IgM, and Wiskott-Aldridge syndrome. ditions (secondary immune deficiencies). Such as those Immune globulin/hyaluronidase co-formulations can be featuring low antibody levels; inflammatory and autoimmune administered subcutaneously to patients with primary diseases; and acute infections. Therapeutic uses include, but 50 immune deficiency diseases with antibody deficiency at are not limited to, immunoglobulin replacement therapy and doses similar to the doses used for intravenous administration immunomodulation therapy for various immunological, of immune globulin. Exemplary doses include, for example, hematological, neurological, inflammatory, dermatological between 100 mg/kg BW and 800 mg/kg BW immune globu and/or infectious diseases and conditions. In some examples, lin, at four-week intervals. The dose can be increased or immune globulin is administered to augment the immune 55 decreased, as can the frequency of the doses, depending on response in healthy patients, such as following possible expo the clinical response. Sure to infectious disease (e.g. accidental needle Stick injury). b. Secondary Immune Deficiency IG co-formulations provided herein also can be used for Secondary, or acquired, immune deficiency is not the result treating multiple Sclerosis (especially relapsing-remitting of inherited genetic abnormalities, but rather occurs in indi multiple sclerosis or RRMS), Alzheimer's disease, and Par 60 viduals in which the immune system is compromised by kinson's disease. It is within the skill of a treating physician to factors outside the immune system. Examples include, but are identify Such diseases or conditions. not limited to, trauma, viruses, chemotherapy, toxins, and Immune globulin/hyaluronidase co-formulations can be pollution. Acquired immunodeficiency syndrome (AIDS) is administered in combination with other agents used in the an example of a secondary immune deficiency disorder treatment of these diseases and conditions. For example, 65 caused by a virus, the human immunodeficiency virus (HIV), other agents that can be administered include, but are not in which a depletion of T lymphocytes renders the body limited to, antibiotics, chemotherapeutics, steroidal anti-in unable to fight infection. US 9,084,743 B2 61 62 Another example, hypogammaglobulinemia, is caused by g/kg BW immune globulin is administered as a single dose a lack of B-lymphocytes, is characterized by low levels of over a 10 hour period. The amount of immune globulin antibodies in the blood, and can occur in patients with chronic administered can be increased or decreased as appropriate. lymphocytic leukemia (CLL), multiple myeloma (MM), non b. Chronic Inflammatory Demyelinating Polyneuropathy Hodgkin’s lymphoma (NHL) and other relevant malignan Chronic inflammatory demyelinating polyneuropathy cies as a result of both leukemia-related immune dysfunction (CIDP) is a neurological disorder characterized by progres and therapy-related immunosuppression. Patients with sive weakness and impaired sensory function in the legs and acquired hypogammaglobulinemia secondary to such hema arms. The disorder, which is sometimes called chronic relaps tological malignancies, and those patients receiving post ing polyneuropathy, is caused by damage to the myelin sheath hematopoietic stem cell transplantation are Susceptible to 10 bacterial infections. The deficiency in humoral immunity is of the peripheral nerves. Although it can occurat any age and largely responsible for the increased risk of infection-related in both genders, CIDP is more common in young adults, and morbidity and mortality in these patients, especially by in men more so than women. It often presents with symptoms encapsulated microorganisms. For example, Streptococcus that include tingling or numbness (beginning in the toes and pneumoniae, Haemophilus influenzae, and Staphylococcus 15 fingers), weakness of the arms and legs, loss of deep tendon aureus, as well as Legionella and Nocardia spp. are frequent reflexes (areflexia), fatigue, and abnormal sensations. CIDP bacterial pathogens that cause pneumonia in patients with is closely related to Guillain-Barré syndrome and is consid CLL. Opportunistic infections such as Pneumocystis carinii, ered the chronic counterpart of that acute disease. There is no fungi, viruses, and mycobacteria also have been observed. specific diagnostic test, but characteristic clinical and labora The number and severity of infections in these patients can be tory findings help distinguish this disorder from other significantly reduced by administration of immune globulin immune mediated neuropathic syndromes. (Griffiths et al. (1989) Blood 73:366-368; Chapeletal. (1994) Studies indicate that treatment with immune globulin Lancet 343:1059-1063). reduces symptoms (van Schaik et al. (2002) Lancet Neurol. Therefore, immune globulin/hyaluronidase co-formula 1:497-498). Thus, immune globulin/hyaluronidase co-for tions can be administered Subcutaneously in Such patients to 25 mulations can be administered Subcutaneously to patients prevent recurrent infections. Exemplary dosages include presenting with CIDP using the methods described herein. those used for intravenous administration of immune globulin Exemplary dosages include those used for intravenous to patients with acquired hypogammaglobulinemia second administration of immune globulin to patients with CIDP. In ary to hematological malignancies. For example, co-formu one example, a patient with, CIDP is administered about 2 lations containing about 400 mg/kg BW immune globulin can 30 g/kg BW of immune globulin Subcutaneously, incombination be administered subcutaneously every 3 to 4 weeks. In a with hyaluronidase. This can be administered, for example, in further example, an additional dose of 400 mg/kg BW can be five doses of 400 mg/kg BW for five consecutive days. The administered in the first month of therapy in cases where the amount of immune globulin administered can be increased or patient’s serum IgG is less than 4 g/L. The amount of immune decreased as appropriate. globulin administered, and the frequency of the doses, can be 35 c. Guillain-Barré Syndrome increased or decreased as appropriate. Guillain-Barré syndrome is a neurologic autoimmune dis 2. Inflammatory and Autoimmune Diseases order involving inflammatory demyelination of peripheral a. Kawasaki Disease nerves. The first symptoms include varying degrees of weak Kawasaki disease is an acute, febrile, multi-system disease ness ortingling sensations in the legs, which can spread to the of children and young infants, often involving the coronary 40 arms and upper body. These symptoms can increase in inten arteries. It also is known as lymph node syndrome, mucocu sity until the muscles cannot be used at all and the patient is taneous node disease, infantile polyarteritis and Kawasaki almost totally paralyzed, resulting in a life-threatening con syndrome. Kawasaki disease is a poorly understood, self dition. Although recovery is generally good or complete in limited vasculitis that affects many organs, including the skin, the majority of patients, persistent disability has been mucous membranes, lymph nodes, blood vessel walls, and 45 reported in about 20% of all patients and death in 4 to 15% of the heart. Coronary artery aneurysms can occur from the patients. Guillain-Barré syndrome can occur a few days or second week of illness during the convalescent stage. weeks after symptoms of a respiratory or gastrointestinal Although the cause of the condition is unknown, there is viral infection. In some instances, Surgery or vaccinations can evidence that the characteristic vasculitis results from an trigger the syndrome. The disorder can develop over the immune reaction characterized by T-cell and macrophage 50 course of hours or days, or it may take up to 3 to 4 weeks. A activation to an unknown antigen, Secretion of cytokines, nerve conduction velocity (NCV) test can give a doctor clues polyclonal B-cell hyperactivity, and the formation of autoan to aid the diagnosis. In some instances, a spinal tap can be tibodies to endothelial cells and smooth muscle cells. In used in diagnosis, as the cerebrospinal fluid in Guillain-Barré genetically Susceptible individuals, one or more uncharacter syndrome patients typically contains more protein than nor ized common infectious agents, possibly with Super-antigen 55 mal subjects. Although there is no known cure for Guillain activity, may trigger the disease. Barré syndrome, treatment with immune globulin can lessen Immune globulin administered early in Kawasaki disease the severity of the illness and accelerate recovery. Immune can prevent coronary artery pathology. Subcutaneous admin globulin/hyaluronidase co-formulations can be administered istration of immune globulin/hyaluronidase co-formulations Subcutaneously to patients at an appropriate dose of IG, Such to patients with ongoing inflammation associated with 60 as, for example, a dose similar to the dose used to administer Kawasaki disease can ameliorate symptoms. Exemplary dos immune globulin intravenously to patients with Guillain ages include those used for intravenous administration of Barré syndrome. For example, a patient with Guillain-Barré immune globulin to patients with Kawasaki disease. For syndrome can be administered about 2 g/kg BW of immune example, a patient with Kawasaki disease can be adminis globulin, in combination with hyaluronidase, Subcutane tered about 1-2 g/kg patient body weight of immune globulin. 65 ously. This can be administered, for example, in five doses of This can be administered, for example, in four doses of 400 400 mg/kg BW for five consecutive days. The amount of mg/kg BW for four consecutive days. In another example, 1 immune globulin administered can be increased or decreased US 9,084,743 B2 63 64 depending on, for example, the severity of the disease and the have difficulty breathing due to muscle failure. As many as clinical response to therapy, which can be readily evaluated one-third of PM patients have muscle pain. The disease by one of skill in the art. affects more women than men, and rarely affects people d. Idiopathic Thrombocytopenic Purpura under the age of 20, although cases of childhood and infant Idiopathic thrombocytopenic purpura (ITP), also known as polymyositis have been reported. primary immune thrombocytopenic purpura and autoim iii. Inclusion Body Myositis mune thrombocytopenic purpura, is a reduction in platelet Inclusion body myositis (IBM) is very similar to polymyo count (thrombocytopenia) resulting from shortened platelet sitis. Onset of muscle weakness in IBM is usually very survival due to anti-platelet antibodies. When platelet counts gradual, taking place over months or years. It differs from PM are very low (e.g., <30x10/L), bleeding into the skin (pur 10 in that both proximal and distal muscles are affected, while pura) and mucous membranes can occur. Bone marrow plate generally only the proximal muscles are affected in PM. let production (megakaryopoiesis) in patients with ITP is Typical findings include weakness of the wrist flexors and morphologically normal. In some instances, there is addi finger flexors. Atrophy of the forearms and the quadriceps tional impairment of platelet function related to antibody muscle is characteristic of the disease, with varying degrees binding to glycoproteins on the platelet Surface. ITP can 15 of weakness in other muscles. Approximately half of the present as chronic and acute forms. Approximately 80% of patients afflicted with IBM have difficulty swallowing. adults with ITP have the chronic form of the disease. The Symptoms of IBM usually begin after age 50, although no age highest incidence of chronic ITP is in women aged 15-50 group is excluded. IBM occurs more frequently in men than years, although some reports suggest increasing incidence women. About one in ten cases of IBM may be hereditary. with age. ITP is relatively common in patients with HIV. Studies indicate that administration of immune globulin While ITP can be found at any stage of the infection, its can benefit patients with these inflammatory myopathies. prevalence increases as HIV disease advances. Immune globulin can improve muscle strength, reduce Studies have demonstrated that immune globulin can be inflammation and reduce disease progression and severity used to treat patients with ITP (Godeau et al. (1993) Blood (Dalakas et al. (1993) N. Engl. J. Med. 329(27): 1993-2000; 82(5):1415-21; Godeau et al. (1999) Br. J. Haematol. 107(4): 25 Dalakaset al. (2001) Neurology 56(3):323-7; Dalakas (2004) 716-9). Immune globulin/hyaluronidase co-formulations can Pharmacol. Ther: 102(3):177-93; Walter et al. (2000).J. Neu be administered subcutaneously to patients at an IG dose rol. 247(1):22-8). Immune globulin/hyaluronidase co-formu similar to the dose used to administer immune globulin intra lations can be administered subcutaneously to patients with venously to treat patients with ITP. For example, a patient DM, PM or IBM at a dose of IG similar to the dose used to with ITP can be administered about 1 to 2 g/kg BW of 30 administer immune globulin intravenously. For example, 2 immune globulin, in combination with hyaluronidase, Subcu g/kg BW of immune globulin can be administered, typically taneously. This can be administered over several days, or can over several days, such as, for example, five doses of 400 be administered in one dose. In some examples, five doses of mg/kg BW on consecutive days. 400 mg/kg BW immune globulin on consecutive days is f. Lambert-Eaton Myasthenic Syndrome administered. In another example, 1 g/kg BW is administered 35 Lambert-Eaton myasthenic syndrome (LEMS) is a rare for 1-2 consecutive days, depending on platelet count and autoimmune disorder of neuromuscular transmission first clinical response. The amount of immune globulin adminis recognized clinically in association with lung cancer, and tered, and the frequency of the doses, can be increased or Subsequently in cases in which no neoplasm was detected. decreased depending on, for example, platelet count and the Patients with LEMS have a presynaptic neuromuscular junc clinical response to therapy, which can be readily evaluated 40 tion defect. The disease is characterized clinically by proxi by one of skill in the art. mal muscle weakness, with augmentation of strength after e. Inflammatory Myopathies exercise, mild oculomotor signs, depressed deep tendon Inflammatory myopathies are a group of muscle diseases reflexes and autonomic dysfunction (dry mouth, constipation, involving the inflammation and degeneration of skeletal erectile failure). muscle tissues. These acquired disorders all present with 45 Subcutaneous administration of immune globulin/hyalu significant muscle weakness and the presence of an inflam ronidase co-formulations to patients with LEMS can amelio matory response within the muscle. rate symptoms. Exemplary dosages of IG in the co-formula i. Dermatomyositis tions include those used for intravenous administration of Dermatomyositis (DM) is the most easily recognized of the immune globulin to patients with LEMS. For example, a inflammatory myopathies due to its distinctive rash, which 50 patient with LEMS can be administered 2 g/kg BW of occurs as a patchy, dusky, reddish or lilac rash on the eyelids, immune globulin over several doses. For example, five doses cheeks, and bridge of the nose, and on the back or upper chest, of 400 mg/kg BW immune globulin can be administered on elbows, knees and knuckles. In some patients, calcified nod five consecutive days. The amount of immune globulin ules or hardened bumps develop under the skin. The rash administered can be increased or decreased as appropriate. often precedes muscle weakness, which typically develops 55 g. Multifocal Motor Neuropathy over a period of weeks, but may develop over months or even Multifocal motor neuropathy (MMN) with conduction days. Dermatomyositis can occurat any age from childhood block is an acquired immune-mediated demyelinating neur to adulthood, and is more common in females than males. opathy with slowly progressive weakness, fasciculations and Approximately one-third of DM patients report difficulty cramping, without significant sensory involvement. The swallowing. More than 50% of children with DM complain of 60 duration of disease prior to diagnosis ranges from several muscle pain and tenderness, while this generally occurs in months to more than 15 years. The precise cause of MMN is less than 25% of adults with DM. unknown. Histopathologic and electrodiagnostic studies ii. Polymyositis demonstrate the presence of both demyelinating and axonal Polymyositis (PM) does not have the characteristic rash of injury. Motor nerves are primarily affected, although mild dermatomyositis, and the onset of muscle weakness usually 65 demyelination has been demonstrated in sensory nerves as progresses slower than DM. Many PM patients present with well. Efficacy of immunomodulatory and immunosuppres difficulty in Swallowing. In some instances, the patients also sive treatment further supports the immune nature of MMN. US 9,084,743 B2 65 66 Titers of anti-GM1 antibodies are elevated in over half of the respiratory muscle involvement leads to breathing difficulty patients with MMN. Although the role of the anti-GM1 anti and facial muscle involvement to a mask-like face. bodies in the disease in unknown, their presence can be used Treatment with immune globulin can effect decreased stiff as a diagnostic marker for MMN. ness and heightened sensitivity Scores in patients with Moer Subcutaneous administration of immune globulin/hyalu sch-Woltmann syndrome (Dalakas et al. (2001) N. Engl. J. ronidase co-formulations to patients with MMN can amelio Med. 345 (26): 1870-6). Immune globulin/hyaluronidase co rate symptoms. Exemplary dosages of IG in the co-formula formulations can be administered subcutaneously to patients tions include those used for intravenous administration of with Moersch-Woltmann syndrome using the methods immune globulin to patients with MMN. For example, a described herein. Exemplary dosages of IG in the co-formu 10 lations include those used for intravenous administration of patient with MMN can be administered 2 g/kg BW of immune immune globulin to patients with Moersch-Woltmann syn globulin over several doses. For example, five doses of 400 drome. For example; immune globulin can be administered at mg/kg BW immune globulin can be administered on five doses of 400 mg/kg BW on five consecutive days. Some consecutive days. In another example, 1 g/kg BW can be patients can be given maintenance therapy, which can administered on 2 consecutive days. Some patients can be 15 include, for example, 1-2 g/kg BW immune globulin every given maintenance therapy, which can include, for example, 4-6 weeks. The amount of immune globulin administered can doses of 400 mg/kg BW to 2 g/kg BW, given every 2-6 weeks. be increased or decreased as appropriate. The amount of immune globulin administered can be 3. Acute Infections increased or decreased as appropriate, taking into account the Immune globulin also has been shown to have antimicro patient's response. bial activity against a number of bacterial, viral and fungal h. Myasthenia Gravis infections, including, but not limited to, Haemophilus influ Myasthenia gravis (MG) is a chronic autoimmune neuro enzae type B; Pseudomonas aeruginosa types A and B: Sta muscular disease characterized by varying degrees of weak phylococcus aureus; group B Streptococcus, Streptococcus ness of the skeletal muscles of the body. It is associated with pneumoniae types 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19, and 23; the presence of antibodies to acetylcholine receptors (AChR) 25 adenovirus types 2 and 5; cytomegalovirus; Epstein-Barr or muscle-specific tyrosine kinase (MuSK) at the neuromus virus VCA; hepatitis A virus; hepatitis B virus; herpes sim cular junction, although some patients are antibody negative. plex virus-1; herpes simplex virus-2; influenza A, measles; The clinical features of MG include fluctuating weakness and parainfluenza types 1, 2 and 3; polio: Varicella Zoster virus; fatigability of Voluntary muscles, particularly levator palpe Aspergillus; and Candida albicans. Thus, immune globulin/ brae, extraocular, bulbar, limb and respiratory muscles. 30 hyaluronidase co-formulations can be administered Subcuta Patients usually present with unilateral or bilateral drooping neously to patients with bacterial, viral and fungal infections of the eyelid (ptosis), double vision (diplopia), difficulty in to augment the patient’s immune system and treat the disease. Swallowing (dysphagia) and proximal muscle weakness. In Some examples, antibiotics or other antimicrobials also are Weakness of respiratory muscles can result in respiratory administered. failure in severe cases, or in acute severe exacerbations (my 35 4. Other Diseases and Conditions asthenic crisis). Myasthenia gravis occurs in all ethnic groups Exemplary of other diseases and conditions treatable by IG and both genders. It most commonly affects young adult therapy and not described above include, but are not limited women under 40 and older men over 60, but it can occurat any to, iatrogenic immunodeficiency; specific antibody defi age. In some instances, thymectomy is performed to reduce ciency; acute disseminated encephalomyelitis; ANCA-posi symptoms. 40 tive systemic necrotizing vasculitis; autoimmune haemolytic Immune globulin can be used, for example, as maintenance anaemia; bullous pemphigoid, cicatricial pemphigoid; Evans therapy for patients with moderate to severe MG, typically syndrome (including autoimmune haemolytic anaemia with when other treatments have been ineffective or caused severe immune thrombocytopenia); feto-maternal/neonatal alloim side effects, and also can be administered prior to thymec mune thrombocytopenia (FMAIT/NAIT); haemophagocytic tomy or during an acute exacerbation of the disease (myas 45 syndrome; high-risk allogeneichaemopoietic stem cell trans thenic crisis). Immune globulin/hyaluronidase co-formula plantation, IgM paraproteinaemic neuropathy; kidney trans tions can be administered Subcutaneously to patients with plantation; multiple Sclerosis; opSoclonus myoclonus ataxia; MG using the methods described herein. Exemplary dosages pemphigus foliaceus; pemphigus Vulgaris; post-transfusion of IG in the co-formulations include those used for intrave purpura; toxic epidermal necrolysis/Steven Johnson syn nous administration of immune globulinto patients with MG. 50 drome (TEN/SJS); toxic shock syndrome; systemic lupus For example, a patient with MG can be administered doses of erythematosus; multiple myeloma; sepsis: bone marrow 400 mg/kg BW to 2 g/kg BW every 4-6 weeks for mainte transplantation; B cell tumors; and Alzheimer's disease. nance therapy. Prior to thymectomy or during myasthenic Alzheimer's disease, for example, includes treatment with crisis, 1-2 g/kg BW can be administered over several doses, intravenous immunoglobulin (see e.g., Dodel et al. (2004) J such as, for example, five doses of 400 mg/kg BW on five 55 Neurol. Neurosurg. Psychiatry 75:1472-4; Solomon et al. consecutive days. In another example, 1 g/kg BW can be (2007) Curr. Opin. Mol. Ther: 9:79-85; Relkin et al. (2008) administered on 2 consecutive days. Neurobiol Aging). IG contains antibodies that bind to beta i. Moersch-Woltmann Syndrome amyloid (AB), which is a central component of the plaque in Moersch-Woltmann syndrome, also known as stiff person the brains of Alzheimer's patients. Thus, IG can help to pro syndrome (SPS) or stiffman syndrome, is a rare neurological 60 mote the clearance of AB from the brain and block AB’s toxic disorder with features of an autoimmune disease. Patients effects on brain cells. Hence, immune globulin/hyaluronidase present with symptoms related to muscular rigidity and co-formulations can be administered Subcutaneously to Superimposed episodic spasms. Muscle rigidity spreads to patients with Alzheimer's disease using the methods involve axial muscles, primarily abdominal and thoracolum described herein. Subjects to be treated include patients hav bar, as well as proximal limb muscles. Typically, co-contrac 65 ing mild, moderate or advanced Alzheimer's disease. It is tion of truncal agonist and antagonistic muscles leads to a within the level of skill of a treating physician to identify board-like appearance with hyperlordosis. Less frequently, patients for treatment. Immune globulin/hyaluronidase co US 9,084,743 B2 67 68 formulations can be administered every week, every two (Horowitz et al. (1994) Blood Coagul. Fibrin. 5(3): S21-S28; weeks, or once a month. Treatment can continue over the Kreil et al. (2003) Transfusion 43:1023-1038), 35 nm nano course of months or years. The co-formulations can be filtration (Hamamoto et al. (1989) Vox Sang. 56:230-236: administered at IG doses at or between 200 mg/kg BW to 2 Yuasaetal. (1991).J. Gen. Virol. 72:2021-2024), and a low pH 5 incubation at elevated temperatures (Kempf et al. (1991) g/kg BW every week or every two weeks, and generally at Transfitsion 31:423-427; Louie et al. (1994) Biologicals least 200 mg/kg to 2 g/kg BW at least once a month. Treat 22:13-19). The S/D procedure included treatment with an ment with immune globulin can effect an increase in a organic mixture of tri-n-butyl phosphate, octoxynol-9 and patients anti-amyloid beta antibody levels compared to lev polysorbate-80 at 18 to 25°C. for a minimum of 60 minutes els before treatment. (Polsler et al., (2008) Vox Sang.94:184-192). I. Articles Of Manufacture And Kits 10 The final preparations used in the studies were 10% liquid Pharmaceutical compositions of immune globulin and preparations of highly purified and concentrated immunoglo hyaluronidase co-formulations can be packaged as articles of bulin G (IG) antibodies formulated in 0.25 mM glycine at pH manufacture containing packaging material, a pharmaceuti 4.6 to 5.1 (as measured in the concentrated Solution). Glycine cal composition which is effective for treatinga IG-treatable serves as a stabilizing and buffering agent, and there were no disease or condition, and a label that indicates that the com 15 added sugars, sodium or preservatives. All lots of 10% IG position is to be used for treating an IG-treatable diseases and (e.g. lots LE12H020, LE12H062, LE12H173, LE12F047) conditions. Exemplary of articles of manufacture are contain were substantially similar. The osmolality was 240 to 300 ers including single chamber and dual chamber containers. mOsmol/kg, which is similar to physiological osmolality. The containers include, but are not limited to, tubes, bottles The distribution of the IG subclasses of the product manufac and Syringes. The containers can further include a needle for tured according to the process described above was similar to Subcutaneous administration. that of normal plasma: at least 98% of the protein preparation The articles of manufacture provided herein contain pack being IgG, the average IgA concentration was 37 ug/mL aging materials. Packaging materials for use in packaging (none of these lots had an IgA concentration of >140 ug/mL) pharmaceutical products are well known to those of skill in and IgM was present only in trace amounts. The Fc and Fab the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,033,252 25 functions were maintained. Pre-kalikrein activator activity and 5,052,558, each of which is incorporated herein in its was not detectable. entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, Example 2 inhalers, pumps, bags, vials, containers, Syringes, bottles, and any packaging material Suitable for a selected formulation 30 Preparation of SUBQ NG 20% (20% IG) and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions A. Producing a Concentrated, Purified IG Composition provided herein are contemplated as are a variety of treat a. Summary ments for any IG-treatable disease or condition. Previously frozen pooled plasma from blood donors was Compositions of immune globulin and a soluble hyalu 35 separated into a cryo-poor plasma sample for isolation of ronidase co-formulations also can be provided as kits. Kits various crude coagulation factors and inhibitors prior to Sub can include a pharmaceutical composition described herein sequent cold alcohol fractionation using a modified Cohn and an item for administration. For example compositions fractionation procedure as described by Teschner et al. (2007) can be supplied with a device for administration, such as a Vox Sang. 92:42-55. The alcohol fractionation procedure Syringe, an inhaler, a dosage cup, a dropper, or an applicator. 40 gave a principal intermediate IG fraction, referred to as Pre The kit can, optionally, include instructions for application cipitate G, which was further processed to the final product including dosages, dosing regimens and instructions for using chromatographic purification. The downstream manu modes of administration. Kits also can include a pharmaceu facturing involved cation exchange (CM-Sepharose fast flow) tical composition described herein and an item for diagnosis. and anion exchange chromatography (ANX-Sepharose fast For example, such kits can include an item for measuring the 45 flow). To provide a high safety margin with respect to poten concentration, amount or activity of IG. tial virus transmission, three dedicated virus inactivation/ removal steps, which complement each other in their mode of J. EXAMPLES action, were integrated in the manufacturing process, namely: solvent/detergent treatment (mixture of 1% Triton X-100, The following examples are included for illustrative pur 50 0.3% tri-n-butyl phosphate and 0.3% polysorbate-80), nano poses only and are not intended to limit the scope of the filtration (Asahi Planova 35 nm), and low pH (4.7) storage for invention. 3 weeks at elevated temperature. b. Separation of Cryoprecipitates Example 1 Previously frozen pooled plasma from blood donors, 55 already checked for safety and quality considerations, was Preparation of Gammagard Liquid (10% thawed at a temperature no higher than 6° C. Centrifugation Immunoglobulin (IG) Formulations) in the cold was performed to separate Solid and liquid, which formed upon the plasma thawing. The liquid portion (also Gammagard Liquid (10% IG) was manufactured from referred to as “cryo-poor plasma, after cold-insoluble pro large pools of human plasma, Screened throughout for infec 60 teins were removed by centrifugation from fresh thawed tious agents. Immune globulins were purified from plasma plasma) was then cooled to 0-1° C., and its pH was adjusted pools using a modified Cohn-Oncley cold ethanol fraction to 7. The cryo-poor plasma was used for isolation of various ation process (Cohn et al. (1946).J. Am. Chem. Soc. 68:459 crude coagulation factors and inhibitors prior to Subsequent 467), as well as cation and anion exchange chromatography cold alcohol fractionation. Seven pathways were chosen for (Teschner et al. (2007) Vox Sang. 92:42-55). The purified 65 batch adsorption of crude coagulation factors and inhibitors protein was further subjected to three independent viral inac from the cryo-poor plasma prior to SUBQNG 20% purifica tivation/removal steps: solvent/detergent (S/D) treatment tion and are referred to as pathways 1 to 7 in Table 3. US 9,084,743 B2 69 70 TABLE 3 Pathways for batch adsorption of coagulation factors and inhibitors from cryo-poor plasma Adsorption Pathways
Step Gel Heparin 1 2 3 4 S 6 7 Cryoprecipitation — X X X X X X X FEIBA 0.5g X X DEAE-Sephadex/L Factor IX 0.5 g DEAE- 2000 IU/mL, X X X X Sephadex/L Factor VII 120 mg 7SOIUAL X X Al(OH)/L Antithrombin 1 g DEAE- 80000 IU/mL, X X X Sephadex/L
For pre-clinical SUBQNG 20% production, Cohn starting vi. Suspension of Precipitate G and Solvent/Detergent materials derived from pathways 1 (US source plasma with- 20 Treatment out adsorption steps), 3 (US source plasma after FEIBA, The precipitate was dissolved and filtered with a depth AT-III adsorption) and 6 (US source plasma after F-IX, F-VII, filter of a nominal pore size of 0.2 Lum (e.g., Cuno VR06 filter AT-III adsorption) were chosen to cover a broad variety of or equivalent) to obtain a clear filtrate which was used for the different adsorption steps prior to alcohol fractionation. Vari- solvent/detergent (S/D) treatment. ous adsorption processes are described in Teschner et al. 25 The first of the steps in viral inactivation is S/D treatment of (2007) Vox Sang. 92:42-55; Polsler et al. (2008) Vox Sang. the re-suspended Precipitate G. The S/D treatment mixture 94:184-192; U.S. Pat. Nos. 6,395,880 and 5,409,990; and contained 1.0% (v/v) Triton X-100, 0.3% (v/v) Tween-80, Prothrombin complex: Brummelhuis in Methods of Plasma and 0.3% (v/v) tri-n-butyl phosphate, and the mixture was Protein Fractionation (J. M. Curling Editor, Academic Press, held at 18 to 25° C. for at least 60 minutes. 1980). 30 d. Cation Exchange Chromatography c. Fractionation The S/D-containing protein Solution was then passed i. Obtain Supernatant of Fractionation I through a cation exchange column (Carboxymethyl (CM)- While the plasma was being stirred, pre-cooled ethanol Sepharose fast flow) to remove the solvent and detergent. was added, to a target concentration of 8% V/v ethanol, and After washing out of S/D reagents, the absorbed proteins the temperature was further lowered to -2 to 0°C. to allow 35 were then eluted with high pH elution buffer (pH 8.5-0.1). precipitation. Supernatant (or Fractionation I) was collected e. Anion Exchange Chromatography after centrifugation. The eluate was then adjusted to pH 6 and diluted to the ii. Precipitate of Fractionations II and III appropriate conductivity before the solution was passed Fractionation I was adjusted to pH 7 and 20 to 25% V/v through the equilibrated anion exchange column (ANX ethanol concentration, while the temperature was further low- 40 Sepharose fast flow). The column flow-through during load ered. Subsequently, centrifugation was performed to separate ing and washing was collected for further processing. liquid (Fractionation II+III Supernatant) and solid. f. Nanofiltration iii. Extraction From Fractionations II and III Precipitate In the second of three virus inactivation steps, the column A cold extraction buffer (5 mM monobasic sodium phos- effluent from the last step was nanofiltered (Asahi Planova 35 phate, 5 mM acetate, pH 4.5+0.2, conductivity of 0.7 to 0.9 45 nm filter) to generate a nanofiltrate. mS/cm) was used to re-suspend Fractionations II+III at a ratio g. Ultrafiltration and Diafiltration of 1:15 precipitate:extraction buffer. The extraction process The glycine concentration of the nanofiltrate was adjusted was performed at 2 to 8°C. to 0.25 M and the nanofiltrate was further concentrated to a iV. Fumed Silica Treatment and Filtration protein concentration of 5+1% w/v by ultrafiltration and pH Fumed silica (e.g., Aerosil 380 or equivalent) was added to 50 was adjusted to 5.2+0.2. In order to reach a higher protein the Suspension to a concentration of about 40 g/kg of suspen concentration for Subcutaneous application, the ultrafiltration sion (or equivalent to 1.8 g/L of cryo-poor plasma) and was was carried out in a cassette with an open channel Screen and mixed at 2 to 8°C. for 50 to 70 minutes. Liquids and solids ultrafiltration membrane (Millipore Pelicon Biomax) with a were separated by filtration at 2 to 8° C. using a filter aid nominal molecular weight cut off (NMWCO) of 50 kDa or (Hyflo Super-Cel, World Minerals Inc., 0.5 kg/kg of suspen 55 less that was especially designed for high viscosity products. sion), followed by post-washing of the filterpress with extrac The concentrate was diafiltered against a 0.25 M glycine tion buffer. solution with a pH of 4.2-0.2. The minimum exchange vol v. Fractionation of Precipitate G ume was 10x the original concentrate Volume. Throughout The filtrate was mixed with polysorbate-80 to a concentra the ultrafiltration/diafiltration operation, the solution was tion of about 0.2% w/v with stirring for at least 30 minutes at 60 maintained at 4 to 20°C. After diafiltration, the solution was 2 to 8° C. Sodium citrate dehydrate was then mixed into the concentrated to a protein concentration of minimum 22% w/v. solution at 8 g/L for another 30 minutes of stirring at 2 to 8° and adjusted to 2 to 8° C. C. The pH was then adjusted to 7.0+0.1 with either 1M In order to recover the complete residual protein in the sodium hydroxide or 1M acetic acid. Cold alcohol was then system, thereby increasing the protein concentration, the added to the solution to a concentration of about 25% V/V, and 65 post-wash of the first bigger ultrafiltration system was done a precipitation step similar to Cohn II was performed (Cohnet with at least 2x the dead volume in re-circulation mode to al. (1946).J. Am. Chem. Soc. 68:459-467). assure that all protein was washed out. Then the post-wash of US 9,084,743 B2 71 72 the first ultrafiltration system was concentrated to a protein Thus, the resulting 20%. IG formulations were highly puri concentration of at least 22% w/v with a second ultra-fidiafil fied, isotonic liquid formulations of immunoglobulin (at least tration system equipped with the same type of membrane 95% gamma globulin) formulated in 0.25 mM glycine at pH which was dimensioned a tenth or less of the first one. The 4.4 to 4.9. The final preparations used in the studies were lots post-wash concentrate was added to the bulk solution. The second ultrafiltration system was then post-washed and the SC00107NG, SC00207NG, and SC00307NG. solution temperature was adjusted to 2 to 8°C. B. Characterization of Pre-Clinical Batches h. Formulation Pre-clinical lots SC00107NG, SC00207NG, and For formulation, the protein concentration of the solution SC00307NG were manufactured on the 200 L. Scale and char was adjusted to 20.4+0.4% w/v with post-wash of the second smaller ultrafiltration system and/or with diafiltration buffer. 10 acterized according to Table 4. At the final bulk level, the The pH was adjusted to 4.4 to 4.9, if necessary. purity of the preparation was illustrated by the low levels of i. Further Sterilization the main impurities, which were well below 0.1% of the total The formulated bulk solution was further sterilized by first IgG. The molecular size distribution (MSD) in the 20% IG filtering through a membrane filter with an absolute pore size product at the final stage of the process was similar to the of 0.2 micron or less, then was aseptically dispensed into final 15 MSD of a 10% IG (Gammagard Liquid) final container. This containers for proper sealing, with samples taken for testing. indicated that increasing the concentration to 20% protein did The final virus inactivation/removal step was performed by not have a negative impact on the integrity of the IgG mol storing the sealed containers at 30 to 32°C. for 21 to 22 days. ecule. TABLE 4 Characterization of SUBQ NG 20% lots Sterile Bulk
Test Method Lot SCOO107NG SCOO2O7NG SCOO3O7NG Total protein g/L Plasma 3.4 3.7 3.7 UV IgG? g/L Plasma 3.0 3.0 3.0 Nephelometric IgA/ELISA g/L Plasma <0.001 <0.001 <0.001 IgM/ELISA g/L Plasma <0.001 <0.001 <0.001 MSD (HPLC) % Aggregates O.1 O.1 O.1 % Oligo/Dimers 4.6 4.5 3.2 % Monomers 95.2 95.4 96.6 % Fragments O.1 O O.1 Lotnumber of Precipitate G Precipitate G Precipitate G starting material WNELG171 WNELG173 LBO7903O1
The preliminary final container release criteria were defined on the basis of the requirements from the U.S. and 40 European authorities (FDA and EMEA) for subcutaneous human immunoglobulins, the final container specifications of the current product for subcutaneous administration (SUB CUVIA, licensed for subcutaneous administration in Europe) and the Gammagard Liquid specifications. Characterization 45 of the relevantantibody spectrum of the three final containers was completed and compared to the results from the pre clinical 10% IG Triple Virally Reduced (TVR) lots. Table 5 compares the results of the antibody titers and the enrichment factors of the three pre-clinical SUBQNG 20% final contain ers and pre-clinical Gammagard Liquid lots. The results are in the same order of magnitude for both lots. TABLE 5 Comparison of SUBQ NG 20% and 10% IGTVR release data
10%. IGTVR
Test SUBQNG 20% POO1OING POO2O1NG POO3OING
System Unit SCOO107NG SCOO2O7NG SCOO3O7NG. O1C21 AN11 OIC21AN21 O1DOSAN11
Bacteria:
Coryne- Guinea IU/mL, 6.O 6.O 6.0 S.O S.O S.O bacterium pigs diphtheriae EUR US 9,084,743 B2 73 74 TABLE 5-continued Comparison of SUBQ NG 20% and 10%. IG TVR release data 10%. IGTVR
Test SUBQ NG 20% POO1 OING POO2O1NG POO3OING
System Unit SCOO 107NG SCOO2O7NG SCOO3O7NG. O1C21 AN11 OIC21AN21 O1DOSAN11 Viruses
HAV ELISA IU/mL, 14.0 14.0 27.O 14 9 16 HBV ELISA IU/mg 40.O 47.0 43.O 35.9 40.1 40.O (antibody to TP hep Bs Ag) Measles virus Hemagglut. 41.O 41.O 24.O na na na EUR Enrich. Factor Measles virus Hemagglut. NIH O.8 O.8 O 1.001 1.O 1.001 US 176 Parvo 619 ELISA IU/mL, 718 78 71 567 442 36 Poliomyelitis NIHU, 1.4 1.7.11 1.S. 1.01 1.11 1.21 virus type I mL
Additional quality control tests were performed to evaluate final containers. The removal of product and process related the level of product and/or process-related impurities. Table 6 impurities is satisfactory, and all product-related preliminary shows the quality control data of the three SUBQ NG 20% specifications are met for all three lots. TABLE 6 Quality control tests of SUBQ NG 20% final container Test System Unit SCOO 107NG SCOO2O7NG Fc functional integrity Bc-binding % of BPR lot 3 15.8 138 164 Anti-complementary EP method % 41.1 41.5 41.2 activity Anti-complementary EP method CH50 U/mg 41.4 4.1.8 41.6 activity Prekallikrein activator chromogenic IU/mL, <0.6 1.004 1.237 activity, EUR Anti-Ahemagglutinins, hemagglut. Dilution: 1 8 16 pH. Eur. Anti-B hemagglutinins, hemagglut. Dilution: 1 4 4 pH. Eur. Anti-D hemagglut. complies complies Complies Exclusion of pyrogenicity, rabbit C. rise pyrogen free pyrogen free pyrogen free pH. Eur, and CFR Bacterial Endotoxins Chromogenic <1.2 1.8 <1.2 Purity by cellulose acetate CAE 99.6 99.8 99.5 electrophoresis Molecular size SE-HPLC % 99.2 99.3 99.2 distribution (Monomer + Dimers) Molecular size SE-HPLC % O.2 O.2 O.3 distribution (Polymers) Molecular size S E-HPLC % O6 O.S distribution (Fragments) IgA-EUR LISA Ig/mL 2O 2O 30 IgM LISA Ig/mL 1.1 1.O 1.2 IgG Nephelometry mg/mL 177 16S 163 Protein (Bulk) UV mg/mL 2O1 2O3 Protein Autom.N2 mg/mL 2O2 208 2O3 Glycine HPLC mg/mL 14.7 14.5 14.7 Polysorbate 80 Spectrophot. Ig/mL <250 <250 Gas-chromat. Ig/mL <0.3 <0.3 Octoxynol 9 on-chromat. Ig/mL <3 <3 Sterility Membrane na sterile sterile sterile iltr. Osmolality mOSmol/kg 298 298 299 pH, undiluted Potentiometry S.1 5.2 5.3 Appearance Visual Inspec. satisfied satisfied satisfied Ethanol Gas-chromat. Ig/mL <2O <2O <2O Isopropanol Gas-chromat. Ig/mL <2O <2O <2O Aluminum AAS Photometry <50 <50 <50 US 9,084,743 B2 75 76 TABLE 6-continued Quality control tests of SUBQ NG 20% final container Test System Unit SCOO 107NG SCOO2O7NG SCOO3O7NG Silicium ICPOES Ion Electr. Jig?L 34.66 17270 2118O Heparin IU/mL, <0.0075 <0.0075 <0.0075
In-process parameters monitored during the pre-clinical 10 TABLE 8-continued production and the characterization of intermediates and the final product showed that there were no obvious differences MSD of the feasibility lot IsGSCO2 at 2 to SC and 28 to SOC detectable between the three lots. All final containers met the MSD (HP-SEC) (% product related preliminary specifications regardless of which kind of starting material (Precipitate G VNELG171, 15 Aggregates Olig Dimers + Fragments VNELG173, or LB0790301) was chosen. Lot o C. Month (>450 KDa) Monomers (<70 Kda) C. Storage Study of 20%. IG Formulations 6 O.3 99.4 O.3 12 0.4 99.3 O.3 In order to evaluate the storage stability of the 20%. IG final 18 0.4 99.2 0.4 containers, the 3 pre-clinical lots described above 28 to 30 O O.2 99.5 O.3 (SC00107NG, SC00207NG, SC00307NG) and one feasibil 1 O.2 99.2 O6 3 O.3 98.7 1.O ity lot (IgGSC 62/1) were stored at 2 to 8° C. and 28 to 30° C. 6 O.6 98.0 1.4 (feasibility lot only) for up to 18 months. High performance 12 1.2 95.6 3.2 size exclusion chromatography was used to determine the 18 1.9 93.5 3.8 molecular size distribution (MSD) and stability of the Release <5 >90 <5 samples. The mainstability indicating parameter is molecular 25 criteria size, and a change in size can be the result of degradation by denaturation, aggregation or fragmentation. D. Stability Study of Various IG Concentrations and Formu The MSD of the pre-clinical final containers after storage lations at 2 to 8° C. up to 12 months are shown in Table 7. Table 8 30 The storage stability of high protein concentration formu gives the MSD of the feasibility lot, IgGSC 62/1, at 2 to 8° C. lations (14-20%) with low pH (0.25 M glycine pH 4.4-4.9) and 28 to 30° C., after storage up to 18 months. The data was compared to high protein concentration formulations confirmed that the product complies to the pre-defined speci with neutral pH (22.5g/L glycine, 3 g/L NaCl, pH 7.0), which fications for the parameters investigated for up to 18 months are currently used for intramuscularly and Subcutaneously storage at 2 to 8° C. and 28 to 30° C. 35 injectable immunoglobulins. TABLE 7 All runs started with concentration of the nanofiltrate to 5% protein. A 10x buffer exchange against 0.15 M glycine (low MSD of pre-clinical 20%. IG batches at 2 to 8 C. est glycine concentration investigated) was performed, fol MSD (HP-SEC) (9/o lowed by the final concentration to a target value above 20% Aggregates Olig Dimers + Fragments 40 protein using a 0.5 m polyethersulfone Millipore membrane Lot Month (>450 KDa) Monomers (<70 Koda) with a molecular cut-off of 30K (standard screen). The final containers were either formulated and stored at low pH (4.7) SCOO 107NG O O.3 99.5 O.2 3 0.4 99.5 O.2 or the low pH storage was done in bulk and afterwards they 4 O.S 99.4 O.2 were formulated at neutral pH (7.0) prior to storage at either 6 O.S 99.3 O.2 45 2 to 8° C. or 28 to 30° C. for 3 months. After 3 months, 12 0.7 99.1 O.3 molecular size distribution was determined by high perfor SCOO2O7NG O O.3 99.5 O.2 3 0.4 99.5 O.1 mance size exclusion chromatography in order to determine 4 O.S 99.3 O.2 aggregate and fragment content. Acceptance criteria was 6 O.6 99.2 O.2 defined as: monomers and oligo-fcdimers, a 90%; aggregates, 12 O.8 99.0 O.2 50 s5%, fragments, s5%. ACA titer was tested as described in SCOO3O7NG O O.3 99.6 O.1 the European Pharmacopoeia. Acceptable ACA titer was 3 O.S 99.3 O.2 4 O.6 99.2 O.1 defined as less than 50% CHSO units consumed per mg 6 0.7 99.1 O.2 protein. 12 O.9 98.8 O.2 Tables 9 and 10 show aggregate and fragment content as Release criteria <5 >90 <5 55 well as ACA titer after 3 months storage at 28 to 30° C. and 2 to 8°C., respectively, for the standard formulations (pH 4.7, TABLE 8 0.25 M glycine; or pH 7.0, 22.5 g/L glycine, 3 g/L NaCl) at different protein concentrations. The data clearly show that MSD of the feasibility lot IgGSC 62/1 at 2 to 8 C. and 28 to 30° C. the low pH formulation had lower aggregates and lower ACA 60 titer after 3 months storage at 28 to 30°C. All ACA titers of the MSD (HP-SEC) (% pH 7.0 formulations were above the acceptance criterion Aggregates Olig Dimers + Fragments defined for this test. Lot o C. Month (>450 KDa) Monomers (<70 Kda) The results at 2 to 8° C. confirm the trend seen at 28 to 30° IgGSC 2 to 8 O O.2 99.5 O.3 C. The ACA titers were all below the limit defined as accep 621 1 O.1 99.7 O.2 65 tance criterion, although the pH 7.0 formulations seem to 3 O.2 99.6 O.2 have higher values. The protein value does not influence the results of the parameters tested. US 9,084,743 B2 77 78 TABLE 9 TABLE 12 Fragment, aggregate and ACA values after 3 months storage at 2 to Fragment, aggregate and ACA values after 3 months storage 8. C. at pH 4.7 with different protein concentration methods at 28 to 30° C. at pH 4.7 and pH 7.0 at different Fragments (% Aggregates (% ACA titer (% protein concentrations standard- open- standard- open- standard- open Protein screen SCCEl SCCEl SCCEl SCCEl SCCEl Fragments % Aggregates% ACA tilter% 14% O.36 0.27 O16 O.17 38.3 39.6 10 16% O.30 O.22 O.11 O.14 37.4 38.3 Protein pH 4.7 pH 7.0 pH 4.7 pH 7.0 pH 4.7 pH 7.0 18% O.33 O.23 O.17 O.18 35.8 39.6 20% O.33 O.22 O.20 O.20 36.1 39.9
14% 1.35 1...SO O.10 O.92 44.1 S2.0 16% 1.24 1.38 O.08 O.91 4O.S 53.1 15 Example 3 18% 1.24 1.60 O.11 O.93 40.3 52.4 20% 1.35 1.52 O.12 O.93 37.5 62.7 Preparation of Soluble Recombinant Human PH20 (rHuPH20) A. Generation of a Soluble rHuPH20-Expressing Cell Line TABLE 10 The HZ24 plasmid (set forth in SEQID NO:52) was used to Fragment, aggregate and ACA values after 3 months storage transfect Chinese Hamster Ovary (CHO cells) (see e.g. appli at 2 to 8° C. at pH 4.7 and pH 7.0 at different cation Nos. 10,795,095, 11/065,716 and 11/238,171). The protein concentrations HZ24 plasmid vector for expression of soluble rhuPH20 Fragments 90 Aggregates 90 ACA tilter 25 contains a pCI vector backbone (Promega), DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ ID Protein pH 4.7 pH 7.0 pH 4.7 pH 7.0 pH 4.7 pH 7.0 NO:49, an internal ribosomal entry site (IRES) from the 14% O.36 18O O16 1.09 38.3 46.5 ECMV virus (Clontech), and the mouse dihydrofolate reduc 16% O.30 O.S1 O.11 1.01 37.4 44.7 tase (DHFR) gene. The pCI vector backbone also includes 18% O.33 1.10 O.17 O.86 35.8 39.8 30 DNA encoding the Beta-lactamase resistance gene (AmpR), 20% O.33 1.98 O.2O 1.06 36.1 46.0 an fl origin of replication, a Cytomegalovirus immediate early enhancer/promoter region (CMV), a chimeric intron, and an SV40 late polyadenylation signal (SV40). The DNA The influence of different concentration procedures on encoding the soluble rHuPH20 construct contains an Nhel MSD and ACA titer was investigated. The first procedure 35 site and a Kozak consensus sequence prior to the DNA encod used a 0.5 m polyethersulfone Millipore membrane with a ing the methionine at amino acid position 1 of the native 35 molecular cut-off of 30K (standard screen), as described amino acid signal sequence of human PH20, and a stop codon above, and the second procedure used a 0.5 m polyethersul following the DNA encoding the tyrosine corresponding to fone Millipore membrane with an open screen, suitable for amino acid position 482 of the human PH20 hyaluronidase solutions with higher viscosity. The post-wash fractions were 40 (set forth in SEQID NO:1), followed by a BamHI restriction concentrated by a second ultra-/diafiltration device with a site. The construct pCI-PH20-IRES-DHFR-SV40pa (HZ24), lower membrane surface (0.1 m, open screen) in order to therefore, results in a single mRNA species driven by the reduce yield losses. CMV promoter that encodes amino acids 1-482 of human Tables 11 and 12 show MSD and ACA titer after 3 months PH20 (set forth in SEQID NO:3) and amino acids 1-186 of storage at 28 to 30° C. or 2 to 8°C., respectively, for the low 45 mouse dihydrofolate reductase (set forth in SEQID NO:53). pH (4.7) formulations at various protein concentrations. The separated by the internal ribosomal entry site (IRES). data showed similar results after 3 months storage for both Non-transfected DG44 CHO cells growing in GIBCO concentration modes. The values obtained at 2 to 8° C. con Modified CD-CHO media for DHFR(-) cells, supplemented firmed the results obtained at 28 to 30°C. The concentration with 4 mM glutamine and 18 mL/L Pluronic F68/L (Gibco), 50 were seeded at 0.5x10° cells/mL in a shake flask in prepara method does not influence the stability of the product, though tion for transfection. Cells were grown at 37°C. in 5% CO in adequate post-wash can only be obtained with open-screen a humidified incubator, shaking at 120 rpm. Exponentially membranes. growing non-transfected DG44 CHO cells were tested for viability prior to transfection. TABLE 11 55 Sixty million viable cells of the non-transfected DG44 Fragment, aggregate and ACA values after 3 months storage at 28 to CHO cell culture were pelleted and re-suspended to a density 30° C. at pH 4.7 with different protein concentration methods of 2x107 cells in 0.7 mL of 2x transfection buffer (2xHeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KC1, 1.4 mM Fragments (% Aggregates (% ACA tilter NaHPO, 12 mM dextrose). To each aliquot of re-suspended 60 standard- open- standard- open- standard- open cells, 0.09 mL (250 ug) of the linear HZ24 plasmid (linearized Protein screen SCCEl SCCEl SCCEl SCCEl SCCEl by overnight digestion with Cla I (New England Biolabs)) was added, and the cell/DNA solutions were transferred into 14% 1.35 O.92 O.10 O.21 44.1 42.6 0.4 cm gap BTX (Gentronics) electroporation cuvettes at 16% 1.24 1.09 O.08 O.20 4.O.S 40.9 18% 1.24 O.96 O.11 O.23 40.3 40.7 room temperature. A negative control electroporation was 20% 1.35 O.98 O.12 O.30 37.5 41.6 65 performed with no plasmid DNA mixed with the cells. The cell/plasmid mixes were electroporated with a capacitor dis charge of 330 V and 960 uF or at 350 V and 960 g. US 9,084,743 B2 79 80 The cells were removed from the cuvettes after electropo methotrexate giving rise to clones producing in excess of ration and transferred into 5 mL of Modified CD-CHO media 1,000 Units/mL in shake flasks (clone 3D35M; or Gen1 for DHFR(-) cells, supplemented with 4 mM glutamine and 3D35M). A master cell bank (MCB) of the 3D35M cells was 18 mL/L Pluronic F68/L (Gibco), and allowed to grow in a then prepared. well of a E-well tissue culture plate without selection for 2 B. Production and Purification of Gen1 Human PH2O days at 37°C. in 5% CO in a humidified incubator. a. 5 L Bioreactor Process Two days post-electroporation, 0.5 mL of tissue culture A vial of 3D35M was thawed and expanded from shake media was removed from each well and tested for the pres flasks through 1 L spinner flasks in CD-CHO media (Invitro ence of hyaluronidase activity, using the microturbidity assay gen, Carlsbad Calif.) supplemented with 100 nM methotrex described in Example 4. 10 ate and GlutaMAXTM-1 (Invitrogen). Cells were transferred from spinner flasks to a 5 L bioreactor (Braun) at an inocula TABLE 13 tion density of 4x10 viable cells/mL. Parameters were: tem Initial hyaluronidase activity of HZ24 transfected DG44 CHO perature setpoint: 37° C.; pH: 7.2 (starting setpoint); dis cells at 40 hours post-transfection 15 solved oxygen setpoint: 25%; and air overlay: 0-100 cc/min. Activity At 168 hrs, 250 mL of Feed #1 Medium (CD CHO with 50g/L Dilution (Units/mL) glucose) was added. At 216 hours, 250 mL of Feed #2 Medium (CD CHO with 50 g/L glucose and 10 mM sodium Transfection 1 (330 V) 1 to 10 O.25 butyrate) was added, and at 264 hours 250 mL of Feed #2 Transfection 2 (350 V) 1 to 10 O.S2 Medium was added. This process resulted in a final produc Negative Control 1 to 10 O.O15 tivity of 1600 Units/mL with a maximal cell density of 6x10 cells/mL. The addition of sodium butyrate was to dramati Cells from Transfection 2 (350V) were collected from the cally enhance the production of soluble rhuPH20 in the final tissue culture well, counted and diluted to 1x10" to 2x10' stages of production. viable cells per mL. A 0.1 mL aliquot of the cell suspension 25 Conditioned media from the 3D35M clone was clarified by was transferred to each well of five, 96-well round bottom depth filtration and tangential flow diafiltration into 10 mM tissue culture plates. One hundred microliters of CD-CHO Hepes pH 7.0. SolublerHuPH20 was thenpurified by sequen media (GIBCO) containing 4 mM GlutaMAXTM-1 supple tial chromatography on Q Sepharose (Pharmacia) ion ment (GIBCOTM, Invitrogen Corporation) and without exchange, Phenyl Sepharose (Pharmacia) hydrophobic inter hypoxanthine and thymidine Supplements were added to the 30 action chromatography, phenyl boronate (Prometics) and wells containing cells (final Volume 0.2 mL). hydroxyapatite chromatography (Bio-Rad, Richmond, Ten clones were identified from the 5 plates grown without Calif.). methotrexate. Soluble rHuPH20 bound to Q Sepharose and eluted at 400 mMNaCl in the same buffer. The eluate was diluted with 2M TABLE 1.4 35 ammonium sulfate to a final concentration of 500 mMammo Hyaluronidase activity of identified clones nium sulfate and passed through a Phenyl Sepharose (low Sub) column, followed by binding under the same conditions Plate? Relative to a phenylboronate resin. The soluble rhuPH20 was eluted We ID Hyaluronidase from the Phenyl Sepharose resin in Hepes pH 6.9 after wash 1C3 261 40 ing at pH 9.0 in 50 mM bicine without ammonium sulfate. 2C2 261 The eluate was loaded onto a ceramic hydroxyapatite resin at 3D3 261 pH 6.9 in 5 mM potassium phosphate and 1 mM CaCl and 3E5 243 3C6 174 eluted with 80 mM potassium phosphate, pH 7.4 with 0.1 mM 2G8 103 CaCl. 1B9 3O4 45 The resultant purified soluble rHuPH20 possessed a spe 2D9 273 cific activity in excess of 65,000 USP Units/mg protein by 4D10 3O2 way of the microturbidity assay (Example 4) using the USP reference standard. Purified soluble rhuPH20 eluted as a Six HZ24 clones were expanded in culture and transferred single peak from 24 to 26 minutes from a Pharmacia 5RPC into shake flasks as single cell suspensions. Clones 3D3,3E5. 50 styrene divinylbenzene column with a gradient between 0.1% 2G8, 2D9, 1E11, and 4D10 were plated into 96-well round TFA/HO and 0.1% TFA/90% acetonitrile/10% H0 and bottom tissue culture plates using a two-dimensional infinite resolved as a single broad 61 kDa band by SDS electrophore dilution strategy in which cells were diluted 1:2 down the sis that reduced to a sharp 51 kDa band upon treatment with plate, and 1:3 across the plate, starting at 5000 cells in the top PNG ASE-F. N-terminal amino acid sequencing revealed that left hand well. Diluted clones were grown in a background of 55 the leader peptide had been efficiently removed. 500 non-transfected DG44 CHO cells per well, to provide b. Upstream Cell Culture Expansion process Into 100 L necessary growth factors for the initial days in culture. Ten Bioreactor Cell Culture plates were made per subclone, with 5 plates containing 50 A scaled-up process was used to separately purify soluble nM methotrexate and 5 plates without methotrexate. rHuPH20 from four different vials of 3D35M cell to produce Clone 3D3 produced 24 visual subclones (13 from the no 60 4 separate batches of soluble rHuPH20 : HUAO406C, methotrexate treatment, and 11 from the 50 nM methotrexate HUAO410C, HUAO415C and HUAO420C. Each vial was treatment). Significant hyaluronidase activity was measured separately expanded and cultured through a 125 L bioreactor, in the supernatants from 8 of the 24 subclones (>50 Units/ then purified using column chromatography. Samples were mL), and these 8 subclones were expanded into T-25 tissue taken throughout the process to assess Such parameters as culture flasks. Clones isolated from the methotrexate treat 65 enzyme yield. The description of the process provided below ment protocol were expanded in the presence of 50 nM meth sets forth representative specifications for Such things as otrexate. Clone 3D35M was further expanded in 500 nM bioreactor starting and feed media Volumes, transfer cell den US 9,084,743 B2 81 82 sities, and wash and elution Volumes. The exact numbers vary and culture temperature was changed to 36°C. At day 11, 3.7 slightly with each batch, and are detailed in Tables 15 to 22. L of Feed #3 (CD CHO+50 g/L glucose--40 mL/L Four vials of 3D35M cells were thawed in a 37° C. water GlutaMAXTM-1+1.1 g/L sodium butyrate) was added, and the bath, CD CHO containing 100 nM methotrexate and 40 mL/L culture temperature was changed to 35.5°C. The reactor was GlutaMAXTM-1 was added and the cells were centrifuged. harvested at 14 days, or when the viability of the cells dropped The cells were re-suspended in a 125 mL shake flask with 20 below 50%. The process resulted in production of soluble mL of fresh media and placed in a 37°C., 7% CO incubator. rHuPH20 with an enzymatic activity of 1600 Units/mL with The cells were expanded up to 40 mL in the 125 mL shake a maximal cell density of 8 million cells/mL. At harvest, the flask. When the cell density reached 1.5-2.5x10° cells/mL, culture was sampled for mycoplasma, bioburden, endotoxin, the culture was expanded into a 125 mL spinner flaskin a 100 10 and virus in vitro and in Vivo, transmission electron micros mL culture volume. The flask was incubated at 37° C., 7% copy (TEM) for viral particles, and enzyme activity. CO. When the cell density reached 1.5-2.5x10° cells/mL, the culture was expanded into a 250 mL spinner flask in 200 The 100 L bioreactor cell culture harvest was filtered mL culture volume, and the flask was incubated at 37°C., 7% CO. When the cell density reached 1.5-2.5x10° cells/mL, 15 through a series of disposable capsule filters having a poly the culture was expanded into a 1 L spinner flask in 800 mL ethersulfone medium (Sartorius): first through a 8.0 um depth culture volume and incubated at 37° C., 7% CO. When the capsule, a 0.65 um depth capsule, a 0.22 um capsule, and cell density reached 1.5-2.5x10° cells/mL, the culture was finally through a 0.22um Sartopore 2000 cm filter and into a expanded into a 6 L spinner flask in 5 L culture Volume and 100 L sterile storage bag. The culture was concentrated 10x incubated at 37° C., 7% CO. When the cell density reached using two TFF with Spiral Polyethersulfone 30 kDa MWCO 1.5-2.5x10° cells/mL, the culture was expanded into a 3.6 L filters (Millipore), followed by a 6x buffer exchange with 10 spinner flask in 20 L culture volume and incubated at 37°C., mM HEPES, 25 mM NaSO pH 7.0, into a 0.22 um final 7% CO. filter into a 20 L sterile storage bag. Table 15 provides moni A 125L reactor was sterilized with steam at 121°C., 20 psi toring data related to the cell culture, harvest, concentration and 65 L of CD CHO media was added. Before use, the and buffer exchange steps. TABLE 1.5 Monitoring data for cell culture, harvest, concentration and buffer exchange steps
Parameter
Time from thaw to inoculate 100 L 21 19 17 18 bioreactor (days) 100 L inoculation density (x10 cells/mL) O45 O.33 0.44 O46 Doubling time in logarithmic 29.8 27.3 29.2 23.5 growth (hr) Max. cell density (x10 cells/mL) 5.65 8.70 6.07 9.70 Harvest viability (%) 41 48 41 41 Harvesttiter (U/mL) 1964 1670 991 1319 Time in 100-L bioreactor (days) 13 13 12 13 Clarified harvest volume (mL) 818OO 93300 91800 891OO Clarified harvest enzyme assay 2385 1768 1039 1425 Concentrate enzyme assay 22954 17091 8561 17785 Buffer exchanged concentrate 15829 11649 9915 86.79 enzyme assay (U/mL) Filtered buffer exchanged 21550 10882 94.71 85.27 concentrate enzyme assay (U/mL) Buffer exchanged concentrate 10699 13578 12727 2OSOO volume(mL) Ratio enzyme units O.87 O.96 1.32 1.4 concentration harvest reactor was checked for contamination. When the cell density A Q Sepharose (Pharmacia) ion exchange column (3 L in the 36 L spinner flasks reached 1.8-2.5x10 cells/mL, 20L resin, Height=20 cm, Diameter=14 cm) was prepared. Wash of cell culture was transferred from the 3.6 L spinner flasks to 55 samples were collected for a determination of pH, conductiv the 125 L bioreactor (Braun), resulting in a final volume of 85 ity and endotoxin (LAL) assay. The column was equilibrated L and a seeding density of approximately 4x10 cells/mL. with 5 column volumes of 10 mM Tris, 20 mM NaSO, pH Parameters were: temperature setpoint: 37°C.; pH: 7.2; dis 7.5. The concentrated, diafiltered harvest was loaded onto the solved oxygen: 25%+10%; impeller speed: 50 rpm; vessel Q columnata flow rate of 100 cm/hr. The column was washed pressure: 3 psi; air sparge: 1 L/min...; air overlay: 1 L/min. The 60 with 5 column volumes of 10 mM Tris, 20 mM NaSO, pH reactor was sampled daily for cell counts, pH verification, 7.5 and 10 mM Hepes, 50 mMNaCl, pH 7.0. The protein was media analysis, protein production and retention. Nutrient eluted with 10 mM Hepes, 400 mMNaCl, pH 7.0, and filtered feeds were added during the run. At Day 6, 3.4 L of Feed #1 through a 0.22 Lum final filter into a sterile bag. Medium (CD CHO+50 g/L glucose-40 mL/L GlutaMAXTM Phenyl Sepharose (Pharmacia) hydrophobic interaction 1) was added, and culture temperature was changed to 36.5° 65 chromatography was next performed. A Phenyl Sepharose C. At day 9, 3.5 L of Feed #2 (CD CHO+50 g/L glucose+40 (PS) column (9.1 L resin, Height=29 cm, Diameter-20 cm) mL/L GlutaMAXTM-1+1.2 g/L sodium butyrate) was added, was prepared. The column was equilibrated with 5 column US 9,084,743 B2 83 84 volumes of 5 mM potassium phosphate, 0.5 M ammonium concentrated protein into the final buffer: 10 mM Hepes, 130 sulfate, 0.1 mM CaCl, pH 7.0. The protein eluate from above mM NaCl, pH 7.0. The concentrated protein was passed was supplemented with 2M ammonium sulfate, 1 M potas though a 0.22 um filter into a 20 L sterile storage bag. The sium phosphate and 1 M CaCl2 stock solutions to final con protein was sampled and tested for protein concentration, centrations of 5 mM, 0.5 M and 0.1 mM, respectively. The enzyme activity, free sulfhydryl groups, oligosaccharide pro protein was loaded onto the PS column at a flow rate of 100 filing and osmolarity. cm/hr. 5 mM potassium phosphate, 0.5 Mammonium sulfate Tables 16 through 22-provide monitoring data related to and 0.1 mM CaCl, pH 7.0, was added at 100 cm/hr. The each of the purification steps described above, for each flow-through was passed through a 0.22 um final filter into a 3D35M cell lot. sterile bag. 10 The PS-purified protein was then loaded onto an ami TABLE 16 nophenyl boronate column (ProMedics) (6.3 L resin, Height=20 cm, Diameter-20 cm) that had been equilibrated Q Sepharose column data with 5 column volumes of 5 mM potassium phosphate, 0.5 M Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC ammonium sulfate. The protein was passed through the col 15 umn at a flow rate of 100 cm/hr, and the column was washed Load volume 10647 13524 12852 20418 with 5 mM potassium phosphate, 0.5 Mammonium sulfate, (mL) Load Volume? 3.1 4.9 4.5 7.3 pH 7.0. The column was then washed with 20 mMbicine, 100 Resin Volume mMNaCl, pH 9.0, and the protein eluted with 50 mM Hepes, ratio 100 mM NaCl, pH 6.9, through a sterile filter and into a 20 L Column 2770 3840 28SO 288O Volume (mL) sterile bag. The eluate was tested for bioburden, protein con Eluate volume 6108 5923 5759 6284 centration and enzyme activity. (mL) A hydroxyapatite (HAP) column (Bio-Rad) (1.6 L resin, Protein Conc. 2.8 3.05 2.80 2.86 Height=10 cm, Diameter=14 cm) was equilibrated with 5 of Eluate mM potassium phosphate, 100 mMNaCl, 0.1 mM CaCl, pH 25 (mg/mL) Eluate Enzyme 24493 26683 18321 21052 7.0. Wash samples were collected and tested for pH, conduc Assay (U/mL) tivity and endotoxin (LAL assay). The aminophenyl bor Enzyme Yield 65 107 87 76 onate-purified protein was Supplemented with potassium (%) phosphate and CaCl to yield final concentrations of 5 mM potassium phosphate and 0.1 mM CaCl2, then was loaded 30 onto the HAP column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate, pH 7.0, 100 TABLE 17 mMNaCl, 0.1 mM CaCl, then 10 mM potassium phosphate, Pheny Sepharose column data pH 7.0, 100 mM. NaCl, 0.1 mM CaCl pH. The protein was eluted with 70 mM potassium phosphate, pH 7.0, and filtered 35 Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC through a 0.22 Lum filter into a 5 L sterile storage bag. The Volume Before 5670 5O15 5694 6251 eluate was tested for bioburden, protein concentration and Stock Solution enzyme activity. Addition (mL) Load Volume 7599 6693 7631 8360 The HAP-purified protein was then pumped through a 20 (mL) nM viral removal filter via a pressure tank. The protein was 40 Column 9106 942O 9340 942O added to the DV20 pressure tank and filter (Pall Corporation), Volume (mL) passing through an Ultipor DV20 Filter with 20 nm pores Load Volume? O.8 O.71 O.82 O.89 Resin Volume (Pall Corporation) into a sterile 20 L storage bag. The filtrate ratio was tested for protein concentration, enzyme activity, oli Eluate volume 16144 18010 16960 17328 gosaccharide, monosaccharide and Sialic acid profiling, and 45 (mL) process-related impurities. The protein in the filtrate was then Protein Cone 0.4 O.33 O.33 O.38 of Eluate concentrated to 1 mg/mL using a 10 kDa molecular weight (mg/mL) cut off (MWCO) Sartocon Slice tangential flow filtration Eluate Enzyme 8806 6585 4472 7509 (TFF) system (Sartorius). The filter was first prepared by Assay (U/mL) washing with a Hepes/saline solution (10 mM Hepes, 130 50 Protein Yield 41 40 36 37 (%) mMNaCl, pH 7.0) and the permeate was sampled for pH and Enzyme Yield 102 88 82 96 conductivity. Following concentration, the concentrated pro (%) tein was sampled and tested for protein concentration and enzyme activity. A 6x buffer exchange was performed on the TABLE 18 Amino phenylboronate column data
Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC
Load Volume (mL) 16136 17958 16931 17884 Load Volume? Resin 2.99 3.15 3.08 2.98 Volume ratio Column Volume (mL) S400 5700 5500 5300 Eluate volume (mL) 17595 22O84 2O686 1914.5 Protein Conc. of Eluate (mg/ O.O O.O3 O.O3 O.04 mL) US 9,084,743 B2 85 86 TABLE 18-continued Amino phenylboronate column data
Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC Protein Conc. of Not tested O.O3 O.OO O.04 Filtered Eluate (mg/mL) Eluate Enzyme Assay 4OSO 2410 1523 4721
Protein Yield (%) O 11 11 12 Enzyme Yield (%) Not determined 41 40 69
TABLE 19 Hydroxyapatite column data
Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC
Volume Before Stock 16345 2O799 2O640 19103 Solution Addition (mL) Load Volume?Resin 10.95 13.58 1419 12.81 Volume ratio Column Volume (mL) 1SOO 1540 1462 1500 Load volume (mL) 16429 20917 2O746 19213 Eluate volume (mL) 4100 24.15 1936 2419 Protein Conc. of Eluate (mg/ Not tested O.24 O.17 O.23 mL) Protein Conc. of NA NA O.17 NA Filtered Eluate (mg/mL) Eluate Enzyme Assay 14051 29089 20424 29826 (U/mL) Protein Yield (%) Not tested 93 53 73 Enzyme Yield (%) 87 118 140 104
TABLE 20
DV20 filtration data
Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC Start volume (mL) 4077 2233 1917 2419 Filtrate Volume (mL) 46O2 3334 2963 3SO4 Protein Conc. of Filtrate (mg/ O.1 NA O.09 NA mL) Protein Conc. of NA O.15 O.09 O16 Filtered Eluate (mg/mL) Protein Yield (%) Not tested 93 82 101
TABLE 21 TABLE 21-continued
Final concentration data Final concentration data 50 Parameter HUAO4O6C HUAO410C HUAO41SC HUAO42OC Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC Protein O.9 1.24 1.16 1.73 Start 4575 3.298 2963 3492 Conc. of volume (mL) Sociate Concentrate S62 407 237 316 55 SS 111 102 103 98 Volume Yield (%) (mL)
TABLE 22 Buffer exchange into final formulation data Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC
Start Volume (mL) S62 407 237 316 Final Volume Buffer 594 S16 310 554 Exchanged Concentrate (mL) US 9,084 ,743 B2 87 88 TABLE 22-continued Buffer exchange into final formulation data Parameter HUAO4O6C HUAO410C HUAO415C HUAO42OC
Protein Conc. of 1.00 0.97 O.98 1.OO Concentrate (mg/mL) Protein Conc. of Filtered O.9S O.92 O.9S 1.02 Concentrate (mg/mL) Protein Yield (%) 118 99 110 101
The purified and concentrated soluble rhuPH20 protein DNA digested with Xba I; and one single hybridizing band of was aseptically filled into sterile vials with 5 mL and 1 mL fill ~1.4 kb observed using 2B2 DNA digested with BamH Volumes. The protein was passed though a 0.22um filter to an I/Hind III. Sequence analysis of the mRNA transcript indi operator controlled pump that was used to fill the vials using 15 cated that the derived cDNA (SEQID NO:56) was identical to a gravimetric readout. The vials were closed with stoppers the reference sequence (SEQID NO:49) except for one base and secured with crimped caps. The closed vials were visu pair difference at position 1131, which was observed to be a ally inspected for foreign particles and then labeled. Follow thymidine (T) instead of the expected cytosine (C). This is a ing labeling, the vials were flash-frozen by Submersion in silent mutation, with no effect on the amino acid sequence. liquid nitrogen for no longer than 1 minute and stored at D. Production of Gent Soluble ruPH2O in 300 L Bioreactor s-15°C. (-20+5° C.). Cell Culture C. Production Gen2 Cells Containing Soluble Human PH20 A vial of HZ24-2B2 was thawed and expanded from shake (rHuPH20) flasks through 36 L spinner flasks in CD-CHO media (Invit The Gen1 3D35M cell line described above was adapted to 25 rogen, Carlsbad, Calif.) supplemented with 20 methotrexate higher methotrexate levels to produce generation 2 (Gen2) and GlutaMAXTM-1 (Invitrogen). Briefly, the vial of cells was clones. 3D35M cells were seeded from established methotr thawed in a 37°C. water bath, media was added and the cells exate-containing cultures into CD CHO medium containing 4 were centrifuged. The cells were re-suspended in a 125 mL mM GlutaMAXTM-1 and 1.0 uM methotrexate. The cells shake flask with 20 mL of fresh media and placed in a 37°C., were adapted to a higher methotrexate level by growing and 30 7% CO, incubator. The cells were expanded up to 40 mL in passaging them 9 times over a period of 46 days in a 37°C., the 125 mL shake flask. When the cell density reached greater 7% CO, humidified incubator. The amplified population of than 1.5x10° cells/mL, the culture was expanded into a 125 cells was cloned out by limiting dilution in 96-well tissue mL spinner flask in a 100 mL culture volume. The flask was culture plates containing medium with 2.0LM methotrexate. incubated at 37° C., 7% CO. When the cell density reached After approximately 4 weeks, clones were identified and 35 greater than 1.5x10° cells/mL, the culture was expanded into clone 3E10B was selected for expansion. 3E10B cells were a 250 mL spinner flask in 200 mL culture volume, and the grown in CD CHO medium containing 4 mM flask was incubated at 37°C., 7% CO. When the cell density GlutaMAXTM-1 and 2.0 LM methotrexate for 20 passages. A reached greater than 1.5x10 cells/mL, the culture was master cell bank (MCB) of the 3E10B cell line was created expanded into a 1 L spinner flask in 800 mL culture volume and frozen and used for Subsequent studies. 40 and incubated at 37° C., 7% CO. When the cell density Amplification of the cell line continued by culturing reached greater than 1.5x10 cells/mL the culture was 3E10B cells in CD CHO medium containing 4 mM expanded into a 6 L spinner flask in 5000 mL culture volume GlutaMAXTM-1 and 4.0 uM methotrexate. After the twelfth and incubated at 37° C., 7% CO. When the cell density passage, cells were frozen in Vials as a research cell bank reached greater than 1.5x10 cells/mL the culture was (RCB). One vial of the RCB was thawed and cultured in 45 expanded into a 3.6 L spinner flaskin 32 L culture volume and medium containing 8.0 LM methotrexate. After 5 days, the incubated at 37°C., 7% CO. methotrexate concentration in the medium was increased to A 400 L reactor was sterilized and 230 mL of CD CHO 16.0 LM, then 20.0 uM 18 days later. Cells from the eighth media was added. Before use, the reactor was checked for passage in medium containing 20.0 LM methotrexate were contamination. Approximately 30 L cells were transferred cloned out by limiting dilution in 96-well tissue culture plates 50 from the 36L spinner flasks to the 400 Lbioreactor (Braun) at containing CD CHO medium containing 4 mM an inoculation density of 40x10 viable cells per mL and a GlutaMAXTM-1 and 20.0 uM methotrexate. Clones were total volume of 260L. Parameters were: temperature setpoint: identified 5-6 weeks later and clone 2B2 was selected for 37° C.; impeller speed 40-55 rpm; vessel pressure: 3 psi; air expansion in medium containing 20.0 LM methotrexate. sparge: 0.5-1.5 L/Min.; air overlay: 3 L/min. The reactor was After the eleventh passage, 2B2 cells were frozen in vials as 55 sampled daily for cell counts, pH verification, media analysis, a research cell bank (RCB). protein production and retention. Also, during the run nutrient The resultant 2B2 cells are dihydrofolate reductase defi feeds were added. At 120 hrs (day 5), 10.4 L of Feed #1 cient (dhfr-) DG44 CHO cells that express soluble recombi Medium (4xCD CHO+33 g/L glucose--160 mL/L nant human PH20 (rhuPH20). The soluble rHuPH20 is GlutaMAXTM-1 mL/L yeastolate--33 mg/L rHuInsulin) was present in 2B2 cells at a copy number of approximately 206 60 added. At 168 hours (day 7), 10.8 L of Feed #2 (2xCD copies/cell. Southern blot analysis of Spe I-, Xba I- and CHO+33 g/L glucose--80 mL/L GlutaMAXTM-1+167 mL/L BamH I/Hind III-digested genomic 2B2 cell DNA using a yeastolate+0.92 g/L sodium butyrate) was added, and culture rHuPH20 -specific probe revealed the following restriction temperature was changed to 36.5°C. At 216 hours (day 9), digest profile: one major hybridizing band of -7.7 kb and four 10.8 L of Feed #3 (1x CD CHO+50 g/L glucose+50 mL/L minor hybridizing bands (~13.9, -6.6, -5.7 and ~4.6 kb) with 65 GlutaMAXTM-1+250 mL/L yeastolate+1.80 g/L sodium DNA digested with Spe I; one major hybridizing band of -5.0 butyrate) was added, and culture temperature was changed to kb and two minor hybridizing bands (~13.9 and -6.5 kb) with 36° C. At 264 hours (day 11), 10.8 L of Feed #4 (1x CD US 9,084,743 B2 89 90 CHO+33 g/L glucose--33 mL/L GlutaMAXTM-1+250 mL/L flow-through was passed through a 0.22 um final filter into a yeastolate+0.92 g/L sodium butyrate) was added, and culture sterile bag. The flow-through was sampled for bioburden, temperature was changed to 35.5°C. The addition of the feed protein concentration and enzyme activity. media was observed to dramatically enhance the production An aminophenyl boronate column (ProMetic) was pre of soluble rhuPH20 in the final stages of production. The 5 pared. The wash was collected and sampled for pH, conduc reactor was harvested at 14 or 15 days or when the viability of tivity and endotoxin (LAL assay). The column was equili the cells dropped below 40%. The process resulted in a final brated with 5 column volumes of 5 mM potassium phosphate, productivity of 17,000 Units/mL with a maximal cell density 0.5 M ammonium sulfate. The PS flow-through containing of 12 million cells/mL. At harvest, the culture was sampled purified protein was loaded onto the aminophenylboronate for mycoplasma, bioburden, endotoxin and viral in vitro and 10 column at a flow rate of 100 cm/hr. The column was washed in vivo, transmission electron microscopy (TEM) and with 5 mM potassium phosphate, 0.5 Mammonium sulfate, enzyme activity. pH 7.0. The column was washed with 20 mM bicine, 0.5 M The culture was pumped by a peristaltic pump through four ammonium sulfate, pH 9.0. The column was washed with 20 Millistak filtration system modules (Millipore) in parallel, mM bicine, 100 mM NaCl, pH 9.0. The protein was eluted each containing a layer of diatomaceous earth graded to 4-8 15 with 50 mM Hepes, 100 mM NaCl, pH 6.9, and passed um and a layer of diatomaceous earth graded to 1.4-1.1 um, through a sterile filter into a sterile bag. The eluted sample followed by a cellulose membrane, then through a second was tested for bioburden, protein concentration and enzyme single Millistak filtration system (Millipore) containing a activity. layer of diatomaceous earth graded to 0.4–0.11um and alayer The hydroxyapatite (HAP) column (Bio-Rad) was pre of diatomaceous earth graded to <0.1 um, followed by a pared. The wash was collected and tested for pH, conductivity cellulose membrane, and then through a 0.22 um final filter and endotoxin (LAL assay). The column was equilibrated into a sterile single use flexible bag with a 350 L capacity. The with 5 mM potassium phosphate, 100 mM NaCl, 0.1 mM harvested cell culture fluid was supplemented with 10 mM CaCl, pH 7.0. The aminophenylboronate-purified protein EDTA and 10 mM Tris to a pH of 7.5. The culture was was Supplemented to final concentrations of 5 mM potassium concentrated 10x with a tangential flow filtration (TFF) appa 25 phosphate and 0.1 mM CaCl and loaded onto the HAP col ratus using four Sartoslice TFF 30 kDa molecular weight umnata flow rate of 100 cm/hr. The column was washed with cut-off (MWCO) polyether sulfone (PES) filter (Sartorious), 5 mM potassium phosphate, pH 7.0, 100 mM. NaCl, 0.1 mM followed by a 10x buffer exchange with 10 mM Tris, 20 mM CaCl. The column was next washed with 10 mM potassium NaSO, pH 7.5, into a 0.22 um final filter into a 50 L sterile phosphate, pH 7.0, 100 mMNaCl, 0.1 mM CaCl.The protein storage bag. 30 was eluted with 70 mM potassium phosphate, pH 7.0, and The concentrated, diafiltered harvest was inactivated for passed through a 0.24 um sterile filter into a sterile bag. The virus. Prior to viral inactivation, a solution of 10% Triton eluted sample was tested for bioburden, protein concentration X-100, 3% tri-n-butyl phosphate (TNBP) was prepared. The and enzyme activity. concentrated, diafiltered harvest was exposed to 1% Triton The HAP-purified protein was then passed through a viral X-100, 0.3% TNBP for 1 hour in a 36 L. glass reaction vessel 35 removal filter. The sterilized Viosart filter (Sartorius) was first immediately prior to purification on the Q column. prepared by washing with 2 L of 70 mM potassium phos E. Purification of Gen2 Soluble ruPH2O phate, pH 7.0. Before use, the filtered buffer was sampled for A Q Sepharose (Pharmacia) ion exchange column (9 L pH and conductivity. The HAP-purified protein was pumped resin, H=29 cm, D=20 cm) was prepared. Wash samples were via a peristaltic pump through the 20 nM viral removal filter. collected for a determination of pH, conductivity and endot 40 The filtered protein in 70 mM potassium phosphate, pH 7.0, oxin (LAL) assay. The column was equilibrated with 5 col was passed through a 0.22um final filter into a sterile bag. The umn volumes of 10 mM Tris, 20 mM NaSO pH 7.5. Fol viral filtered sample was tested for protein concentration, lowing viral inactivation, the concentrated, diafiltered harvest enzyme activity, oligosaccharide, monosaccharide and Sialic was loaded onto the Q column at a flow rate of 100 cm/hr. The acid profiling. The sample also was tested for process-related column was washed with 5 column volumes of 10 mM Tris, 45 impurities. 20 mMNaSO pH 7.5, and 10 mMHepes, 50 mMNaCl, pH The protein in the filtrate was then concentrated to 10 7.0. The protein was eluted with 10 mM Hepes, 400 mM mg/mL using a 10 kD molecular weight cut off (MWCO) NaCl, pH 7.0, into a 0.22 um final filter into sterile bag. The Sartocon Slice tangential flow filtration (TFF) system (Sarto eluate sample was tested for bioburden, protein concentration rius). The filter was first prepared by washing with 10 mM and hyaluronidase activity. A280 absorbance readings were 50 histidine, 130 mM NaCl, pH 6.0, and the permeate was taken at the beginning and end of the exchange. sampled for pH and conductivity. Following concentration, Phenyl Sepharose (Pharmacia) hydrophobic interaction the concentrated protein was sampled and tested for protein chromatography was next performed. A Phenyl Sepharose concentration and enzyme activity. A 6x buffer exchange was (PS) column (19-21 L resin, H=29 cm, D-30 cm) was pre performed on the concentrated protein into the final buffer: 10 pared. The wash was collected and sampled for pH, conduc 55 mM histidine, 130 mM. NaCl, pH 6.0. Following buffer tivity and endotoxin (LAL assay). The column was equili exchange, the concentrated protein was passed though a 0.22 brated with 5 column volumes of 5 mM potassium phosphate, um filter into a 20 L sterile storage bag. The protein was 0.5 Mammonium sulfate, 0.1 mM CaCl, pH 7.0. The protein sampled and tested for protein concentration, enzyme activ eluate from the Q Sepharose column was supplemented with ity, free Sulfhydryl groups, oligosaccharide profiling and 2M ammonium sulfate, 1 M potassium phosphate and 1 M 60 osmolarity. CaCl stock solutions to yield final concentrations of 5 mM, The sterile filtered bulk protein was then aseptically dis 0.5 M and 0.1 mM, respectively. The protein was loaded onto pensed at 20 mL into 30 mL sterile Teflon vials (Nalgene). the PS column at a flow rate of 100 cm/hr and the column The vials were then flash frozen and stored at -20+5° C. flow-through collected. The column was washed with 5 mM F. Comparison of Production and Purification of Gen1 potassium phosphate, 0.5 Mammonium sulfate and 0.1 mM 65 Soluble ruPH2O and Gen2 Soluble ruPH2O CaCl, pH 7.0, at 100 cm/hr and the wash was added to the The production and purification of Gen2 soluble rhuPH20 collected flow-through. Combined with the column wash, the in a 300L bioreactor cell culture contained some changes in US 9,084,743 B2 91 92 the protocols compared to the production and purification of Table 23 sets forth exemplary differences, in addition to Gen1 Soluble ruPH2O in a 100 L bioreactor cell culture. simple scale-up changes, between the methods. TABLE 23 Comparison of Gen1 and Gen2 methods Process Difference Gen1 soluble rEuPH2O Gen2 Soluble rEHuPH2O
Cell line 2B2 Media used to expand cell Contains 0.10 M Contains 20 IM methotrexate inoculum methotrexate (0.045 mg/L) (9 mg/L) Media in 6 L cultures Contains 0.10 M Contains no methotrexate onwards methotrexate 36 L spinner flask No instrumentation Equipped with 20 L operating volume instrumentation that monitors and controls pH, dissolved oxygen, sparge and overlay gas flow rate. 32 L operating volume Final operating volume in Approx. 100 L in a 125 L Approx. 300 L in a 400 L bioreactor bioreactor bioreactor (initial culture (initial culture volume + 65 L) volume + 260 L) Culture media in final No ruInsulin 5.0 mg/L rHuInsulin bioreactor Media feed volume Scaled at 4% of the bioreactor Scaled at 4% of the bioreactor cell culture volume i.e. 3.4, cell culture volume i.e. 10.4, 3.5 and 3.7 L, resulting in a 10.8, 11.2 and 11.7 L, target bioreactor volume of resulting in a target bioreactor --92 L volume of -303 L Media feed Feed #1 Medium: CD CHO + Feed #1 Medium: 4x CD 50 g/L glucose + 8 mM CHO + 33 g/L glucose + 32 mM GltaMAXTM-1 GlutaMAXTM-1 + 16.6 g/L Feed #2 (CD CHO + 50 g/L yeastolate + 33 mg/L. glucose + 8 mM rHuInsulin GlutaMAXTM-1 + 1.1 g/L Feed #2: 2x CD CHO + 33 g/L sodium butyrate glucose + 1.6 mM Feed #3: CD CHO + 50 g/L GlutaMAXTM-1 + 33.4 g/L glucose + 8 mM yeastolate + 0.92 g/L sodium GlutaMAXTM-1 + 1.1 g/L butyrate sodium butyrate Feed #3: 1 x CD CHO + 50 g/L glucose + 10 mM GlutaMAXTM-1 + 50 g/L yeastolate + 1.80 g/L sodium butyrate Feed #4: 1 x CD CHO + 33 g/L glucose + 6.6 mM GlutaMAXTM-1 + 50 g/L yeastolate + 0.92 g/L sodium butyrate Filtration of bioreactor cell Four polyethersulfone filters 1 stage - Four modules in culture (8.0 m, 0.65 m, 0.22 Im parallel, each with a layer of and 0.22 m) in series diatomaceous earth graded to 100 L storage bag 4–8 m and a layer of diatomaceous earth graded to 1.4-1.1 m, followed by a cellulose membrane. 2” stage - single module containing a layer of diatomaceous earth graded to 0.4–0.11 Lim and a layer of diatomaceous earth graded to <0.1 m, followed by a cellulose membrane. 3 stage - 0.22 Lim polyethersulfone filter 300 L storage bag Harvested cell culture is supplemented with 10 mM EDTA, 10 mM Tris to a pH of 7.5 Concentration and buffer Concentrate with 2 TFF with Concentrate using four exchange prior to Millipore Spiral Sartorius SartOSlice TFF 30K chromatography Polyethersulfone 30K MWCO Filter MWCO Filter Buffer Exchange the Buffer Exchange the Concentrate 6x with 10 mM Concentrate 10x with 10 mM Hepes, 25 mM NaCl, pH 7.0 Tris, 20 mM Na2SO, pH 7.5 20 L sterile storage bag 50 L sterile storage bag Viral inactivation prior to None Viral inactivation performed chromatography with the addition of a 1% US 9,084,743 B2 93 94 TABLE 23-continued Comparison of Gen1 and Gen2 methods Process Difference Gen1 soluble rEHuPH2O Gen2 Soluble rEuPH2O Triton X-100, 0.3% tri-n- butyl phosphate, pH 7.5 1 purification step (Q No absorbance reading A280 measurements at the Sepharose) beginning and end Viral filtration after Pall DV-20 filter (20 nm) Sartorius Virosart filter (20 nm) chromatography Concentration and buffer Hepessaline, pH 7.0 buffer Histidine?saline, pH 6.0 exchange after Protein concentrated to 1 mg/mL buffer chromatography Protein concentrated to 10 mg/mL
Example 4 15 shaken for 10 seconds. After shaking, the plate was returned to the heat block and the MULTIDROP384 Liquid Handling Determination of Hyaluronidase Activity of Soluble Device was primed with the warm 0.25 mg/mL sodium hyalu rHuPH20 Using a Microturbidity Assay ronate solution (prepared by dissolving 100 mg of Sodium hyaluronate (LifeCore Biomedical) in 20.0 mL of SWFI. This Hyaluronidase activity of soluble recombinant human was mixed by gently rotating and/or rocking at 2-8°C. for 2-4 PH20 (rhuPH2O) in samples such as cell cultures, purifica hours, or until completely dissolved. The substrate solution tion fractions and purified Solutions was determined using a was prepared by mixing 9 mL SWFI, 10 mL PIPES and 1 mL turbidometric assay, which is based on the formation of an of 5 mg/mL hyaluronate). The reaction plate was transferred insoluble precipitate when hyaluronic acid binds with serum 25 albumin. The activity is measured by incubating soluble to the MULTIDROP 384 and the reaction was initiated by rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set pressing the start key to dispense 30 ul, Sodium hyaluronate period of time (10 minutes) and then precipitating the undi substrate solution into each well. The plate was then removed gested sodium hyaluronate with the addition of acidified from the MULTIDROP384 and shaken for 10 seconds before serum albumin. The turbidity of the resulting sample is mea 30 being transferred to a heat block with the plate cover replaced. sured at 640 nm after a 30 minute development period. The The plate was incubated at 37° C. for 10 minutes. decrease in turbidity resulting from enzyme activity on the The MULTIDROP 384 was prepared to stop the reaction Sodium hyaluronate Substrate is a measure of the soluble by priming the machine with serum working solution (25 mL rHuPH20 hyaluronidase activity. The method is performed of serum stock Solution 1 Volume of horse serum (Sigma) using a calibration curve generated with dilutions of a soluble 35 was diluted with 9 volumes of 500 mM acetate buffer solu rHuPH20 assay working reference standard, and sample tion, pH 4.3, and the pH was adjusted to 3.1 with hydrochloric activity measurements are made relative to this calibration acid in 75 mL of 500 mMacetate buffer solution, pH 4.3) and CUV. changing the Volume setting to 240 uL. The plate was Dilutions of the sample were prepared in Enzyme Diluent 40 removed from the heat block and placed onto the MULTI Solutions. The Enzyme Diluent Solution (EDS) was prepared DROP 384 and 240 uL of serum working solution was dis by dissolving 33.0+0.05 mg of hydrolyzed gelatin in 25.0 mL pensed into the wells. The plate was removed and shaken on of the 50 mM PIPES Reaction Buffer (140 mM. NaCl, 50 mM a plate reader for 10 seconds. After a further 15 minutes, the PIPES, pH 5.5) and 25.0 mL of Sterile Water for Irrigation turbidity of the samples was measured at 640 nm and the (SWFI), and diluting 0.2 mL of 25% human serum albumin 45 hyaluronidase activity (in U/mL) of each sample was deter solution into the mixture and vortexing for 30 seconds. This mined by fitting to the standard curve. was performed within 2 hours of use and stored on ice until Specific activity (Units/mg) was calculated by dividing the needed. The samples were diluted with EDS to an estimated hyaluronidase activity (U/mL) by the protein concentration 1-2 U/mL. Generally, the maximum dilution per step did not (mg/mL). exceed 1:100 and the initial sample size for the first dilution 50 was not less than 20 uL. The minimum sample Volumes needed to perform the assay were: In-process Samples, FPLC Example 5 Fractions: 80 uL. tissue culture Supernatants: 1 mL, concen trated material:80 LL; purified or final step material:80 uL. 55 Effect of Sodium Chloride on the Stability of The dilutions were made in triplicate in a Low Protein Bind rHLPH2O ing 96-well plate, and 30 uL of each dilution was transferred to Optilux black/clear bottom plates (BD BioSciences). The rhuPH20 was in a solution at pH 6.5 containing 10 Dilutions of known soluble rFuPH20 with a concentration mg/mL in histidine/HCl and 130 mM sodium chloride of 2.5 U/mL were prepared in Enzyme Diluent Solution to 60 (NaCl). As shown in Table 24, a total of 6 different formula generate a standard curve and added to the Optilux plate in tions containing the following components were prepared: 25 triplicate. The dilutions included 0 U/mL, 0.25 U/mL, 0.5 mM Tris, pH 7.3, 100 ug/mL rHuPH20, 0.01% Tween 80 and U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL. NaCl (0, 50, 100, 150, 200 or 250 mM). The solutions were “Reagent blank” wells that contained 60 uL of Enzyme Dilu aliquotted into 2 mL type I glass vials with rubber stoppers ent Solution were included in the plate as a negative control. 65 and sealed with aluminum caps. One set of vials was stored at The plate was then covered and warmed on a heat block for 5 40° C. for four days, and the other set was kept in the refrig minutes at 37°C. The cover was removed and the plate was erator at 2 to 8° C. US 9,084,743 B2 95 96 TABLE 24 (NaCl) at pH 6.0. rHuPH20 was diluted to 100000 U/mL using 10 mM histidine--130 mM NaCl, pH 6.0, prior to mix Formulation of rEuPH20 with NaCl ing with immunoglobulin. For this purpose, 200 uL of rHuPH20 stock solution was diluted with 1896 uL of histi Formulation # NaCl dine/NaCl buffer, pH 6.0. OmM The pre-diluted rHuPH20 was added to different IG for 50 mM 100 mM mulations formulated in 0.25 M glycine at pH 4.4 to 4.9 to 150 mM give final concentrations of 100 U/mL or 300 U/mL in the 200 mM solution. One of three different 10% IG lots from large scale 250 mM 10 manufacturing (LE12H020, LE12H062, and LE12H173) or one of three different pre-clinical 20% IG lots (SC00107NG, After 4 days of storage, each of the formulations mentioned SC00207NG, and SC00307NG) was utilized according to in Table 24 was tested for hyaluronidase enzymatic activity Table 26. The solutions were filtered through a 0.2 um filter using the microturbidity assay described in Example 4. Size and transferred in 1 mL portions into sterile 5 mL glass vials. exclusion chromatography (SEC) was performed to evaluate 15 The vials were stored at 2 to 8°C. or 28 to 32° C. Hence, the resulting co-formulations of rhuPH20 and IG were formu the level of aggregates using the following conditions: lated in 0.25 M glycine at pH 4.4 to 4.9. 1xRBS, Toso BioScience G2000 SWXL column, flow rate=1 TABLE 26 mL/min. Table 25 shows the results of the study, including hyalu Co-formulations of rEuPH2O and 10%. IG or 20% IG ronidase activity (U/mL), 96 main peak area (percentage of the rHuPH20 that was contained in the main peak area) and% Amount Amount of rEuPH2O aggregate peak area (percentage of rhuPH20 that was con of 10% diluted to tained in the peak area attributed to aggregates) for each Sample name IG or 20% IG 100000 UmL formulation. The results indicate that the stability of 10%. IG 50.00 mL. O rHuPH20, when incubated at 40°C., was dependent on NaCl 25 10%. IG + 100 UmL rEHuPH2O 49.95 mL. 50 L concentration: an increase in NaCl concentration led to 10%. IG - 300 UnL HPH2O 49.85 mL 150 L increased enzymatic activity of rhuPH20. The samples 20%. IG 50.00 mL. O stored at 2 to 8°C. retained similar levels of rHuPH20 enzy 20%. IG - 100 UnL HPH2O 49.95 mL. 50 L matic activity throughout the course of the study, regardless 20%. IG - 300 UnL HPH2O 49.85 mL 150 L of the formulation. In the absence of NaCl at elevated tem 30 peratures (40°C.), the entire enzymatic activity of rhuPH20 After 0 (start), 1, 3, 6, 12, 24 and 36 weeks (2 to 8°C. only) was lost. of storage, one sample from each of the 6 formulations men The results in Table 25 also show the effect of NaCl con tioned in Table 26 and from each of the storage chambers (2 centration on the aggregate levels of ruPH20. Aggregate to 8°C. and 28 to 32°C.) was withdrawn from the incubation levels increased with decreasing NaCl concentration in 35 and analyzed for hyaluronidase activity using the microtur samples Stored at 40°C. There was essentially no change in bidity assay described in Example 4. To assess effects on IG, the samples stored at 2 to 8° C. molecular size distribution of the IG informulations contain Thus, the results show that within the NaCl concentration ing 20%. IG was determined at 0 (start) and 6 months by high range tested (0-250 nM), there was a direct relationship performance size exclusion chromatography (HP-SEC) using between NaCl concentration and increased ruPH20 stabil a TSKG3000 SW 600x7.5 mm column (Tosoh Bioscience) ity, Suggesting that the NaCl concentration be maintained as 40 and a DMSO-containing buffer system (Kolarich et al. (2006) high as possible within solubility and tonicity limits in order Transfusion, 46:1959-1977). to increase the stability of rhuPH20 at elevated temperature. Table 27 shows hyaluronidase activity (U/mL) at 7 time TABLE 25 points (0, 1, 3, 6, 12, 24 and 36 weeks) for each co-formula tion stored at 2 to 8°C. Table 28 shows hyaluronidase activity Enzymatic activities and SEC results of the samples stored 4 days at 45 40° C. and 28°C. (U/mL) at 6 time points (0, 1, 3, 6, 12 and 2 weeks) for the co-formulations stored at 28 to 32° C. A significant, steady Enzymatic % Main % Aggregate loss of hyaluronidase activity was observed in the presence of Activity Peak Peak 10% and 20%. IG co-formulations Stored at 28 to 32° C. after Formulation 2-8°C. 40°C. 2-8°C. 40°C. 2-8°C. 40° C. 24 weeks, indicating rHuPH20 instability. The 10% IG co 50 formulations were stable after 9 months of storage at 2 to 8° 0 mMNaCl 10430 103.1 95.9 97.3 107 98 99 106 10 LE12HO20 + 100 UmL 99.2 84.9 59.6 36 22 295.0 291.2 281.8 293 282 296 292 LE12HO20+300 UmL 298.5 259.3 185.4 104 57 19 LE12HO62 + 100 UmL 108.5 88.2 60.1 43 29 10 94.0 97.8 81.4 85 87 78 66 LE12HO62 + 300 UmL 325 266.2 185.6 129 76 28 LE12H173 + 100 UmL 103.1 70.5 39.6 24 13 284.3 264.O 261 245 223 210 15 LE12H173 + 300 UmL 295.0 210.1 122.O 60 31 99.7 93.1 91.0 86 83 84 69 SCOO 107NG - 100 UL 94.O 83.1 57.4 43 49 32 SCOO 107NG - 300 UL 284.3 242.2 182.O 124 148 96 286 277 266.2 244 263 227 197 SCOO2O7NG - 100 UL 99.7 84.5 61.1 46 51 35 92.8 95.0 82.7 87 83 82 68 SCOO2O7NG - 300 UL 286 251 198.1 131 145 1 O6 SCOO3O7NG - 100 UL 92.8 82.7 67.9 48 52 34 254.3 2814 274.3 245 247 230 SCOO3O7NG - 300 UL 2S4.3 2S3.6 209.7 140 157 1 O6
TABLE 29
Molecular size distribution of IG in 20%. IG co-formulated with rHuPH20 after storage at 2-8 C.
O (start 6 months
>450 kDa -350kDa -160 kDa <60 kDa >450 kDa -350 kDa ~160 kDa
2.56 86.50 0.27 O.70 3.SO 85.50 O.30 2.39 86.75 O.24 O.70 3.59 85.43 O.28
O.65 2.38 86.70 O.26 3.80 85.19 O.32
0.73 3.25 85.76 O.26 O.86 4.52 8434 O.28 0.75 3.22 85.74 O.29 O.86 4.61 84.21 O.32
COO2O7NG O.77 3.39 85.63 O.21 O.83 4.57 84.30 O.30 OO OmL
O.93 1.76 87.06 O.25 1.01 2.78 85.96 O.25 O.96 1.91 86.94 O.20 1.03 3.04 85.62 O.31
2.00 86.86 O.23 O.99 2.88. 85.85 0.27
TABLE 30
Molecular size distribution of IG in 20%. IG co-formulated with rHuPH20 after storage at 28-32 C. O (start 6 months
Sample >450 kDa -350kDa -160 kDa <60 kDa >450 kDa -350 kDa ~160 kDa SCOO 107NG 12.56 86.50 0.27 OSO 12.53 85.94 1.02 SCOO 107NG 12.39 86.75 O.24 O.47 12.41 86.10 1.02 100 UmL rEHPH2O SCOO 107NG O.65 12.38 86.70 O.26 12.41 85.97 1.09 300 UmL rEHPH2O SCOO2O7NG 0.73 13.25 85.76 O.26 0.44 1321 85.42 US 9,084,743 B2 99 100 TABLE 30-continued
Molecular size distribution of IG in 20% IG co-formulated with rHuPH20 after storage at 28-32 C. O (start 6 months
Sample >450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350 kDa -160 kDa <60 kDa
SCOO2O7NG. -- 0.75 1322 85.74 O.29 O42 13.15 85.52 O.91 100 UmL rEHPH2O SCOO2O7NG. -- O.77 13.39 85.63 O.21 O.47 13.O1 85.62 O.90 300 UmL rEHPH2O SCOO3O7NG O.93 11.76 87.06 O.25 O.47 11.91 86.78 O.84 SCOO3O7NG. -- O.96 11.91 86.94 O.20 OSO 11.85 86.78 O.87 100 UmL rEHPH2O SCOO3O7NG. -- O.91 12.00 86.86 O.23 O4O 11. SO 87.21 O.89 300 UmL rEHPH2O
B. Stability of Co-Formulated 10% IG with rHuPH20 and aggregation (>450 kDa) at 28 to 32°C., and all values remain Sodium Chloride (0-150 mM) within the MSD specification limits (>90% monomer/dimers, To improve rHuPH20 stability in the co-formulations, the s5% aggregates, s5% fragments) after 6 months. effect of sodium chloride (NaCl) addition was investigated. Although the addition of NaCl negatively impacted (in Co-formulations of 300 U/mL rhuPH20 (lot HUB0702CA: 25 creased) the anticomplementary activity (ACA) titer of IG generated using Gen2 production described in Example 3) in formulations stored at 28 to 32°C., ACA titer is a specifica 10% IG (lot LE12F047) were prepared as described in tion indicator for intravenous (IV) administration and is not Example 7A above, with the addition of NaCl at 4 different relevant for Subcutaneous administration of the co-formula concentrations (0.50, 100 and 150 mM). The co-formulations tions. were stored at 2 to 8° C. or 28 to 32°C. Thus, the resulting 30 TABLE 31 co-formulations of rPuPH20 and IG were formulated in 0.25 M glycine at pH 4.6 to 5.1 (as measured in the diluted solu Hyaluronidase activity (U/mL) of 10% IGirHuPH2O co tion) in the presence of varying amounts of NaCl. formulations with NaCl after storage at 2-8 C. After 0 (start), 1, 3, 6, 12, 18 and 24 weeks of storage, one Weeks sample from each of the co-formulations (with NaCl concen 35 trations of 0, 50, 100, and 150 mM) and from each of the O storage chambers (2 to 8°C. and 28 to 32°C.) was withdrawn Salt Conc. (start) 1 2 3 6 12 18 24 from the incubation and analyzed for hyaluronidase activity 0 mMNaCl 276 288 269 289 317 264 276 274 using the microturbidity assay described in Example 4. 50 mM 292 286 296 306 320 287 276 295 Aggregation of IG was determined by molecular size distri 100 mM 28S 295 273 31S 319 287 281 288 bution (MSD) by high performance size exclusion chroma 40 150 mM 294 280 301 3OS 327 294 277 298 tography (HP-SEC) using a TSKG 3000 SW 600x7.5 mm column and a DMSO-containing buffer system (Kolarich et TABLE 32 al. (2006) Transfusion, 46:1959-1977). Tables 31 and 32 show hyaluronidase activity (U/mL) at 7 Hyaluronidase activity (U/mL) of 10% IGirHuPH2O co-formulations time points (0, 1, 3, 6, 12, 18 and 24 weeks) for each co 45 with NaCl after storage at 28-32 C. formulation. The results show that the stability of rhuPH20 co-formulated with 10% IG in the presence of 50, 100 or 150 Salt Weeks mMNaCl remained unchanged for up to 24 weeks of storage Conc. O (start) 1 2 3 6 12 18 24 at 2 to 8° C., while the rHuPH20 stability improved for those OmM 276 232 237 216 201 121 109 81 samples stored at 28 to 32°C. However, hyaluronidase activ 50 50 mM 292 288 280 301 3O2 247 225 223 ity rapidly decreased in the co-formulations having a NaCl 100 mM 285 286 28O 292 315 277 253 258 concentration of 0 mM when stored at 28 to 32° C. 150 mM 294 314 272 298 323 221 253 276 Tables 33 and 34 show that NaCl slightly enhanced IG dimerization (-350 kDa) at both storage temperatures and IG TABLE 33
Molecular size distribution of IG in 10% IGirHuPH2O co formulations with NaCl after storage at 2-8 C. O (start 6 months
Sample >450 kDa -350kDa -160 kDa <60 kDa >450 kDa -350kDa -160 kDa <60 kDa
0 mMNaCl O16 8.21 91.01 O.61 O16 1129 87.98 O.S8 50 mMNaCl O.17 8.99 9024 O.60 O.22 12.54 86.62 O.62 100 mMNaCl O.19 9.03 90.13 O.64 O.23 12.97 86.17 O.63 150 mMNaCl O.19 9.08 90.13 O.61 O.24 12.93 86.30 O.S3 US 9,084,743 B2 101 102 TABLE 34
Molecular size distribution of IG in 10% IGirHuPH2O co formulations with NaCl after storage at 28-32 C. O (start 6 months
Sample >450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350kDa -160 kDa <60 kDa 0 mMNaCl 0.16 8.21 91.01 O.61 O3S 9.37 88.77 1.51 SO MNaCl O.17 8.99 9024 O.60 0.75 10.83 86.85 1.57 100 mMNaCl 0.19 9.03 90.13 O.64 O.87 11.20 86.38 1.55 150 mMNaCl 0.19 9.08 90.13 O.61 1.02 11.15 86.18 1.66
C. Stability of Co-Formulated 10% IG or 20% IG with TABLE 35 rHuPH20 and Sodium Chloride (0-50 mM) 15 The effect of sodium chloride addition to co-formulations of 10% IG or 20%. IG with ruPH2O Stored at 28 to 32°C. was Hyaluronidase activity (U/mL) of 10% IGirHuPH2O co investigated. Co-formulations of 300 U/mL rHuPH20 (lot formulations with NaCl after storage at 28-32° C. HUB0702CA; generated using Gen2 production described in Example 1) in 10% IG (lot LE12F047) and 300 U/mL 20 Salt Weeks rHuPH20 (lot HUB07020A; generated using Gen2 produc tion described in Example 1) in 20% IG (lot SC00108NG) were prepared as described in Example 6B above, using NaCl Concentration O 1 3 6 12 24 concentrations of 0, 5, 10, 20, 30, 40 and 50 mM. Thus, the resulting co-formulations of rHuPH20 and IG were formu- 25 OmM 292 260 225 211 135 lated in 0.25 M glycine at pH 4.6 to 5.1 (as measured in the 5 mM 294 247 242 225 162 diluted solution) in the presence of varying amounts of NaCl. 10 mM 272 255 242 240 177 91 After 0 (start), 1, 3, 6, 12 and 24 weeks of storage one 20 mM 281 3O2 261 259 232 sample from each of the co-formulations (with NaCl concen 154 trations of 0, 5, 10, 20, 30, 40 and 50 mM) was withdrawn 30 30 mM 279 273 2S6 261 229 18O from the incubation and analyzed for hyaluronidase activity 40 mM 274 254 266 275 246 196 using the microturbidity assay described in Example 4. IG 50 mM 275 254 278 281 252 200 aggregation was determined by molecular size distribution by high performance size exclusion chromatography (HP-SEC) using a TSKG3000 SW 600x7.5 mm column and a DMSO 35 containing buffer system. TABLE 36 Tables 35 and 36 show hyaluronidase activity (U/mL) at various time points (0, 1, 3, 6 and 12 and 24 weeks) for each Hyaluronidase activity (U/mL) of 20% IGirHuPH2O co co-formulation. The results show that the stability of formulations with NaCl after storage at 28-32° C. rHuPH20 co-formulated with 10% IG in the presence of 40 higher NaCl concentrations (20,30,40 and 50 mM) remained Salt Weeks relatively unchanged through 24 weeks of storage at 28 to 32 C. Hyaluronidase activity rapidly decreased in the co-formu- Concentration O 1 3 6 12 24 lations having a NaCl concentration of less than 20 mM when OmM 267 264 251 238 212 138 stored at 28 to 32°C. The stability of rhuPH20 co-formulated 45 5 mM 290 261 249 242 214 143 with 20%. IG remained relatively unchanged through 24 10 mM 276 264 262 232 2O7 141 weeks of storage at 28 to 32° C. at all NaCl concentrations. 20 mM 314 249 274 239 222 155 Sodium chloride slightly enhanced IG dimerization (-350 30 mM 252 253 276 241 211 162 kDa) and aggregation in both 10% and 20%. IG co-formula- 40 mM 273 240 275 242 216 170 tions at 28 to 32°C. The effect is less pronounced in 20% IG 50 50 mM 289 238 266 234 232 16S (i.e., higher IG concentration) on IG aggregation (Tables 37 and 38). TABLE 37
Molecular size distribution of IG in 10%. IGirHuPH2O co formulations with NaCl after storage at 28-32° C.
O (start) 6 months
Sample >450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350 kDa -160 kDa
OmM O16 9.35 90.01 O.48 O.19 7.08 91.69 1.04 NaCl 5 mM O16 9.53 89.71 O.60 O.21 7.66 91.11 1.02 NaCl US 9,084,743 B2 103 104 TABLE 37-continued
Molecular size distribution of IG in 10% IGirHuPH2O co formulations with NaCl after storage at 28-32 C. O (start 6 months Sample >450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350 kDa -160 kDa <60 kDa 10 mM O16 9.77 89.52 O.S6 O.22 8.20 90.52 1.OS NaCl 20 mM O.17 9.96 89.27 O.6O O.26 8.42 90.27 1.OS NaCl 30 mM O.17 10.25 89.06 O.S3 O.30 9.07 89.59 1.04 NaCl 40 mM O.17 10.48 88.82 O.S3 O.34 9.06 89.56 1.OS NaCl 50 mM O.18 10.55 88.72 O.S4 O.39 9.22 89.33 1.07 NaCl
TABLE 38
Molecular size distribution of IG in 20% IGirHuPH2O co formulations with NaCl after storage at 28-32 C. O (start 6 months
Sample >450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350 kDa -160 kDa <60 kDa
OmM O.32 14.65 84.72 O.31 O.34 11.77 87.18 O.71 NaC 5 mM O.32 14.70 84.70 0.27 O.34 11.57 87.35 O.74 NaC 10 mM O.35 14.86 84.48 O.31 O.35 12.05 86.94 O.67 NaC 20 mM O.30 14.95 84.48 0.27 0.37 12.17 86.76 O.69 NaC 30 mM O.32 15.12 84.29 0.27 O.40 12.60 86.32 O.68 NaC 40 mM O.32 14.92 84.48 0.27 O.47 12.68 86.16 O.69 NaC 50 mM O.33 1S.OO 84.36 O.30 O.45 12.56 86.34 O.65 NaC
40 D. Stability of rhuPH20 in Co-Formulations with 10% IG using a TSKG3000 SW 600x7.5 mm column and a DMSO or 20%IG in the Presence of Sodium Chloride (100-250 mM) containing buffer system (Kolarich et al. (2006) Transfusion, or Amino Acids (500 mM) 46:1959-1977). The effect on rhuPH20 stability of co-formulations con- as Tables 39 and 41 show hyaluronidase activity (U/mL) at 5 taining 10% IG or 20% IG with rHuPH20 and sodium chlo time points (0, 1, 2, 3 and 6 weeks) for co-formulations ride oramino acid stabilizers was studied. Co-formulations of containing 100 U/mL rHuPH20 and 10% or 20% IG, respec 100 U/mL or 300 U/mL rHuPH20 (lot HUB0702CA; gener tively. Tables 40 and 42 show hyaluronidase activity (U/mL) ated using Gen2 production described in Example 3) in 10% at 6 time points (0, 1, 2, 3, 6 and 12 weeks) for co-formula IG (with 0.25Mglycine at pH4.4) (lot LE12F047) or 20% IG 50 tions containing 300 U/mL rhuPH20 and 10% or 20% IG, (lot SC00108NG) were prepared as described in Example 6A respectively. The results show that high amino acid concen above. Samples contained either NaCl (concentrations of trations (500 mM glycine or 500 mM proline) were less 100, 150 or 250 mM), glycine (500 mM) or proline (500 effective then NaCl instabilizing rHuPH20 in 10% IG or 20% mM). The co-formulations were stored at 2 to 8°C. or 28 to IG co-formulations with ruPH2O. 32° C. Thus, the resulting co-formulations of rHuPH20 and 55 Sodium chloride, at all concentrations studied, enhanced IG were formulated in 0.25 M glycine at pH 4.6 to 5.1 in the IG aggregation (>450 kDa) after storage at 28 to 32°C. in all presence of varying amounts of NaCl, glycine or proline. co-formulations. All co-formulations containing 500 mM After 0 (start), 1, 2, 3, 6 and 12 (300 U/mL only) weeks of proline have a reduced IG dimer content (~350 kDa) and an storage, one sample from each of the co-formulations (with 60 increased monomer content (~160 kDa) after 6 weeks of either NaCl concentrations of 100, 150 or 250 mM, glycine storage at 28 to 32°C. IG dimer content was also reduced in concentration of 500 mM or proline concentration of 500 co-formulations with glycine, though not as pronounced as in mM) was withdrawn from the incubation and analyzed for the proline co-formulations (Tables 43 and 44). High concen hyaluronidase activity using the microturbidity assay trations of proline have proven to be effective at inhibiting described in Example 4. Aggregation of IG was determined 65 protein aggregation during refolding by effectively blocking by molecular size distribution at 0 (start) and 12 weeks by non-specific hydrophobic interactions between proteins (Ku high performance size exclusion chromatography (HP-SEC) mar et al. (1998) Biochem. Mol. Biol. Int. 4:59-517). US 9,084,743 B2 105 106 TABLE 39 TABLE 41 Hyaluronidase activity (U/mL) of 10% IG and 100 U/mL rHuPH2O Hyaluronidase activity (U/mL) of 20% IG and 100 U/mL rHuPH2O co-formulations with stabilizers after storage at 28-32 C. co-formulations with stabilizers after storage at 28-32 C. Stabilizer Weeks 5 Stabilizer Weeks
Concentration O (start) 1 2 3 6 12 Concentration O (start) 1 2 3 6 12 100 mMNaCl 97 97 88 99 85 84 100 mMNaCl 268 313 262 256 223 214 150 mMNaCl 99 91 102 93 94 85 150 mMNaCl 252 292 249 260 232 2O2 250 mMNaCl 89 105 93 88 91 89 10 250 mMNaCl 262 3O2 270 254 236 213 500 mM glycine 94 105 85 84 77 56 500 mM glycine 285 286 291 244 221 191 500 mM proline 88 96 83 8O 88 59 500 mM proline 3O8 303 242 248 230 197
TABLE 40 15 TABLE 42 Hyaluronidase activity (U/mL) of 10% IG and 300 U/mL rHuPH2O Hyaluronidase activity (U/mL) of 20% IG and 300 U/mL co-formulations with stabilizers after storage at 28-32 C. rHuPH2O co-formulations with Stabilizers after storage at 28-32 C. Stabilizer Weeks Stabilizer Weeks
Concentration O (start) 1 2 3 6 12 2O Concentration O (start) 1 2 3 6 12 100 mMNaCl 294 303 284 266 260 233 100 mMNaCl 268 266 264 226 237 255 150 mMNaCl 301 282 28O 272 288 246 150 mMNaCl 252 256 270 220 231 261 250 mMNaCl 28O 290 275 278 255 250 250 mMNaCl 262 243 273 246 243 273 500 mM glycine 254 296 246 256 229 194 500 mM glycine 285 257 289 211 230 267 500 mM proline 242 304 266 244 226 204 25 500 mM proline 3O8 257 268 231 229 259
TABLE 43
Molecular size distribution of IG in 10%. IGirHuPH2O co formulations with NaCl, glycine or proline after storage at 28-32° C.
O (start) 6 months
Sample >450 kDa -350kDa -160 kDa <60 kDa >450 kDa -350kDa -160 kDa <60 kDa
O%IG + 100 UmL O.15 O.92 88.35 O.S9 OSO 9.58 89.13 O.80 rEHPH2O - 50 mMNaC O%IG + 300 UmL O.14 1.OS 88.27 O.S4 O46 9.59 89.11 O.84 rEHPH2O - 50 mMNaC O%IG + 100 UmL O.14 1.07 88.15 O.65 O45 9.71 88.97 O.87 rEHPH2O - 50 mMNaC O%IG + 300 UmL O.14 1.42 87.82 O.62 O45 9.76 89.09 O.70 rEHPH2O - 50 mMNaC O%IG + 100 UmL O.18 1.29 87.91 O.63 O.38 9.36 89.53 O.74 rEHPH2O - OO mMNaC O%IG + 300 UmL O.13 1.43 87.89 0.55 O.38 9.32 89.52 O.78 rEHPH2O - OO mMNaC O%IG + 100 UmL O16 0.67 88.SS O.62 O.12 8.12 90.92 O.84 rEHPH2O - 500 mM glycine O%IG + 300 UmL O16 O.80 88.43 O.61 O16 8.17 90.95 0.73 rEHPH2O - 00 mM glycine O%IG + 100 UmL O.14 9.55 89.75 O.S6 O.11 5.53 93.58 O.78 rEHPH2O - 500 mM proline O%IG + 300 UmL O.14 9.43 89.86 0.57 O.12 5.65 93.52 O.71 rEHPH2O - 00 mM proline US 9,084,743 B2 107 108 TABLE 44
Molecular size distribution of IG in 20% IGirHuPH2O co formulations with NaCl, glycine or proline after storage at 28-32 C. O (start 6 months
>450 kDa -350 kDa -160 kDa <60 kDa >450 kDa -350 kDa -160 kDa <60 kDa
OmL O.25 S.O.3 84.28 0.44 2.55 86.37 O60
OmL O.26 S.12 84.16 O46 2.53 86.36 O.S9
OmL O.26 5.32 83.97 O.45 O45 2.74 86.12 O.69
OmL O.25 S.21 84.08 O46 O.47 2.78 86.13 O.61
OmL O.24 S.40 83.87 O.SO 2.69 86.24 O.65
OmL O.25 5.53 83.81 O.42 O48 2.72 86.17 O.63
O.21 4.40 84.99 O.39 O.22 2.31 86.90 O.S6
O.21 4.38 85.00 O41 O.22 2.47 86.73 O.S8
O.25 547 83.83 O.45 O.24 O.18 88.92 O.66 20 + mM proline O%. IG - 300 O.25 5.72 83.54 O.49 O.24 HPH2O - 00 mM proline
Example 7 35 Eighteen male Yucatan Mini Pigs weighing 18.4-23.2 kg (SNS Farms) were assigned to one or two of eleven treatment groups as shown in Table 45 so that each group utilized three Effects of Co-Formulated ruPH2O and 10% IG or pigs. All formulations were administered Subcutaneously 20% IG in Yucatan Mini Pigs with 10-gauge 90 degree soft bend Huber needles on the backs of anesthetized male pigs. For Leading Edge dosing, A. Experimental Design 40 rHuPH20 followed by IgG was injected consecutively using The feasibility of dosing rhuPH20 co-formulated with the same needle in the exact location, employing a simple 10% or 20% immune globulin (IG) solution (130 mM. NaCl, syringe switch. No delay between dosing rhuPH20 and IgG 10 mM histidine, pH 6.6) subcutaneously in Yucatan Mini was required or employed. Up to two different formulations, Pigs was determined and compared to Leading Edge dosing each from a different treatment group, were tested on each pig (successive dosing of rHuPH20 followed by IG solution). A 45 at a maximum Volume of 110 mL per injection site. Infusions dose response utilizing several concentrations of rhuPH20 lasted approximately 20 minutes for co-formulations and was also evaluated for each IG solution. 22-28 minutes for Leading Edge formulations. TABLE 45 Summary of experimental design
Total Dose Volume Group Treatment Dose Type (mL)
1 100 mL 10%. IG Co-formulation 1OO 2 100 mL 10% IG/rHuPH2O (50 U/mL) Co-formulation 1OO 3 100 mL 10% IG/rHuPH2O (100 U/mL) Co-formulation 1OO 4 100 mL 10% IG/rHuPH2O (300 U/mL) Co-formulation 1OO 5 SOL 20%. IG Co-formulation 50 6 50 mL 20% IG/rHuPH2O (50 U/mL) Co-formulation 50 7 50 mL 20% IG/rHuPH2O (100 U/mL) Co-formulation 50 8 50 mL 20% IG/rHuPH2O (300 U/mL) Co-formulation 50 9 10 mL rHuPH2O (150 U/mL) + 100 mL 10% IG Leading Edge 110 10 20 mL rHuPH2O (150 U/mL) + 50 mL 20% IG Leading Edge 60 11 20 mL rHuPH2O (150 U/mL) + 50 mL 20% IG Leading Edge 70 US 9,084,743 B2 109 110 Injection site observations were assessed following dosing. TABLE 47 Transducers were utilized to measure the continuous pressure (mmHg) exerted to administer each formulation, and blood Mean pressure measurement analysis was collected for Complete Blood Count (CBC) and gamma immunoglobulin (IgG) analysis. At study termination, 3 days Mean Rising Max Pressure Rising Time post-dosing, all animals were euthanized and two sample Pressure Pressure Max Max Time Max sections (A and B) were collected from each of Injection Site Group N (mmHg) (mmHg) (mmHg) (min) (min) 1. Injection Site 2, and Control (collected from a site distant from the two injection sites) and preserved in 10% neutral 1 2 242 266 281 2.7 S.1 buffered formalin, and evaluated by light microscopy (Nova 10 2 2 209 NA* 223 NA* 4.0 Pathology, PC, San Diego, Calif.). Site A was a 2-3 mm thick 3 3 164 O.3 223 O.3 4.1 section through the center of the injection site and Site B was 4 3 289 O.S 255 O.S 2.3 a 2-3 mm thick section taken from the end of the harvested 5 O NA NA NA NA NA injection site. 6 1 164 250 250 1.6 1.6 15 7 2 179 215 215 0.7 0.7 8 2 194 188 2O3 1.6 4.6 9 3 117 119 125 1.9 4.9 B. Injection Site Observations 10 3 241 232 261 3.8 12.9 Within 5 minutes of dosing 10% IG alone (-25 mL into 11 3 241 281 264 4.7 15.2 infusion; Group 1), a distinct bleb was visible on all three pigs. Approximately 10 minutes into dosing 20%. IG alone NA = Not Available, >460 mmHg (-25 mL into infusion; Group 5), a distinct bleb was visible. NA* = Rising curve of pressure recording unclear to interpret Observed bleb formation area increased with all formulations containing rHuPH20 (including Leading Edge) compared to D. Complete Blood Count and IgG Plasma Analysis IG dosing alone, signifying greater dispersion of fluids when utilizing rHuPH20 (Table 46). 25 Blood was collected into KEDTA tubes at pre-dose (-2.0 Co-formulations of ruPH20 with 10% and 20%. IG mL) and at 30 minutes post-dosing (-2.0 mL) for Complete resulted in significantly reduced hardening of skin at all Blood Count (CBC) analysis. Samples were stored at 4°C. rHuPH20 concentrations (sites remained soft), and reduced until analysis (Bioquant, Inc., San Diego, Calif.). CBC results pink/redness of the skin in all rhuPH20 concentrations. Lead do not give any product related specific safety concerns. The ing Edge comparison dosing resulted in similar pink/redness 30 majority of pigs remained within normal CBC levels (normal observations as co-formulations. Occurrences of pink/red CBC range referenced from SNS farms). Five of eighteen ness at injection sites observed post-dosing showed full pigs had non-visible clots in the samples and could not be recovery within 24 hours for all groups (Table 46). evaluated. TABLE 46 Iniection site appearance and analysis Mean Bleb Mean Bleb Group Treatment Area (cm) Observation 1 100 mL 10%. IG 97.5 Slightly pink; Hard 2 100 mL 10% IG/rHuPH2O (50 U/mL) 91.7 Slightly pink Soft 3 100 mL 10% IG/rHuPH2O (100 U/mL) 1803 Slightly pink/pink; Soft 4 100 mL 10% IG/rHuPH2O (300 U/mL) 178.0 Slightly pink/pink; Soft 5 SOL 20%. IG 95.2 Pink/red: Hard 6 50 mL 20% IG/rHuPH2O (50 U/mL) 102.6 Pink/red; So 7 50 mL 20% IG/rHuPH2O (100 U/mL) 111.9 Slightly pink/pink; Soft 8 50 mL 20% IG/rHuPH2O (300 U/mL) 111.1 Normal; So 9 10 mL rHuPH2O (150 U/mL) + 100 mL 10% IG 173.5 Normal; So 10 20 mL rHuPH2O (150 U/mL) + 50 mL. 116.8 Normal/Slightly 20%. IG Pink; Soft 11 20 mL rHuPH2O (150 U/mL) + 50 mL. 131.4 Normal/Slightly 20%. IG Pink; Soft
C. Pressure Measurement Observations Blood for gamma immunoglobulin (IgG) analysis was col lected into Sodium Citrate tubes at pre-dose (-2.0 mL) and at Table 47 Summarizes the mean pressure measurements. At study termination (-4.0 mL) to confirm systemic availability 2.5 minutes or sooner post-dosing 20% IG alone (Group 5), 60 after Subcutaneous administration of human IgG. Samples pressures were out of measurable range (>460 mmHg) for all were centrifuged at 4°C. for 10 minutes at 3000 rpm, plasma three pigs. Two of three pigs were out of the measurable was aliquotted, and samples were stored at -20° C. until pressure range in Group 6, and one pig was out of range for analysis. A general increase in IgG was observed in all ani each of Groups 7 and 8. Groups 1 and 2 each had one pig out mals 3 days after administration, as shown in Table 48. IgG of range. The results show that the pressure needed to accom 65 plasma levels for each pig reflect the mean of the two different plish the injections decreased with all co-formulations con treatments each pig was administered (with the exception of taining rHuPH20. pigs 7-9 that received a single treatment only). US 9,084,743 B2 111 112 TABLE 48 TABLE 50 IgG analysis Summary of histologic findings: 20% IG + rHuPH2O IgG (gfL
Pig # Treatment Group(s) Predose Termination Treatment 1 1 and 2 3.46 8.53 Group 2 1 and 2 2.97 9.27 3 1 and 2 4.35 9.03 Histologic Findings 5 6 7 8 4 3 and 4 6.67 1O.S1 10 5 3 and 4 3.81 10.15 6 3 and 4 4.79 9.83 7 5 4.96 6.06 Inflammation, Mixed Leukocyte, 66* 66 5, 6 66 8 5 3.SO 5.94 Subcutaneous 9 5 3.73 6.86 Mean Group Severity Score** 1.00 1.17 1.00 2.17 10 6 and 7 2.83 8.19 15 11 6 and 7 3.47 10.08 Edema, Subcutaneous 66 66 5, 6 5.6 12 6 and 7 4.08 1112 13 8 and 9 5.07 9.62 Mean Group Severity Score 1.17 1.17 1.17 2.OO 14 8 and 9 4.O2 8.82 Hemorrhage, Subcutaneous Of6 2.6 Of 6 1.6 15 8 and 9 3.94 8.63 16 10 and 11 3.97 9.25 Mean Group Severity Score O.OO O.33 O.OO O.17 17 10 and 11 4.6O 9.68 18 10 and 11 4.76 9.51 Sum of Mean Group Severity Scores 2.17 2.67 2.17 4.34 E. Histopathology Results Histologic findings were present in the epidermis, dermis Number of Sections Affected Number of Sections Evaluated 25 **Sum of severity scores in the group divided by the number of sections evaluated in the and Subcutaneous tissue, and contained a mixed leukocyte group inflammation, edema and hemorrhage. Each histologic find ing was assigned a severity grade based on the following scheme: Not Present: 0; Present, Not Graded: 0; Minimal: 1; TABLE 51 Mild: 2: Moderate: 3; Marked: 4 Histologic findings are sum Summary of histologic findings: Leading Edge dosing marized by incidence and mean group severity score in Tables 30 49-51. Treatment Group TABLE 49 Histologic Findings 9 10 11 Summary of histologic findings: 10% IG + rhuPH20 35 Inflammation, Mixed Leukocyte, Subcutaneous 66* 66 66 Mean Group Severity Score** 1.17 1.17 1.17 Treatment Edema, Subcutaneous 5.6 66 66 Group Mean Group Severity Score 1...SO 1.67 1.83 Hemorrhage, Subcutaneous 16 3f6 1.6 Histologic Findings 1 2 3 4 Mean Group Severity Score O.17 0.67 O.17 40 Inflammation, Mixed Leukocyte, 66* 66 5.6 66 Sum of Mean Group Severity Scores 2.84 3.51 3.17 Subcutaneous Mean Group Severity Score** 1.83 1.00 1.OO 1.17 Number of Sections Affected Number of Sections Evaluated Edema, Subcutaneous 66 5.6 66 5.6 **Sum of severity scores in the group divided by the number of sections evaluated in the Mean Group Severity Score 2.00 O.83 1.OO 1.17 group Hemorrhage, Subcutaneous 3f6 3f6 2.6 1.6 45 Mean Group Severity Score 0.67 O.SO O.33 O.33 The response to the administration of IG and rhuPH20 was qualitatively similar in each dose group in this study. These Sum of Mean Group Severity Scores 4SO 2.33 2.33 2.67 responses were characterized by mixed leukocyte inflamma Number of Sections Affected Number of Sections Evaluated tion, edema and hemorrhage in the Subcutaneous tissue in the **Sum of severity scores in the group divided by the number of sections evaluated in the group injection sites. Table 52 compares the mean group severity score in all of the dose groups. TABLE 52 Summary of mean group severity scores
Sum of Mean Group Severity Group Treatment Dose Type Scores
100 mL 10%. IG Co-formulation 4...SO 100 mL 10% IG/rHuPH2O (50 U/mL) Co-formulation 2.33 100 mL 10% IG/rHuPH2O (100 U/mL) Co-formulation 2.33 100 mL 10% IG/rHuPH2O (300 U/mL) Co-formulation 2.67 SOL 20%. IG Co-formulation 2.17 50 mL 20% IG/rHuPH2O (50 U/mL) Co-formulation 2.67 50 mL 20% IG/rHuPH2O (100 U/mL) Co-formulation 2.17 50 mL 20% IG/rHuPH2O (300 U/mL) Co-formulation 4.34 US 9,084,743 B2 113 114 TABLE 52-continued Summary of mean group Severity Scores Sum of Mean Group Severity Group Treatment Dose Type Scores 9 10 mL rHuPH2O (150 U/mL) + 100 mL 10% IG Leading Edge 2.84 10 20 mL rHuPH2O (150 U/mL) + 50 mL 20% IG Leading Edge 3.51 11 20 mL rHuPH2O (150 U/mL) + 50 mL 20% IG Leading Edge 3.17
Based on mean group severity Scores, the most severe Yucatan Mini Pigs. IG (10% or 20%) administered alone is injection site responses were associated with administration feasible, although a moderate to severe degree of hardening of 100 mL of 1 9% IG alone (Group1) and with administration 15 and pink/redness of the skin resulted. Co-formulations with R O As f %. IG East with SeeSh Elsie rHuPH20 resulted in a decrease in pressure needed to accom (Group 8). e response to administration o O O plish the injections, significantly reduced hardening of the IG co-formulated with rHuPH20 at 50, 100 and 300 U/mL of skin, and reduced pink/redness of the skin. Observed bleb 10%. IG (Groups 2-4) was similar to the response to admin- c. pink . istration of 50 mL of 20% IG alone (Group 5), co-formulated 20 formation area was similar or increased with all formulations with ruPH20 at 50 and 100U/mL of 20%, IG (Groups 6 and that contained ruPH20 compared to IG dosing alone, con 7), and Leading Edge dosing with 10 mL of rhuPH20 (150 firming greater dispersion of fluids when rHuPH20 was uti U/mL) followed by 100 mL of 10% IG (Group 9). However, lized. Leading Edge dosing was feasible, and similar pres Leading Edge dosing with 10 or 20 mL of rHuPH20 (150 sure, pink/redness and bleb areas are observed as with U/mL) followed by 50 mL of 20% IG (Groups 10 and 11) 2s co-formulations. Histopathological findings present in the resulted in a more severe response than did similar co-formu- deep Subcutaneous tissue attributed to dosing included mixed lations (Groups 6 and 7). Sections of control skin contained leukocyte inflammation, edema and hemorrhage, with the few histological findings, which can be attributed to diffusion most severe responses associated with administration of 10% of the injected formulations from the test article injection IG alone and 20% IG co-formulated with rhuPH20 (300 sites, and incidental findings unrelated to the formulations. 30 U/mL). F. SUMMARY Since modifications will be apparent to those of skill in this The results confirmed the feasibility of dosing rhuPH20 art, it is intended that this invention be limited only by the co-formulated with 10% and 20%. IG subcutaneously in Scope of the appended claims.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 56
<21 Os SEQ ID NO 1 &211s LENGTH: 509 212s. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: precursor human PH2O <4 OOs SEQUENCE: 1 Met Gly Val Lieu Lys Phe Llys His Ile Phe Phe Arg Ser Phe Val Lys 1. 5 1O 15 Ser Ser Gly Val Ser Glin Ile Val Phe Thr Phe Leu Lieu. Ile Pro Cys 2O 25 3 O Cys Lieu. Thir Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro 35 4 O 45 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lieu. Gly Llys Phe SO 55 60 Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg
Ile Asn Ala Thr Gly Glin Gly Val Thr Ile Phe Tyr Val Asp Arg Lieu. 85 90 95 Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 1OO 105 110 Gly Ile Pro Gln Lys Ile Ser Lieu. Glin Asp His Lieu. Asp Lys Ala Lys 115 12O 125
Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Lieu. Gly Met Ala Val US 9,084,743 B2 115 116 - Continued
13 O 135 14 O
Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160
Asp Wall Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s
Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Lys Ala Glin Glu Phe 18O 185 19 O
Glu Ala Gly Lys Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195
Lell Luell Arg Pro Asn His Lell Trp Gly Lell Phe Pro Asp 21 O 215
Tyr Asn His His Tyr Lys Pro Gly ASn Gly Ser Phe Asn 225 23 O 235 24 O
Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255
Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O
Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 285
Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO
Arg Ile Wall Phe Thir Asp Glin Wall Luell Phe Lell Ser Glin Asp Glu 3. OS 310 315
Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 325 330 335
Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Ser Luell 34 O 345 35. O
Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365
Wall Thir Luell Ala Ala Met Ser Glin Wall Lell Glin Glu Glin 37 O 375
Gly Wall Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO
Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Gly Gly Phe Thir 4 OS 415
Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu 425 43 O
Phe Cys Ser Cys Ser Thir Luell Ser Glu Ala 435 44 O 445
Wall Lys Asp Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall 450 45.5 460
Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile 465 470 47s 48O
Phe Asn Ala Ser Pro Ser Thir Luell Ser Ala Thir Met Phe Ile Wall 485 490 495
Ser Ile Luell Phe Lell Ile Ile Ser Ser Wall Ala Ser Lell SOO 505
SEQ ID NO 2 LENGTH: 474 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: Mature PH2O US 9,084,743 B2 117 118 - Continued
<4 OOs, SEQUENCE: 2 Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1. 5 1O 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Lieu. Gly Llys Phe Asp Glu Pro 2O 25 3O
Lieu. Asp Met Ser Lieu. Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45 Thr Gly Glin Gly Val Thir Ile Phe Tyr Val Asp Arg Lieu. Gly Tyr SO 55 6 O
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro 65 70 7s
Glin Lys Ile Ser Lieu. Glin Asp His Lieu. Asp Lys Ala Lys Lys Asp Ile 85 90 95 Thr Phe Tyr Met Pro Val Asp Asn Lieu. Gly Met Ala Val Ile Asp Trp 1OO 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Llys Pro Lys Asp Wall 115 12 O 125
Tyr Lys Asn Arg Ser Ile Glu Lieu Val Glin Glin Glin Asn Val Glin Luell 13 O 135 14 O
Ser Lieu. Thr Glu Ala Thr Glu Lys Ala Lys Glin Glu Phe Glu Lys Ala 145 150 155 160 Gly Lys Asp Phe Lieu Val Glu Thir Ile Llys Lieu. Gly Llys Lieu. Lieu. Arg 1.65 17O 17s
Pro Asn His Leu Trp Gly Tyr Tyr Lieu Phe Pro Asp Cys Tyr Asn His 18O 185 19 O
His Tyr Lys Llys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile 195 2OO 2O5
Lys Arg Asn Asp Asp Lieu. Ser Trp Lieu. Trp Asn. Glu Ser Thir Ala Luell 21 O 215 22O
Tyr Pro Ser Ile Tyr Lieu. Asn Thr Glin Glin Ser Pro Val Ala Ala Thir 225 23 O 235 24 O
Lieu. Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Wall 26 O 265 27 O Phe Thr Asp Glin Val Lieu Lys Phe Lieu. Ser Glin Asp Glu Lieu Val 27s 28O 285 Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp 29 O 295 3 OO Gly. Thir Lieu. Ser Ile Met Arg Ser Met Lys Ser Cys Lieu. Lieu. Lieu. Asp 3. OS 310 315 32O
Asn Tyr Met Glu Thir Ile Lieu. Asn Pro Tyr Ile Ile Asn Val Thr Luell 3.25 330 335
Ala Ala Lys Met Cys Ser Glin Val Lieu. Cys Glin Glu Glin Gly Val 34 O 345 35. O Ile Arg Lys Asn Trp Asn. Ser Ser Asp Tyr Lieu. His Lieu. Asn. Pro Asp 355 360 365
Asn Phe Ala Ile Glin Lieu. Glu Lys Gly Gly Llys Phe Thr Val Arg Gly 37 O 375 38O
Llys Pro Thr Lieu. Glu Asp Lieu. Glu Glin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 4 OO
Ser Cys Tyr Ser Thr Lieu. Ser Cys Lys Glu Lys Ala Asp Wall Lys Asp 4 OS 41O 415 US 9,084,743 B2 119 120 - Continued
Thir Asp Ala Val Asp Wall Ile Ala Asp Gly Val Cys Ile Asp Ala 42O 425 43 O
Phe Luell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile Phe Asn 435 44 O 445
Ala Ser Pro Ser Thir Lell Ser Ala Thir Met Phe Ile Wall Ser Ile Luell 450 45.5 460
Phe Luell Ile Ile Ser Ser Wall Ala Ser Luell 465 470
SEQ ID NO 3 LENGTH: 482 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: precursor soluble rHuPH2O <4 OOs, SEQUENCE: 3
Met Gly Val Lieu Lys Phe His Ile Phe Phe Arg Ser Phe Wall 1. 5 15
Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lell Lell Ile Pro 2O 25
Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45
Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 6 O
Asp Glu Pro Lieu. Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 75
Ile Asn Ala Thr Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Luell 85 90 95
Gly Tyr Pro Tyr Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly 1OO 105 11 O
Gly Ile Pro Gln Lys Ile Ser Luell Glin Asp His Lell Asp Ala 115 12 O 125
Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O
Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160
Asp Wall Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s
Wall Glin Luell Ser Luell Thir Glu Ala Thir Glu Lys Ala Glin Glu Phe 18O 185 19 O
Glu Ala Gly Lys Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 2O5
Lell Luell Arg Pro Asn His Lell Trp Gly Tyr Lell Phe Pro Asp 21 O 215 22O
Tyr Asn His His Tyr Lys Pro Gly Tyr ASn Gly Ser Phe Asn 225 23 O 235 24 O
Wall Glu Ile Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255
Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O
Ala Ala Thir Leu Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 28O 285
Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO US 9,084,743 B2 121 122 - Continued
Arg Ile Val Phe Thr Asp Glin Val Lieu Lys Phe Lieu. Ser Glin Asp Glu 3. OS 310 315
Lieu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile 3.25 330 335
Val Ile Trp Gly Thr Lieu. Ser Ile Met Arg Ser Met Ser Cys Luell 34 O 345 35. O
Lieu. Lieu. Asp Asn Tyr Met Glu Thir Ile Lieu. Asn. Pro Tyr Ile Ile Asn 355 360 365
Val Thir Lieu Ala Ala Lys Met Cys Ser Glin Val Lieu. Glin Glu Glin 37 O 375 38O
Gly Val Cys Ile Arg Lys Asn Trp Asn. Ser Ser Asp Luell His Luell 385 390 395 4 OO
Asn Pro Asp Asn. Phe Ala Ile Glin Lieu. Glu Lys Gly Gly Phe Thir 4 OS 41O 415
Val Arg Gly Llys Pro Thr Lieu. Glu Asp Lieu. Glu Glin Phe Ser Glu 42O 425 43 O
Phe Tyr Cys Ser Cys Tyr Ser Thr Lieu Ser Cys Lys Glu Ala 435 44 O 445
Val Lys Asp Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Wall 450 45.5 460
Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Glin Ile 465 470 47s 48O Phe Tyr
<210s, SEQ ID NO 4 &211s LENGTH: 447 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223 is OTHER INFORMATION: soluble rulPH2O 1 - 447
<4 OOs, SEQUENCE: 4
Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Luell Trp 1. 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Lieu. Gly Lys Phe Asp Glu Pro 2O 25
Lieu. Asp Met Ser Lieu. Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thr Gly Glin Gly Val Thir Ile Phe Tyr Val Asp Arg Lell Gly SO 55 6 O
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro 65 8O
Glin Lys Ile Ser Lieu. Glin Asp His Lieu. Asp Lys Ala Asp Ile 85 90 95
Thr Phe Tyr Met Pro Val Asp Asn Lieu. Gly Met Ala Wall Ile Asp Trp 1OO 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Llys Pro Asp Wall 115 12 O 125
Tyr Lys Asn Arg Ser Ile Glu Lieu Val Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O
Ser Lieu. Thr Glu Ala Thr Glu Lys Ala Lys Glin Glu Phe Glu Ala 145 150 155 160
Gly Lys Asp Phe Lieu Val Glu Thir Ile Llys Lieu. Gly Luell Luell Arg 1.65 17O 17s US 9,084,743 B2 123 124 - Continued
Pro Asn His Luell Trp Gly Tyr Luell Phe Pro Asp Cys Tyr Asn His 18O 185 19 O
His Lys Pro Gly Tyr Asn Gly Ser Phe Asn Wall Glu Ile 195
Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285
Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thir Luell Ser Ile Met Arg Ser Met Lys Ser Lell Luell Luell Asp 3. OS 310 315 32O
Asn Tyr Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall 34 O 345 35. O
Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Luell His Lell Asn Pro Asp 355 360 365
Asn Phe Ala Ile Gln Lieu Glu Gly Gly Lys Phe Thr Wall Arg Gly 37 O 375
Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO
Ser Ser Thir Lell Ser Glu Lys Ala Wall Lys Asp 4 OS 415
Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O
Phe Luell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile Phe 435 44 O 445
SEO ID NO 5 LENGTH: 446 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: soluble ruPH2O 1 - 4.46
<4 OOs, SEQUENCE: 5
Lell Asin Phe Arg Ala Pro Pro Wall Ile Pro ASn Wall Pro Phe Luell Trp 1. 5 1O 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Phe Asp Glu Pro 25 3O
Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Lell Gly Tyr SO 55 6 O
Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70 8O
Glin Ile Ser Lell Glin Asp His Luell Asp Lys Ala Asp Ile 85 90 95 US 9,084,743 B2 125 126 - Continued
Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Lys Pro Lys Asp Wall 115 12 O 125
Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O
Ser Luell Thir Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160
Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17s
Pro Asn His Luell Trp Gly Tyr Luell Phe Pro Asp Tyr Asn His 18O 185 19 O
His Lys Pro Gly Tyr Asn Gly Ser Phe Asn Wall Glu Ile 195
Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285
Thr Phe Gly Glu Thr Wall Ala Lieu Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thir Luell Ser Ile Met Arg Ser Met Lys Ser Lell Luell Luell Asp 3. OS 310 315 32O
Asn Tyr Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall 34 O 345 35. O
Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Luell His Lell Asn Pro Asp 355 360 365
Asn Phe Ala Ile Glin Lell Glu Gly Gly Lys Phe Thir Wall Arg Gly 37 O 375
Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO
Ser Ser Thir Lell Ser Glu Lys Ala Asp Wall Lys Asp 4 OS 415
Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Cys Ile Asp Ala 425 43 O
Phe Luell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile Phe 435 44 O 445
SEQ ID NO 6 LENGTH: 445 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: soluble ruPH2O 1 - 445
<4 OOs, SEQUENCE: 6 Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1. 5 15 US 9,084,743 B2 127 128 - Continued
Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Lys Phe Asp Glu Pro 25 3O
Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Lell Gly SO 55 6 O
Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70
Glin Ile Ser Lell Glin Asp His Luell Asp Ala Asp Ile 85 90 95
Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro Asp Wall 115 12 O 125
Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O
Ser Luell Thir Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160
Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17s
Pro Asn His Luell Trp Gly Luell Phe Pro Asp Tyr Asn His 18O 185 19 O
His Lys Pro Gly Asn Gly Ser Phe Asn Wall Glu Ile 195
Arg Asn Asp Asp Lieu Ser Trp Lieu Trp Asn Glu Ser Thr Ala Lieu 21 O 215
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285
Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thir Luell Ser Ile Met Arg Ser Met Ser Lell Luell Luell Asp 3. OS 310 315 32O
Asn Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall 34 O 345 35. O
Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Lell Asn Pro Asp 355 360 365
Asn Phe Ala Ile Glin Lell Glu Gly Gly Phe Thir Wall Arg Gly 37 O 375
Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO
Ser Ser Thir Lell Ser Glu Ala Wall Lys Asp 4 OS 415
Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O
Phe Luell Pro Pro Met Glu Thir Glu Glu Pro Glin Ile US 9,084,743 B2 129 130 - Continued
435 44 O 445
SEO ID NO 7 LENGTH: 444 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: soluble ruPH2O 1 - 444
<4 OOs, SEQUENCE: 7
Lell Asin Phe Arg Ala Pro Pro Wall Ile Pro ASn Wall Pro Phe Luell Trp 1. 5 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Phe Asp Glu Pro 25
Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Lell Gly SO 55 6 O
Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70
Glin Ile Ser Lell Glin Asp His Luell Asp Ala Asp Ile 85 90 95
Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro Asp Wall 115 12 O 125
Lys Asn Arg Ser Ile Glu Lieu Wall Gln Gln Asn Wall Gln Lieu 13 O 135 14 O
Ser Luell Thir Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160
Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17O 17s
Pro Asn His Luell Trp Gly Luell Phe Pro Asp Tyr Asn His 18O 185 19 O
His Lys Pro Gly Asn Gly Ser Phe Asn Wall Glu Ile 195
Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall Tyr 28O 285
Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thir Luell Ser Ile Met Arg Ser Met Ser Lell Luell Luell Asp 3. OS 310 315
Asn Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall Cys 34 O 345 35. O
Ile Arg Asn Trp Asn Ser Ser Asp Luell His Lell Asn Pro Asp US 9,084,743 B2 131 132 - Continued
355 360 365
Asn Phe Ala Ile Glin Lell Glu Gly Gly Lys Phe Thir Wall Arg Gly 37 O 375
Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Lys Phe Cys 385 390 395 4 OO
Ser Ser Thir Lell Ser Glu Lys Ala Asp Wall Lys Asp 4 OS 415
Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O
Phe Luell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin 435 44 O
SEQ ID NO 8 LENGTH: 443 TYPE : PRT ORGANISM: Homo sapiens FEATURE: OTHER INFORMATION: soluble ruPH2O 1 - 443
<4 OOs, SEQUENCE: 8
Lieu. Asn. Phe Arg Ala Pro Pro Wall Ile Pro ASn Wall Pro Phe Luell Trp 1. 5 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Phe Asp Glu Pro 25 3O
Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thr Gly Gln Gly Wall Thr Ile Phe Wall Asp Arg Lieu Gly SO 55 6 O
Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70
Glin Ile Ser Lell Glin Asp His Luell Asp Ala Asp Ile 85 90 95
Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro Asp Wall 115 12 O 125
Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O
Ser Luell Thir Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160
Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17O 17s
Pro Asn His Luell Trp Gly Luell Phe Pro Asp Tyr Asn His 18O 185 19 O
His Lys Pro Gly Asn Gly Ser Phe Asn Wall Glu Ile 195
Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215 22O
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall Tyr US 9,084,743 B2 133 134 - Continued
27s 28O 285
Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thr Lieu Ser Ile Met Arg Ser Met Lys Ser Cys Lell Luell Luell Asp 3. OS 310 315 32O
Asn Tyr Met Glu Thir Ile Lieu. Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Lys Met Cys Ser Glin Val Lieu. Cys Glin Glu Glin Gly Wall 34 O 345 35. O
Ile Arg Lys Asn Trp Asn. Ser Ser Asp Tyr Lieu. His Lell Asn Pro Asp 355 360 365
Asn Phe Ala Ile Glin Lieu. Glu Lys Gly Gly Llys Phe Thir Wall Arg Gly 37 O 375 38O
Llys Pro Thir Lieu. Glu Asp Lieu. Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO
Ser Cys Tyr Ser Thr Lieu. Ser Cys Lys Glu Lys Ala Wall Lys Asp 4 OS 41O 415
Thir Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Ile Asp Ala 42O 425 43 O Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro 435 44 O
SEO ID NO 9 LENGTH: 442 TYPE PRT ORGANISM; Homo sapiens FEATURE: OTHER INFORMATION: soluble ruPH2O. 1-442
SEQUENCE: 9
Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Luell Trp 1. 15
Ala Trp Asn Ala Pro Ser Glu Phe Cys Lieu. Gly Lys Phe Asp Glu Pro 2O 25
Lieu. Asp Met Ser Lieu. Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45
Thr Gly Glin Gly Val Thir Ile Phe Tyr Val Asp Arg Lell Gly SO 55 6 O
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro 65
Glin Lys Ile Ser Lieu. Glin Asp His Lieu. Asp Lys Ala Asp Ile 85 90 95
Thr Phe Tyr Met Pro Val Asp Asn Lieu. Gly Met Ala Wall Ile Asp Trp 1OO 105 11 O
Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Llys Pro Asp Wall 115 12 O 125
Tyr Lys Asn Arg Ser Ile Glu Lieu Val Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O
Ser Lieu. Thr Glu Ala Thr Glu Lys Ala Lys Glin Glu Phe Glu Ala 145 150 155 160
Gly Lys Asp Phe Lieu Val Glu Thir Ile Llys Lieu. Gly Luell Luell Arg 1.65 17O 17s
Pro Asn His Leu Trp Gly Tyr Tyr Lieu Phe Pro Asp Tyr Asn His 18O 185 19 O
His Tyr Lys Llys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Wall Glu Ile US 9,084,743 B2 135 136 - Continued
195 2O5
Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215 22O
Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O
Lell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O
Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285
Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO
Gly Thir Luell Ser Ile Met Arg Ser Met Lys Ser Lell Luell Luell Asp 3. OS 310 315 32O
Asn Tyr Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall 34 O 345 35. O
Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Luell His Lell Asn Pro Asp 355 360 365
Asn Phe Ala Ile Glin Lell Glu Gly Gly Lys Phe Thir Wall Arg Gly 37 O 375
Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO
Ser Ser Thir Lell Ser Glu Lys Ala Wall Lys Asp 4 OS 415
Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O
Phe Luell Lys Pro Pro Met Glu Thir Glu Glu 435 44 O
SEQ ID NO 10 LENGTH: 450 TYPE : PRT ORGANISM: Bos talurus FEATURE: OTHER INFORMATION: hyaluronidase
<4 OOs, SEQUENCE: 10
Met Arg Pro Phe Ser Lell Glu Wall Ser Luell His Lell Pro Trp Ala Met 1. 5 1O 15
Ala Ala His Luell Lell Pro Wall Thir Luell Phe Lell Asn Luell Luell Ser 25 3O
Met Thir Glin Gly Ser Arg Asp Pro Wall Wall Pro Asn Glin Pro Phe Thir 35 4 O 45
Thir Ile Trp Asn Ala Asn Thir Glu Trp Met Lys His Gly Wall SO 55 6 O
Asp Wall Asp Ile Ser Ile Phe Asp Wall Wall Thir Asn Pro Gly Glin Thir 65 70 7s
Phe Arg Gly Pro Asn Met Thir Ile Phe Tyr Ser Ser Glin Luell Gly Thir 85 90 95
Pro Tyr Thir Ser Ala Gly Glu Pro Wall Phe Gly Gly Luell Pro 1OO 105 11 O
Glin Asn Ala Ser Lell Asn Ala His Luell Ala Arg Thir Phe Glin Asp Ile US 9,084,743 B2 137 138 - Continued
115 12 O 125
Lell Ala Ala Met Pro Glu Pro Arg Phe Ser Gly Lell Ala Wall Ile Asp 13 O 135 14 O
Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe ASn Trp Asp Thir Asp 145 150 155 160
Ile Arg Glin Arg Ser Arg Ala Luell Wall Glin Glin His Pro Asp 1.65 17O 17s
Trp Luell Ala Pro Arg Wall Glu Ala Ala Ala Glin Asp Glin Phe Glu Gly 18O 185 19 O
Ala Ala Glu Glu Trp Met Ala Gly Thir Luell Lell Gly Glin Ala Luell 195
Arg Pro Glin Gly Lell Trp Gly Phe Asn Phe Pro Glu Asn 21 O 215 22O
Tyr Asp Phe Ser Pro Asn Thir Gly Arg Pro Luell Asn Ile 225 23 O 235 24 O
Ala Glin Asn Asp Glin Lell Gly Trp Luell Trp Gly Glin Ser Arg Ala 245 250 255
Lell Pro Ser Ile Lell Pro Ala Ala Luell Glu Gly Thir Lys 26 O 265 27 O
Thir Glin Met Phe Wall Glin His Arg Wall Ala Glu Ala Phe Arg Wall Ala 285
Ala Gly Ala Gly Asp Pro Lys Luell Pro Wall Luell Pro Met Glin Luell 29 O 295 3 OO
Phe Asp Met Thir Asn His Phe Luell Pro Ala Glu Glu Luell Glu His 305 310 315
Ser Luell Gly Glu Ser Ala Ala Glin Gly Ala Ala Gly Wall Wall Luell Trp 3.25 330 335
Wall Ser Trp Luell Ser Thir Ser Thir Lys Glu Ser Glin Ala Ile Lys 34 O 345 35. O
Glu Wall Asp Thir Thir Lell Gly Pro Ser Ile Lell Asn Wall Thir Ser 355 360 365
Gly Ala Arg Luell Cys Ser Glin Wall Luell Cys Ser Gly His Gly Arg Cys 37 O 375 38O
Ala Arg Arg Pro Ser Tyr Pro Ala Arg Luell Ile Lell Asn Ser Thir 385 390 395 4 OO
Ser Phe Ser Ile Lys Pro Thir Pro Gly Gly Gly Pro Lell Thir Luell Glin 4 OS 415
Gly Ala Luell Ser Lell Glu Asp Arg Luell Arg Met Ala Wall Glu Phe Glu 42O 425 43 O
Arg Cys Gly Trp Arg Gly Thir Arg Glu Glin Trp Gly 435 44 O 445
Met Trp 450
SEQ ID NO 11 LENGTH: 553 TYPE : PRT ORGANISM: Bos taurus FEATURE: OTHER INFORMATION: PH2O
SEQUENCE: 11 Met Arg Met Lieu. Arg Arg His His Ile Ser Phe Arg Ser Phe Ala Gly 1. 5 15
Ser Ser Gly Thr Pro Glin Ala Val Phe Thr Phe Leu Lleu Lleu Pro Cys US 9,084,743 B2 139 140 - Continued
25
Luell Ala Luell Asp Phe Arg Ala Pro Pro Luell Ile Ser Asn Thir Ser 35 4 O 45
Phe Luell Trp Ala Trp Asn Ala Pro Wall Glu Arg Cys Wall Asn Arg Arg SO 55 6 O
Phe Glin Luell Pro Pro Asp Lell Arg Luell Phe Ser Wall Gly Ser Pro 65 70
Glin Ser Ala Thir Gly Glin Phe Ile Thir Luell Phe Ala Asp Arg 85 90 95
Lell Gly Tyr Pro His Ile Asp Glu Thir Gly Thir Wall Phe 105 11 O
Gly Gly Ile Pro Glin Lell Gly Asn Luell Ser His Met Glu Ala 115 12 O 125
Asn Asp Ile Ala Tyr Ile Pro Asn Asp Ser Wall Gly Luell Ala 13 O 135 14 O
Wall Ile Asp Trp Glu Asn Trp Arg Pro Thir Trp Ala Arg Asn Trp Lys 145 150 155 160
Pro Asp Wall Tyr Arg Asp Glu Ser Wall Glu Lell Wall Luell Glin Lys 1.65 17O 17s
Asn Pro Glin Luell Ser Phe Pro Glu Ala Ser Ile Ala Lys Wall Asp 18O 185 19 O
Phe Glu Thir Ala Gly Ser Phe Met Glin Glu Thir Lell Luell Gly 195
Luell Luell Arg Pro Asn His Luell Trp Gly Tyr Lell Phe Pro Asp 210 215 220
Cys Asn His Asn His Asn Glin Pro Thir Tyr Asn Gly Asn Pro 225 23 O 235 24 O
Asp Wall Glu Arg Arg Asn Asp Asp Luell Glu Trp Lell Trp Lys Glu 245 250 255
Ser Thir Ala Luell Phe Pro Ser Wall Tyr Luell ASn Ile Arg Luell Ser 26 O 265 27 O
Thir Glin Asn Ala Ala Lell Wall Arg Asn Arg Wall Glin Glu Ala Ile 27s 285
Arg Luell Ser Ile Ala Ser Wall Glu Ser Pro Lell Pro Wall Phe Wall 29 O 295 3 OO
Tyr Ala Arg Pro Wall Phe Thir Asp Gly Ser Ser Thir Luell Ser Glin 3. OS 310 315
Gly Asp Luell Wall Asn Ser Wall Gly Glu Ile Wall Ser Lell Gly Ala Ser 3.25 330 335
Gly Ile Ile Met Trp Gly Ser Luell Asn Luell Ser Lell Ser Met Glin Ser 34 O 345 35. O
Met Asn Luell Gly Thir Luell Asn Thir Thir Lell Asn Pro Ile 355 360 365
Ile Asn Wall Thir Lell Ala Ala Met Ser Glin Wall Luell His 37 O 375
Asn Glu Gly Wall Cys Thir Arg His Trp ASn Ser Ser Asp Luell 385 390 395 4 OO
His Luell Asn Pro Met Asn Phe Ala Ile Glin Thir Gly Glu Gly Gly Lys 4 OS 41O 415
Thir Wall Pro Gly Thir Wall Thir Luell Glu Asp Lell Glin Lys Phe Ser 42O 425 43 O
Asp Thir Phe Cys Ser Tyr Ala Asn Ile His Cys 435 44 O 445 US 9,084,743 B2 141 142 - Continued
Wall Asp Ile Lys Asn Val His Ser Wall Asn. Wall Cys Met Ala Glu Asp 450 45.5 460
Ile Cys Ile Asp Ser Pro Val Llys Lieu. Glin Pro Ser Asp His Ser Ser 465 470 47s 48O
Ser Glin Glu Ala Ser Thr Thir Thir Phe Ser Ser Ile Ser Pro Ser Thir 485 490 495
Thir Thir Ala Thir Wal Ser Pro Cys Thr Pro Glu His Ser Pro Glu SOO 505
Lieu Lys Val Arg Cys Ser Glu Wall Ile Pro Asn Wall Thir Glin 52O 525
Ala Cys Glin Ser Wall Lys Lieu Lys Asn. Ile Ser Tyr Glin Ser Pro Ile 53 O 535 54 O
Glin Asn. Ile Lys Asn Glin Thr Thr Tyr 5.45 550
<210s, SEQ ID NO 12 &211s LENGTH: 331 212. TYPE: PRT <213> ORGANISM: Vespula vulgaris 22 Os. FEATURE: 223 OTHER INFORMATION: hyaluronidase A
<4 OOs, SEQUENCE: 12
Ser Glu Arg Pro Lys Arg Val Phe Asin Ile Tyr Trp Asn Wall Pro Thir 1. 5 1O 15
Phe Met Cys His Glin Tyr Asp Lieu. Tyr Phe Asp Glu Wall Thir Asn Phe 2O 25 30
Asn Ile Lys Arg Asn. Ser Lys Asp Asp Phe Glin Gly Asp Ile Ala 35 4 O 45
Ile Phe Tyr Asp Pro Gly Glu Phe Pro Ala Lieu. Lell Ser Luell Asp SO 55 6 O
Gly Asn Gly Gly Val Pro Glin Glu Gly Asn Ile 65 70 7s
Thir Ile His Lieu Gln Lys Phe Ile Glu Asn Lieu. Asp Ile Tyr Pro 85 90 95
Asn Arg Asn. Phe Ser Gly Ile Gly Val Ile Asp Phe Glu Arg Trp Arg 1OO 105 11 O
Pro Ile Phe Arg Glin Asn Trp Gly Asn Met Lys Ile His Asn Phe 115 12 O 125
Ser Ile Asp Lieu Val Arg ASn Glu. His Pro Thir Trp Asn Met 13 O 135 14 O
Ile Glu Lieu. Glu Ala Ser Lys Arg Phe Glu Lys Ala Arg Phe Phe 145 150 155 160
Met Glu Glu Thir Lieu Lys Lieu Ala Lys Llys Thr Arg Glin Ala Asp 1.65 17O 17s
Trp Gly Tyr Tyr Gly Tyr Pro Tyr Cys Phe Asn Met Ser Pro Asn Asn 18O 185 19 O
Lell Val Pro Glu. Cys Asp Wall Thir Ala Met His Glu Asn Asp Met 195 2OO
Ser Trp Lieu. Phe Asn. Asn Glin Asn. Wall Lieu. Lieu Pro Ser Wall Wall 21 O 215 22O
Arg Glin Glu Lieu. Thir Pro Asp Glin Arg Ile Gly Lell Wall Glin Gly Arg 225 23 O 235 24 O
Wall Lys Glu Ala Val Arg Ile Ser Asn. Asn Lieu His Ser Pro 245 250 255 US 9,084,743 B2 143 144 - Continued
Wall Luell Ser Tyr Trp Trp Tyr Val Tyr Glin Asp Glu Thir Asn Thir Phe 26 O 265 27 O
Lell Thir Glu Thir Asp Wall Lys Llys Thr Phe Glin Glu Ile Wall Ile Asn 27s 28O 285
Gly Gly Asp Gly Ile Ile Ile Trp Gly Ser Ser Ser Asp Wall Asn Ser 29 O 295 3 OO
Lell Ser Lys Arg Lieu. Glin Asp Tyr Lieu Lell Thir Wall Luell Gly 3. OS 310 315 32O
Pro Ile Ala Ile Asn Wall Thr Glu Ala Wall Asn 3.25 330
SEQ ID NO 13 LENGTH: 34 O TYPE : PRT ORGANISM: Vespula vulgaris FEATURE: OTHER INFORMATION: hyaluronidase B
SEQUENCE: 13
Asp Arg Thir Ile Pro Llys Lys Gly Phe Ser Ile Trp Asn Ile 1. 1O 15
Pro Thir His Phe His Asn Phe Gly Val Tyr Phe Glu Luell 25
Glin Phe Asn Ile Asn. Ser Met Asn. Asn Phe Arg Gly Glu Thir 35 4 O 45
Ile Ser Luell Phe Tyr Asp Pro Gly Asn Phe Pro Ser Met Wall Luell Luell 50 55 60
Lys Asn Gly Thir Tyr Glu Ile Arg Asn. Glu Gly Wall Pro Glin Gly 65 70 7s
Asn Luell Thir Ile His Lell Glu Glin Phe Thr Lys Glu Lell Asp Glu Ile 85 90 95
Pro Lys Ile Ala Gly Gly Ile Gly Val Ile His Phe His Asn 105 11 O
Trp Arg Pro Ile Phe Arg Arg Asn Val Asp Asn Lell Lys Ile Asn 115 12 O 125
Asp Ile Ser Ile Asp Lell Val Arg Lys Glu. His Pro Trp Asp 13 O 135 14 O
Ser Met Ile Glu Glu Ala Ser Asn Arg Phe Glu Thir Ser Ala Lys 145 150 155 160
Ile Phe Met Glu Lys Thir Lieu Lys Lieu Ala Lys Glu Ile Arg Lys Lys 1.65 17O 17s
Thir Glu Trp Gly Tyr His Gly Tyr Pro His Cys Lell Ser Gly Ser Thir 18O 185 19 O
Asp Pro Ser Phe Asp Cys Asp Ala Lieu. Ser Met Ser Glu Asn Asp 195 2OO
Met Ser Trp Lell Phe Asn Asn. Glin Asn. Wall Lell Lell Pro Ser Ile 21 O 215
Tyr Luell Asn Wall Lell Llys Pro Asp Glu Lys Ile His Luell Wall Glin 225 23 O 235 24 O
Glu Arg Luell Glu Ala Ile Arg Ile Ser Lys Asn Phe His Luell 245 250 255
Pro Wall Luell Pro Trp Trp Tyr Thr Tyr Glin Asp Lys Glu Ser 26 O 265 27 O
Ile Phe Luell Thir Glu Ala Asp Wall Lys Asn Thr Phe Lys Glu Ile Luell 27s 28O 285 US 9,084,743 B2 145 146 - Continued
Thir Asn Gly Ala Asp Gly Ile Ile Ile Trp Gly Wall Ser Tyr Glu Luell 29 O 295 3 OO
Thir Asp Arg Lys Arg Cys Glu Lys Lieu Lys Glu Tyr Lieu Met Lys Ile 3. OS 310 315 32O
Lieu. Gly Pro Ile Ala Phe Llys Val Thr Lys Ala Wall Lys Glu Asn Thir 3.25 330 335
Pro Lieu. Asn. Phe 34 O
<210s, SEQ ID NO 14 &211s LENGTH: 382 212. TYPE: PRT <213> ORGANISM: Apis mellifera 22 Os. FEATURE: <223s OTHER INFORMATION: hyaluronidase
<4 OOs, SEQUENCE: 14
Met Ser Arg Pro Lell Wall Ile Thir Glu Gly Met Met Ile Gly Wall Luell 1. 5 15
Lell Met Luell Ala Pro Ile Asn Ala Luell Luell Luell Gly Phe Wall Glin Ser 2O 25
Thir Pro Asp Asn Asn Thir Wall Arg Glu Phe Asn Wall Trp Asn 35 4 O 45
Wall Pro Thir Phe Met His Gly Luell Arg Phe Glu Glu Wall SO 55 6 O
Ser Glu Gly Ile Lell Glin Asn Trp Met Asp Phe Arg Gly 65 70 75
Glu Glu Ile Ala Ile Lell Tyr Asp Pro Gly Met Phe Pro Ala Luell Luell 85 90 95
Asp Pro Asn Gly Asn Wall Wall Ala Arg ASn Gly Gly Wall Pro Glin 105 11 O
Lell Gly Asn Luell Thir His Luell Glin Wall Phe Arg Asp His Luell Ile 115 12 O 125
Asn Glin Ile Pro Asp Ser Phe Pro Gly Wall Gly Wall Ile Asp Phe 13 O 135 14 O
Glu Ser Trp Arg Pro Ile Phe Arg Glin Asn Trp Ala Ser Luell Glin Pro 145 150 155 160
Luell Ser Wall Glu Wall Wall Arg Arg Glu His Pro Phe Trp 1.65 17s
Asp Asp Glin Arg Wall Glu Glin Glu Ala Lys Arg Arg Phe Glu Tyr 18O 185 19 O
Gly Glin Luell Phe Met Glu Glu Thir Luell Ala Ala Lys Arg Met Arg 195
Pro Ala Ala Asn Trp Gly Tyr Ala Tyr Pro Tyr Asn Luell 21 O 215 22O
Thir Pro Asn Glin Pro Ser Ala Glin Glu Ala Thir Thir Met Glin Glu 225 23 O 235 24 O
Asn Asp Met Ser Trp Lell Phe Glu Ser Glu Asp Wall Luell Luell Pro 245 250 255
Ser Wall Luell Arg Trp Asn Luell Thir Ser Gly Glu Arg Wall Gly Luell 26 O 265 27 O
Wall Gly Gly Arg Wall Glu Ala Luell Arg Ile Ala Arg Glin Met Thir 27s 285
Thir Ser Arg Lys Wall Lell Pro Tyr Trp Tyr Glin Asp 29 O 295 3 OO US 9,084,743 B2 147 148 - Continued
Arg Arg Asp Thir Asp Lell Ser Arg Ala Asp Luell Glu Ala Thir Luell Arg 3. OS 310 315 32O
Ile Thir Asp Lell Gly Ala Asp Gly Phe Ile Ile Trp Gly Ser Ser 3.25 330 335
Asp Asp Ile Asn Thir Ala Cys Luell Glin Phe Arg Glu Luell 34 O 345 35. O
Asn Asn Glu Luell Gly Pro Ala Wall Ile Ala Lell Asn Asn Asn 355 360 365
Ala Asn Asp Arg Lell Thir Wall Asp Wall Ser Wall Asp Glin Wall 37 O 375 38O
SEO ID NO 15 LENGTH: 331 TYPE : PRT ORGANISM: Dolichovespula maculata FEATURE: OTHER INFORMATION: hyaluronidase
SEQUENCE: 15
Ser Glu Arg Pro Lys Arg Wall Phe Asn Ile Trp Asn Wall Pro Thir 1. 5 15
Phe Met Cys His Glin Gly Luell Tyr Phe Asp Glu Wall Thir Asn Phe 2O 25
Asn Ile Lys His Asn Ser Asp Asp Phe Glin Gly Asp Ile Ser 35 4 O 45
Ile Phe Asp Pro Gly Glu Phe Pro Ala Luell Lell Pro Luell Glu 50 55 60
Gly Asn Ile Arg Asn Gly Gly Wall Pro Glin Glu Gly Asn Ile 65 70
Thir Ile His Luell Glin Arg Phe Ile Glu Asn Luell Asp Thir Tyr Pro 85 90 95
Asn Arg Asn Phe Asn Gly Ile Gly Wall Ile Asp Phe Glu Arg Trp Arg 105 11 O
Pro Ile Phe Arg Glin Asn Trp Gly Asn Met Met Ile His Phe 115 12 O 125
Ser Ile Asp Luell Wall Arg Asn Glu His Pro Phe Trp Asp Met 13 O 135 14 O
Ile Glu Luell Glu Ala Ser Arg Phe Glu Lys Ala Arg Luell Phe 145 150 155 160
Met Glu Glu Thir Lell Lell Ala Lys Thir Arg Glin Ala Asp 1.65 17s
Trp Gly Tyr Gly Pro Cys Phe ASn Met Ser Pro Asn Asn 18O 185 19 O
Lell Wall Pro Asp Cys Asp Ala Thir Ala Met Luell Glu Asn Asp Met 195
Ser Trp Luell Phe Asn Asn Glin Asn Wall Luell Luell Pro Ser Wall Ile 21 O 215 22O
Arg His Glu Luell Thir Pro Asp Glin Arg Wall Gly Lell Wall Glin Gly Arg 225 23 O 235 24 O
Wall Glu Ala Wall Arg Ile Ser Asn Asn Luell His Ser Pro Lys 245 250 255
Wall Luell Ser Tyr Trp Trp Wall Tyr Glin Asp Asp Thir Asn Thir Phe 26 O 265 27 O
Lell Thir Glu Thir Asp Wall Lys Thir Phe Glin Glu Ile Ala Ile Asn 27s 28O 285 US 9,084,743 B2 149 150 - Continued
Gly Gly Asp Gly Ile Ile Ile Trp Gly Ser Ser Ser Asp Wall Asn. Ser 29 O 295 3 OO Lieu. Ser Lys Cys Lys Arg Lieu. Arg Glu Tyr Lieu. Lieu. Thr Val Lieu. Gly 3. OS 310 315
Pro Ile Thir Wall Asn. Wall. Thir Glu. Thir Wall ASn 3.25 330
<210s, SEQ ID NO 16 &211s LENGTH: 367 212. TYPE: PRT <213> ORGANISM: Polistes annularis 22 Os. FEATURE: <223> OTHER INFORMATION: hyaluronidase
<4 OOs, SEQUENCE: 16
Tyr Wal Ser Lieu. Ser Pro Asp Ser Wall Phe ASn Ile Ile Thir Asp Asp 1. 5 15
Ile Ser His Glin Ile Lell Ser Arg Ser Asn Cys Glu Arg Ser 2O 25
Pro Arg Wall Phe Ser Ile Tyr Trp Asn Wall Pro Thir Phe Met 35 4 O 45
His Glin Tyr Gly Met Asn Phe Asp Glu Wall Thir Asp Phe Asn Ile SO 55 6 O
His Asn Ser Asp Asn Phe Arg Gly Glu Thir Ile Ser Ile Tyr 65 70
Asp Pro Gly Phe Pro Ala Luell Met Pro Luell Lys Asn Gly Asn Tyr 85 90 95
Glu Glu Arg Asn Gly Gly Wall Pro Glin Arg Gly ASn Ile Thir Ile His 1OO 105 11 O
Lell Glin Glin Phe Asn Glu Asp Luell Asp Met Thir Pro Asp Asn 115 12 O 125
Phe Gly Gly Ile Gly Wall Ile Asp Phe Glu Arg Trp Llys Pro Phe 13 O 135 14 O
Arg Glin Asn Trp Gly Asn Thir Glu Ile His Lys Ser Glu 145 150 155 160
Lell Wall Arg Glu His Pro Trp Ser Glu Ser Met Ile Ala 1.65 17O
Glu Ala Thir Lys Lys Phe Glu Tyr Ala Arg Tyr Phe Met Glu 18O 185 19 O
Thir Luell Lys Luell Ala Thir Arg Arg Ala Lys Trp 195 2OO 2O5
Gly Phe Pro Tyr Tyr Asn Wall Thir Pro Asn. Asn Pro Pro 21 O 215 22O
Asp Asp Ala Ala Thir Ile Glu Asn Asp Arg Lieu. Ser Trp Met 225 23 O 235 24 O
Asn Asn Glin Glu Ile Lell Phe Pro Ser Wall Tyr Val Arg His Glu 245 250 255
Glin Pro Glu Glu Arg Wall Luell Wall Glin Gly Arg Ile Glu 26 O 265 27 O
Ala Wall Arg Ile Ser Asn Asn Luell Glu His Ser Pro Ser Wall Luell Ala 28O 285
Trp Trp Wall Glin Asp Met Asp Ile Tyr Luell Ser Glu 29 O 295 3 OO
Thir Asp Wall Glu Thir Phe Glin Glu Ile Wall Thir Asn Gly Gly Asp 3. OS 310 315 32O US 9,084,743 B2 151 152 - Continued
Gly Ile Ile Ile Trp Gly Ser Ser Ser Asp Wall Asn. Ser Luell Ser 3.25 330 335
Cys Lys Arg Lieu. Arg Glu Tyr Lieu. Lieu. Asn. Thir Lieu. Gly Pro Phe Ala 34 O 345 35. O
Wall Asn. Wall Thr Glu Thr Val Asn Gly Arg Ser Ser Lieu. Asn Phe 355 360 365
SEO ID NO 17 LENGTH: 462 TYPE : PRT ORGANISM: Mus musculus FEATURE: OTHER INFORMATION: hyaluronidase
<4 OOs, SEQUENCE: 17
Met Lieu. Gly Lieu Thir Glin His Ala Glin Lys Wall Trp Arg Met Lys Pro 1. 5 15
Phe Ser Pro Glu Wall Ser Pro Gly Ser Ser Pro Ala Thir Ala Gly His 25
Lell Luell Arg Ile Ser Thir Lell Phe Luell Thir Luell Lell Glu Luell Ala Glin 35 4 O 45
Wall Cys Arg Gly Ser Wall Wall Ser Asn Arg Pro Phe Ile Thir Wall Trp SO 55 6 O
Asn Gly Asp Thir His Trp Luell Thir Glu Tyr Gly Wall Asp Wall Asp 65 70
Wall Ser Wall Phe Asp Wall Wall Ala Asn Lys Glu Glin Ser Phe Glin Gly 85 90 95
Ser Asn Met Thir Ile Phe Arg Glu Glu Luell Gly Thir Tyr Pro 105 11 O
Thir Pro Thir Gly Glu Pro Wall Phe Gly Gly Lell Pro Glin Asn Ala 115 12 O 125
Ser Luell Wall Thir His Lell Ala His Thir Phe Glin Asp Ile Ala Ala 13 O 135 14 O
Met Pro Glu Pro Asp Phe Ser Gly Luell Ala Wall Ile Asp Trp Glu Ala 145 150 155 160
Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ser Asp Ile Tyr Arg 1.65 17O 17s
Glin Arg Ser Met Glu Lell Wall Glin Ala Glu His Pro Asp Trp Pro Glu 18O 185 19 O
Thir Luell Wall Glu Ala Ala Ala Lys Asn Glin Phe Glin Glu Ala Ala Glu 195
Ala Trp Met Ala Gly Thir Lell Glin Luell Gly Glin Wall Lell Arg Pro Arg 21 O 215 22O
Gly Luell Trp Gly Tyr Tyr Gly Phe Pro Asp Cys Asn Asn Asp Phe 225 23 O 235 24 O
Lell Ser Luell Asn Tyr Thir Gly Glin Pro Wall Phe Wall Arg Asp Glin 245 250 255
Asn Asp Glin Luell Gly Trp Lell Trp Asn Glin Ser Ala Luell Tyr Pro 26 O 265 27 O
Ser Ile Tyr Luell Pro Ala Ala Luell Met Gly Thir Gly Lys Ser Glin Met 27s 285
Wall Arg His Arg Wall Glin Glu Ala Luell Arg Wall Ala Ile Wall Ser 29 O 295 3 OO
Arg Asp Pro His Wall Pro Wall Met Pro Tyr Wall Glin Ile Phe Glu 3. OS 310 315 32O US 9,084,743 B2 153 154 - Continued
Met Thr Asp Tyr Lieu Lieu Pro Lieu. Glu Glu Lieu. Glu His Ser Luell Gly 3.25 330 335
Glu Ser Ala Ala Glin Gly Val Ala Gly Ala Val Lell Trp Luell Ser Ser 34 O 345 35. O
Asp Lys. Thir Ser Thr Lys Glu Ser Cys Glin Ala Ile Lys Ala Met 355 360 365
Asp Ser Thr Lieu. Gly Pro Phe Ile Wall Asn. Wall Thir Ser Ala Ala Luell 37 O 375
Lell Cys Ser Glu Ala Lieu. Cys Ser Gly His Gly Arg Wall Arg His 385 390 395 4 OO
Pro Ser Tyr Pro Glu Ala Lieu. Lieu. Thir Lieu. Asn Pro Ala Ser Phe Ser 4 OS 41O 415
Ile Glu Lieu. Thir His Asp Gly Arg Pro Pro Ser Lell Gly Thir Luell 42O 425 43 O
Ser Lieu Lys Asp Arg Ala Gln Met Ala Met Lys Phe Arg Arg 435 44 O 445
Arg Gly Trp Arg Gly Llys Trp Cys Asp Llys Arg Gly Met 450 45.5 460
<210s, SEQ ID NO 18 &211s LENGTH: 473 212. TYPE: PRT &213s ORGANISM: Mus musculus 22 Os. FEATURE: OTHER INFORMATION: Hyaluronidase 2 < 4 OO SEQUENCE: 18
Met Arg Ala Gly Lieu Gly Pro Ile Ile Thr Lieu. Ala Lell Wall Luell Glu 1. 5 1O 15
Wall Ala Trp Ala Gly Glu Lieu Lys Pro Thr Ala Pro Pro Ile Phe Thir 2O 25
Gly Arg Pro Phe Val Val Ala Trp Asn Val Pro Thir Glin Glu Ala 35 4 O 45
Pro Arg His Llys Val Pro Lieu. Asp Lieu. Arg Ala Phe Asp Wall Ala SO 55 6 O
Thir Pro Asn Glu Gly Phe Phe Asn. Glin Asn. Ile Thir Thir Phe Tyr 65 70 7s
Asp Arg Lieu. Gly Lieu Tyr Pro Arg Phe Asp Ala Ala Gly Thir Ser Wall 85 90 95
His Gly Gly Val Pro Glin Asn Gly Ser Lieu. Cys Ala His Luell Pro Met 1OO 105 11 O
Lell Lys Glu Ser Val Glu Arg Tyr Ile Glin Thr Glin Glu Pro Gly Gly 115 12 O 125
Lell Ala Val Ile Asp Trp. Glu Glu Trp Arg Pro Wall Trp Wall Arg Asn 13 O 135 14 O
Trp Glin Glu Lys Asp Val Tyr Arg Glin Ser Ser Arg Glin Luell Wall Ala 145 150 155 160
Ser Arg His Pro Asp Trp Pro Ser Asp Arg Val Met Glin Ala Glin 1.65 17O 17s
Glu Phe Glu Phe Ala Ala Arg Glin Phe Met Lell Asn Thir Luell Arg 18O 185 19 O
Val Lys Ala Val Arg Pro Gln His Lieu. Trp Gly Phe Luell Phe 195 2OO 2O5
Pro Asp Cys Tyr Asn His Asp Tyr Val Glin Asn Trp Glu Ser Thir 21 O 215 22O US 9,084,743 B2 155 156 - Continued
Gly Arg Cys Pro Asp Val Glu Val Ala Arg Asn Asp Glin Luell Ala Trp 225 23 O 235 24 O
Lell Trp Ala Glu Ser Thir Ala Lieu. Phe Pro Ser Wall Luell Asp Glu 245 250 255
Thir Lieu Ala Ser Ser Val His Ser Arg Asn Phe Wall Ser Phe Arg Wall 26 O 265 27 O
Arg Glu Ala Lieu. Arg Wall Ala His Thir His His Ala Asn His Ala Luell 27s 28O 285
Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Thr Arg Gly Luell Thir Gly 29 O 295 3 OO
Lell Ser Glin Val Asp Lieu. Ile Ser Thr Ile Gly Glu Ser Ala Ala Luell 3. OS 310 315
Gly Ser Ala Gly Val Ile Phe Trp Gly Asp Ser Glu Asp Ala Ser Ser 3.25 330 335
Met Glu Thr Cys Glin Tyr Lieu Lys Asn Tyr Lieu. Thir Glin Luell Luell Wall 34 O 345 35. O
Pro Tyr Ile Val Asn Val Ser Trp Ala Thr Glin Cys Ser Trp Thir 355 360 365
Glin Cys His Gly His Gly Arg Cys Val Arg Arg Asn Pro Ser Ala Asn 37 O 375
Thir Phe Lieu. His Lieu. Asn Ala Ser Ser Phe Arg Lell Wall Pro Gly His 385 390 395 4 OO
Thir Pro Ser Glu Pro Glin Lieu. Arg Pro Glu Gly Glin Lell Ser Glu Ala 4 OS 41O 415
Asp Lieu. Asn Tyr Lieu. Glin Llys His Phe Arg Cys Glin Tyr Luell Gly 42O 425 43 O
Trp Gly Gly Glu Glin Cys Glin Arg Asn Tyr Lys Gly Ala Ala Gly Asn 435 44 O 445
Ala Ser Arg Ala Trp Ala Gly Ser His Lieu. Thr Ser Lell Luell Gly Luell 450 45.5 460
Wall Ala Wall Ala Lieu Thir Trp Thr Lieu. 465 470
<210s, SEQ ID NO 19 &211s LENGTH: 412 212. TYPE: PRT &213s ORGANISM: Mus musculus 22 Os. FEATURE: OTHER INFORMATION: hyalurinidase 3
<4 OOs, SEQUENCE: 19
Met Ile Met His Lieu. Gly Lieu Met Met Val Val Gly Lell Thir Luell 1. 5 1O 15
Lell Met His Gly Glin Ala Lieu. Lieu. Glin Wall Pro Glu His Pro Phe Ser 2O 25 3O
Wall Val Trp Asn Val Pro Ser Ala Arg Cys Llys Ala His Phe Gly Wall 35 4 O 45
His Lieu Pro Lieu. Asp Ala Lieu. Gly Ile Val Ala Asn His Gly Glin His SO 55 6 O
Phe His Gly Glin Asn Ile Ser Ile Phe Tyr Lys Asn Glin Phe Gly Luell 65 70 7s
Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly Ile Pro 85 90 95
Glin Ala Wal Ser Lieu Asp His His Lieu Ala Arg Ala Ala His Glin Ile 1OO 105 11 O US 9,084,743 B2 157 158 - Continued
Lell His Ser Luell Gly Ser Ser Phe Ala Gly Luell Ala Wall Luell Asp Trp 115 12 O 125
Glu Glu Trp Pro Lell Trp Ala Gly Asn Trp Gly Pro His Arg Glin 13 O 135 14 O
Wall Luell Ala Ala Ser Trp Wall Trp Thr Glin Glin Met Phe Pro Gly 145 150 155 160
Lell Asp Pro Glin Glu Glin Lell His Lys Ala His Thir Ser Phe Glu Glin 1.65 17O 17s
Ala Ala Arg Ala Lell Met Glu Tyr Thir Lieu. Glin Lell Gly Arg Thir Luell 18O 185 19 O
Arg Pro Ser Gly Lell Trp Gly Phe Pro Ala Gly Asn 195
Gly Trp His Met Ala Ser Asn Tyr Thr Gly His His Ala Ala 21 O 215 22O
Ile Thir Thir Glin Asn Thir Glin Luell Arg Trp Luell Trp Ala Ala Ser Ser 225 23 O 235 24 O
Ala Luell Phe Pro Ser Ile Luell Pro Pro Arg Lell Pro Luell Ala Tyr 245 250 255
Arg Glin Ala Phe Wall Arg His Arg Lieu. Glu Glu Ala Phe Arg Wall Ala 26 O 265 27 O
Lell Luell Glu His Ser His Pro Luell Pro Wall Luell Ala Tyr Ser Arg Luell 285
Thir His Arg Ser Ser Gly Arg Phe Luell Ser Luell Asp Asp Luell Met Glin 29 O 295 3 OO
Thir Ile Gly Wall Ser Ala Ala Luell Gly Thr Ala Gly Wall Wall Luell Trp 3. OS 310 315
Gly Asp Luell Ser Phe Ser Ser Ser Glu Glu Lys Trp Arg Luell His 3.25 330 335
Asp Luell Wall Gly Thir Lell Gly Pro Tyr Wall Ile Asn Wall Thir 34 O 345 35. O
Ala Asp Met Ala Ser His Glin Arg Cys His Gly His Gly Arg 355 360 365
Ala Arg Asp Pro Gly Glin Met Glu Ala Phe Lell His Luell Glin Pro 37 O 375 38O
Asp Asp Ser Luell Gly Ala Trp Asn Ser Phe Arg His Ser 385 390 395 4 OO
Gly Trp Ala Gly Pro Thir Luell Glu Pro Pro 4 OS 41O
SEQ ID NO 2 O LENGTH: 435 TYPE : PRT ORGANISM: Sus scrofa FEATURE: OTHER INFORMATION: hyalauronidase
<4 OOs, SEQUENCE:
Met Ala Ala His Lieu. Lieu. Pro Ile Cys Thr Luell Phe Lell Asn Luell Luell 1. 5 1O 15
Ser Val Ala Glin Gly Ser Arg Asp Pro Wall Wall Lell Asn Arg Pro Phe 25 3O
Thir Thir Ile Trp Asn Ala Asn Thir Gln Trp Cys Lell Lys Arg His Gly 35 4 O 45
Val Asp Val Asp Val Ser Wall Phe Glu Wall Wall Wall Asn Pro Gly Glin SO 55 6 O US 9,084,743 B2 159 160 - Continued
Thir Phe Arg Gly Pro Asn Met Thir Ile Phe Tyr Ser Ser Glin Luell Gly 65 70 8O
Thir Pro Tyr Thir Ser Ala Gly Glu Pro Wall Phe Gly Gly Luell 85 90 95
Pro Glin Asn Ala Ser Lell Asp Wall His Luell ASn Arg Thir Phe Asp 105 11 O
Ile Luell Ala Ala Met Pro Glu Ser Asn Phe Ser Gly Lell Ala Wall Ile 115 12 O 125
Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ala 13 O 135 14 O
Asp Ile Arg Glin Arg Ser Arg Ala Luell Wall Glin Glin His Pro 145 150 155 160
Asp Trp Pro Ala Pro Trp Wall Glu Ala Ala Ala Glin Asp Glin Phe Glin 1.65 17s
Glu Ala Ala Glin Thir Trp Met Ala Gly Thir Luell Lell Gly Glin Thir 18O 185 19 O
Lell Arg Pro His Gly Lell Trp Gly Phe Gly Phe Pro Asp Tyr 195
Asn Tyr Asp Phe Glin Ser Ser Asn Tyr Thir Gly Glin Pro Pro Gly 21 O 215 22O
Wall Ser Ala Glin Asn Asp Glin Luell Gly Trp Luell Trp Gly Glin Ser Arg 225 23 O 235 24 O
Ala Luell Tyr Pro Ser Ile Luell Pro Ser Ala Lell Glu Gly Thir Asn 245 250 255
Thir Glin Luell Tyr Wall Glin His Arg Wall ASn Glu Ala Phe Arg Wall 26 O 265 27 O
Ala Ala Ala Ala Gly Asp Pro Asn Luell Pro Wall Lell Pro Ala Glin 285
Ile Phe His Asp Met Thir Asn Arg Luell Luell Ser Arg Glu Glu Luell Glu 29 O 295 3 OO
His Ser Luell Gly Glu Ser Ala Ala Glin Gly Ala Ala Gly Wall Wall Luell 3. OS 310 315
Trp Wall Ser Trp Glu Asn Thir Arg Thir Lys Glu Ser Glin Ser Ile 3.25 330 335
Glu Tyr Wall Asp Thir Thir Luell Gly Pro Phe Ile Lell Asn Wall Thir 34 O 345 35. O
Ser Gly Ala Luell Lell Ser Glin Ala Wall Cys Ser Gly His Gly Arg 355 360 365
Wall Arg Arg Pro Ser His Thir Glu Ala Luell Pro Ile Luell Asn Pro 37 O 375
Ser Ser Phe Ser Ile Lys Pro Thir Pro Gly Gly Gly Pro Luell Thir Luell 385 390 395 4 OO
Glin Gly Ala Luell Ser Lell Asp Arg Wall Glin Met Ala Glu Glu Phe 4 OS 41O 415
Glin Arg Cys Tyr Pro Gly Trp Arg Gly Thir Trp Glu Glin Glin 42O 425 43 O
Gly Thir Arg 435
SEQ ID NO 21 LENGTH: 419 TYPE : PRT ORGANISM: Sus scrofa FEATURE: US 9,084,743 B2 161 162 - Continued <223> OTHER INFORMATION: hyaluronidase 3 <4 OOs, SEQUENCE: 21 Met Thr Met Glin Lieu. Gly Lieu Ala Lieu Val Lieu. Gly Val Ala Met 1. 5 1O 15 Lieu. Gly Cys Gly Glin Pro Lieu. Lieu. Arg Ala Pro Glu Arg Pro Phe 2O 25 3O
Val Lieu. Trp Asn Val Pro Ser Ala Arg Cys Lys Ala Arg Phe Gly Wall 35 4 O 45 His Lieu Pro Lieu. Glu Ala Lieu. Gly Ile Thir Ala Asn His Gly Glin Arg SO 55 6 O
Phe His Gly Glin Asn Ile Thr Ile Phe Tyr Lys Ser Gln Leu Gly Luell 65 70 7s
Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly Ile Pro 85 90 95
Glin Ala Val Ser Lieu. Asp His His Lieu Ala Arg Ala Ala Tyr Glin Ile 1OO 105 11 O His Arg Ser Lieu. Arg Pro Gly Phe Thr Gly Lieu Ala Val Lieu. Asp Trp 115 12 O 125
Glu Glu Trp Cys Pro Lieu. Trp Ala Gly Asn Trp Gly Arg Arg Glin Ala 13 O 135 14 O
Tyr Glin Ala Ala Ser Cys Ala Trp Ala Glin Arg Val Tyr Pro Asn Luell 145 150 155 160
Asp Pro Glin Glu Gln Lieu. Cys Lys Ala Arg Ala Gly Phe Glu Glu Ala 1.65 17O 17s Ala Arg Ala Lieu Met Glu Asp Thir Lieu. Arg Lieu. Gly Arg Met Lieu. Arg 18O 185 19 O Pro His Gly Leu Trp Gly Phe Tyr His Tyr Pro Ala Cys Gly Asn Gly 195 2OO 2O5
Trp. His Gly Thr Ala Ser Asn Tyr Thr Gly His Cys His Ala Ala Ala 21 O 215 22O
Lieu Ala Arg Asn. Thr Glin Lieu. Tyr Trp Lieu. Trp Ala Ala Ser Ser Ala 225 23 O 235 24 O
Lieu. Phe Pro Ser Ile Tyr Lieu Pro Pro Gly Lieu Pro Pro Ala Tyr His 245 250 255
Glin Ala Phe Val Arg Tyr Arg Lieu. Glu Glu Ala Phe Arg Val Ala Luell 26 O 265 27 O
Val Gly His Pro His Pro Lieu Pro Val Lieu Ala Tyr Ala Arg Lieu. Thir 27s 28O 285
His Arg Asn. Ser Gly Arg Phe Lieu. Ser Glin Asp Glu Lieu Val Glin Thir 29 O 295 3 OO Ile Gly Val Ser Ala Ala Lieu. Gly Ala Ser Gly Val Val Lieu. Trip 3. OS 310 315 Asp Lieu. Ser Phe Ser Ser Ser Glu Glu Glu. Cys Trp His Lieu. Arg 3.25 330 335
Tyr Lieu Val Gly Thr Lieu. Gly Pro Tyr Val Ile Asn Val Thr Arg 34 O 345 35. O Ala Met Ala Cys Ser His Glin Arg Cys His Gly His Gly Arg Cys 355 360 365 Trp Glin Asp Pro Gly Glin Lieu Lys Val Phe Lieu. His Lieu. His Pro 37 O 375 38O
Gly Ser Pro Gly Ala Trp Glu Ser Phe Ser Cys Arg Cys Tyr Trp 385 390 395 US 9,084,743 B2 163 164 - Continued Trp Ala Gly Pro Thr Cys Glin Glu Pro Arg Pro Glu Lieu. Gly Pro Glu 4 OS 415
Glu Ala Thr
SEQ ID NO 22 LENGTH: 449 TYPE : PRT ORGANISM: Rattus norvegicus FEATURE: OTHER INFORMATION: hyaluronidase 1
<4 OOs, SEQUENCE: 22
Met Llys Pro Phe Ser Pro Glu Wall Ser Pro Asp Pro Pro Ala Thir 1. 5 15
Ala Ala His Luell Lell Arg Thir Thir Luell Phe Lell Thir Luell Luell Glu 25
Lell Ala Glin Gly Cys Arg Gly Ser Met Wall Ser Asn Arg Pro Phe Ile 35 4 O 45
Thir Wall Trp Asn Ala Asp Thir His Trp Cys Luell Lys Asp His Gly Wall SO 55 6 O
Asp Wall Asp Wall Ser Wall Phe Asp Wall Wall Ala Asn Glu Glin Asn 65 70
Phe Glin Gly Pro Asn Met Thir Ile Phe Tyr Arg Glu Glu Luell Gly Thir 85 90 95
Pro Tyr Thir Pro Thir Gly Glu Pro Wall Phe Gly Gly Luell Pro 105 11 O
Asn Ser Lieu Wall Thr His Lieu Ala His Ala Phe Gln Asp Ile 12 O 125
Ala Met Pro Glu Pro Asp Phe Ser Gly Lell Ala Wall Ile Asp 13 O 135 14 O
Trp Glu Trp Arg Pro Arg Trp Ala Phe ASn Trp Asp Ser Asp 145 150 155 160
Ile Glin Arg Ser Met Glu Luell Wall Arg Ala Glu His Pro Asp 1.65 17O 17s
Trp Pro Thir Lell Wall Glu Ala Glu Ala Glin Gly Glin Phe Glin Glu 18O 185 19 O
Ala Ala Ala Trp Met Ala Gly Thir Luell Glin Lell Gly Glin Wall Luell 2OO
Arg Pro Arg Gly Lell Trp Gly Gly Phe Pro Asp Asn 21 O 215 22O
Tyr Asp Phe Luell Ser Pro Asn Thir Gly Glin Ser Luell Ser Ile 225 23 O 235 24 O
His Asp Glin Asn Asp Glin Lell Gly Trp Luell Trp Asn Glin Ser Tyr Ala 245 250 255
Lell Pro Ser Ile Lell Pro Ala Ala Luell Met Gly Thir Gly Lys 26 O 265 27 O
Ser Glin Met Wall Arg Arg Wall Glin Glu Ala Phe Arg Luell Ala 285
Lell Wall Ser Asp Pro His Wall Pro Ile Met Pro Wall Glin Ile 29 O 295 3 OO
Phe Glu Thir Asp Luell Luell Pro Luell Glu Glu Luell Glu His 3. OS 310 315 32O
Ser Luell Gly Glu Ser Ala Ala Glin Gly Ala Ala Gly Ala Wall Luell Trp 3.25 330 335
Ile Ser Ser Glu Lys Thir Ser Thir Glu Ser Glin Ala Ile Lys US 9,084,743 B2 165 166 - Continued
34 O 345 35. O
Ala Tyr Met Asp Ser Thr Lieu. Gly Pro Phe Ile Lell Asn Wall Thir Ser 355 360 365
Ala Ala Lieu. Lieu. Cys Ser Glu Ala Lieu. Cys Ser Gly Arg Gly Arg 37 O 375
Wall Arg His Pro Ser Tyr Pro Glu Ala Lieu. Lieu. Thir Lell Ser Pro Ala 385 390 395 4 OO
Ser Phe Ser Ile Glu Pro Thr His Asp Gly Arg Pro Lell Ser Luell 4 OS 41O 415
Gly Thir Lieu. Ser Lieu Lys Asp Arg Ala Glin Met Ala Met Lys Phe 42O 425 43 O
Arg Cys Tyr Arg Gly Trp Ser Gly Glu Trp Lys Glin Asp 435 44 O 445
Met
<210s, SEQ ID NO 23 &211s LENGTH: 473 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus 22 Os. FEATURE: <223> OTHER INFORMATION: hyaluronidase 2
<4 OOs, SEQUENCE: 23
Met Arg Ala Gly Lieu. Gly Pro Ile Ile Thir Lieu. Ala Lell Wall Luell Glu 1. 5 1O 15
Wall Ala Trp Ala Ser Glu Lieu Lys Pro Thir Ala Pro Pro Ile Phe Thir 2O 25 30
Gly Arg Pro Phe Val Val Ala Trp Asn Val Pro Thir Glin Glu Ala 35 4 O 45
Pro Arg His Llys Val Pro Lieu. Asp Lieu. Arg Ala Phe Asp Wall Glu Ala SO 55 6 O
Thir Pro Asn Glu Gly Phe Phe Asin Glin Asn Ile Thir Thir Phe Tyr 65 70 7s
Asp Arg Lieu. Gly Lieu. Tyr Pro Arg Phe Asp Ala Ala Gly Met Ser Wall 85 90 95
His Gly Gly Val Pro Glin Asn Gly Ser Lieu. Cys Ala His Luell Pro Met 1OO 105 11 O
Lell Lys Glu Ala Val Glu Arg Tyr Ile Glin Thr Glin Glu Pro Ala Gly 115 12 O 125
Lell Ala Val Ile Asp Trp Glu Glu Trp Arg Pro Wall Trp Wall Arg Asn 13 O 135 14 O
Trp Glin Glu Lys Asp Val Tyr Arg Glin Ser Ser Arg Glin Luell Wall Ala 145 150 155 160
Ser Arg His Pro Asp Trp Pro Ser Asp Arg Ile Wall Glin Ala Glin 1.65 17O
Glu Phe Glu Phe Ala Ala Arg Glin Phe Met Lell Asn Thir Luell Arg 18O 185 19 O
Val Lys Ala Val Arg Pro Gln His Lieu. Trp Gly Phe Luell Phe 195 2OO 2O5
Pro Asp Cys Tyr Asn His Asp Tyr Val Glin Asn Trp Asp Ser Thir 21 O 215 22O
Gly Arg Cys Pro Asp Val Glu Val Ala Glin Asn Asp Glin Luell Ala Trp 225 23 O 235 24 O
Lell Trp Ala Glu Asn Thr Ala Leu Phe Pro Ser Wall Luell Asp Lys 245 250 255 US 9,084,743 B2 167 168 - Continued
Thir Lieu Ala Ser Ser Llys His Ser Arg Asn. Phe Wall Ser Phe Arg Wall 26 O 265 27 O
Glin Glu Ala Lieu. Arg Val Ala His Thr His His Ala Asn His Ala Luell 27s 28O 285
Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Thr Arg Arg Luell Thir Glu 29 O 295 3 OO
Lell Asin Glin Met Asp Lieu. Ile Ser Thr Ile Gly Glu Ser Ala Ala Luell 3. OS 310 315
Gly Ser Ala Gly Val Ile Phe Trp Gly Asp Ser Wall Ala Ser Ser 3.25 330 335
Met Glu Asn. Cys Glin Asn Lieu Lys Llys Tyr Lieu. Thir Glin Thir Luell Wall 34 O 345 35. O
Pro Tyr Ile Val Asn Val Ser Trp Ala Thr Glin Cys Ser Trp Thir 355 360 365
Glin Cys His Gly. His Gly Arg Cys Val Arg Arg Asn Pro Ser Ala Ser 37 O 375
Thir Phe Lieu. His Leu Ser Pro Ser Ser Phe Arg Lell Wall Pro Gly Arg 385 390 395 4 OO
Thir Pro Ser Glu Pro Gln Lieu. Arg Pro Glu Gly Glu Lell Ser Glu Asp 4 OS 41O 415
Asp Leu Ser Tyr Lieu Gln Met His Phe Arg Cys His Tyr Luell Gly 42O 425 43 O
Trp Gly Gly Glu Gln Cys Gln Trp Asn His Lys Arg Ala Ala Gly Asp 435 44 O 445
Ala Ser Arg Ala Trp Ala Gly Ala His Lieu Ala Ser Lell Luell Gly Luell 450 45.5 460
Wall Ala Met Thr Lieu. Thir Trp Thr Lieu. 465 470
<210s, SEQ ID NO 24 &211s LENGTH: 412 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus 22 Os. FEATURE: <223> OTHER INFORMATION: hyaluronidase 3
<4 OOs, SEQUENCE: 24
Met Ile Thr Gln Leu Gly Lieu. Thir Lieu Val Val Gly Lell Thir Luell 1. 5 1O 15
Lell Wal His Val Glin Ala Lieu. Lieu. Glin Val Pro Glu Phe Pro Phe Ser 2O 25
Wall Lieu. Trp Asn Val Pro Ser Ala Arg Cys Llys Thir Arg Phe Gly Wall 35 4 O 45
His Lieu Pro Lieu. Asp Ala Lieu. Gly Ile Ile Ala Asn His Gly Glin Arg SO 55 6 O
Phe His Gly Glin Asn Ile Thr Ile Phe Tyr Lys Asn Glin Phe Gly Luell 65 70 7s
Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly Ile Pro 85 90 95
Glin Ala Val Ser Lieu. Asp His His Lieu Ala Glin Ala Ala His Glin Ile 1OO 105 11 O
Lell His Asn Lieu. Gly Ser Ser Phe Ala Gly Lieu. Ala Wall Luell Asp Trp 115 12 O 125
Glu Glu Trp Tyr Pro Leu Trp Ala Gly Asn Trp Gly Thir His Arg Glin 13 O 135 14 O US 9,084,743 B2 169 170 - Continued
Wall Glin Ala Ala Ser Trp Ala Trp Ala Glin Glin Met Phe Pro Asp 145 150 155 160
Lell Asn Pro Glin Glu Glin Lell His Ala Glin Thir Gly Phe Glu Glin 1.65 17O
Ala Ala Arg Ala Lell Met Glu His Thir Luell Arg Lell Gly Glin Met Luell 18O 185 19 O
Arg Pro His Gly Lell Trp Gly Phe Arg Pro Wall Gly Asn 195
Gly Trp His Asn Met Ala Ser Asn Thir Gly His His Pro Ala 21 O 215 22O
Ile Ile Thir Arg Asn Thir Glin Luell Arg Trp Luell Trp Ala Ala Ser Ser 225 23 O 235 24 O
Ala Luell Phe Pro Ser Ile Luell Pro Pro Arg Lell Pro Pro Ala Tyr 245 250 255
His Glin Thir Phe Wall Arg His Arg Luell Glu Glu Ala Phe Arg Wall Ala 26 O 265 27 O
Lell Thir Gly His Ala His Pro Luell Pro Wall Luell Ala Tyr Wall Arg Luell 285
Thir His Arg Ser Ser Gly Arg Phe Luell Ser Luell Asp Asp Luell Met Glin 29 O 295 3 OO
Thir Ile Gly Wall Ser Ala Ala Luell Gly Ala Ala Gly Wall Wall Luell Trp 3. OS 310 315
Gly Asp Luell Ser Wall Ser Ser Ser Glu Glu Glu Trp Arg Luell His 3.25 330 335
Asp Luell Wall Gly Thir Lell Gly Pro Wall Ile Asn Wall Thir Lys 34 O 345 35. O
Ala Ala Thir Ala Cys Ser His Glin Arg His Gly His Gly Arg Cys 355 360 365
Ser Trp Asp Pro Gly Glin Met Glu Ala Phe Lell His Luell Glin Pro 37 O 375 38O
Asp Asp Asn Luell Gly Ala Trp Ser Phe Arg Arg Luell 385 390 395 4 OO
Gly Trp Ser Gly Pro Thir Luell Glu Pro Pro 4 OS 41O
SEO ID NO 25 LENGTH: 545 TYPE : PRT ORGANISM: Oryctolagus cuniculus FEATURE: OTHER INFORMATION: PH2O
<4 OOs, SEQUENCE: 25
Met Gly Val Lieu. Lys Phe His Ile Phe Phe Gly Ser Ala Wall Glu 1. 5 15
Lell Ser Gly Wall Phe Glin Ile Wall Phe Ile Phe Lell Lell Ile Pro Cys 25 3O
Luell Thir Ala Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45
Phe Luell Trp Ala Trp Asn Ala Pro Thir Glu Phe Cys Lell Gly Ser SO 55 6 O
Gly Glu Pro Luell Asp Met Ser Luell Phe Ser Luell Phe Gly Ser Pro Arg 65 70
Asn Thir Gly Glin Gly Ile Thir Ile Phe Wall Asp Arg Luell 85 90 95 US 9,084,743 B2 171 172 - Continued
Gly yr Pro Tyr Ile Asp Pro His Thir Gly Ala Ile Wall His Gly 1OO 105 11 O
Arg Ile Pro Glin Lell Gly Pro Luell Glin Glin His Lell Thir Luell Arg 15 12 O 125
Glin Glu le Luell Tyr Met Pro Asp ASn Wall Gly Luell Ala Wall 13 O 135 14 O
Ile Asp rp Glu Glu Trp Lell Pro Thir Trp Luell Arg Asn Trp Pro 145 150 155 160
Asp le Arg Ile Ser Ile Glu Luell Wall Ser Glin His 1.65 17O 17s
Pro Glin Asn His Ser Ala Thir Glu Ala Arg Asp Phe 18O 185 19 O
Glu Gly Lys Asp Phe Met Glu Glu Thir Lell Lys Luell Gly Arg
Lell Luell Arg Pro Asn His Lell Trp Gly Lell Phe Pro Asp 21 O 215
Tyr Asn His His Tyr Asp Pro Asn Luell Tyr Gly Ser Phe 225 23 O 235 24 O
Asp Ile Glu Lys Arg Asn Asp Asp Luell Ser Trp Lell Trp Lys Glu 245 250 255
Ser Thir Ala Luell Phe Pro Ser Wall Tyr Luell Thir Ser Arg Ala Arg Ser 26 O 265 27 O
Ala Thir Ala Luell Ser Lell Tyr Wall Wall Arg Asn Arg Wall His Glu 28O 285
Ala Ile Arg Wall Ser Ile Pro Asp Asp Ser Pro Luell Pro Asn 29 O 295 3 OO
Phe Wall Thir Lell Wall Phe Thir Asp Glin Ile Phe Glin Phe Luell 3. OS 310 315
Ser His His Asp Lell Wall Thir Ile Gly Glu Ile Wall Ala Luell Gly 3.25 330 335
Ala Ser Gly Ile Wall Wall Trp Gly Ser Glin Ser Lell Ala Arg Ser Met 34 O 345 35. O
Ser Cys Luell His Lell Asp Asn Met Thir Ile Luell Asn Pro 355 360 365
Luell Ile Asn Wall Thir Lell Ala Ala Met Cys Asn Glin Wall Luell 37 O 375
Cys Glin Glu Glin Gly Wall Thir Arg ASn Trp Asn Pro Asn Asp 385 390 395 4 OO
Luell His Luell Asn Pro Gly Asn Phe Ala Ile Glin Lell Gly Ser Asn 4 OS 41O 415
Gly Thir Lys Wall Asp Gly Pro Thir Luell Thir Asp Luell Glu Glin 425 43 O
Phe Ser Lys Asn Phe Glin Ser Thir Asn Lell Asn Lys 435 44 O 445
Glu Arg Thir Asp Met Asn Asn Wall Arg Thir Wall Asn Wall Ala Wall 450 45.5 460
Glu Asn Wall Ile Asp Thir Asn Wall Gly Pro Glin Ala Wall Thir Tyr 465 470 48O
Ala Pro Glu Lys Asp Wall Ala His Ile Lell Ser Asn Thir Thir 485 490 495
Ser Ile Asn Ser Ser Thir Thir Met Ser Luell Pro Phe Pro Arg His SOO 505 51O US 9,084,743 B2 173 174 - Continued
Wall Ser Gly Cys Lieu. Leu Val Lieu. Cys Met Tyr Ser Glin Tyr Lieu. Asn 515 525
Ile Cys Tyr Arg Lieu Val Ala Ile Gly Ile Gln His Gly Tyr Tyr Lieu. 53 O 535 54 O Lys 5.45
SEQ ID NO 26 LENGTH: 476 TYPE : PRT ORGANISM: Ovis airies FEATURE: OTHER INFORMATION: hyaluronidase 2
<4 OOs, SEQUENCE: 26
Met Trp Thir Gly Lell Gly Pro Ala Wall Thir Luell Ala Lell Wall Luell Wall 1. 5 1O 15
Wall Ala Trp Ala Thir Glu Lell Pro Thir Ala Pro Pro Ile Phe Thir 2O 25
Gly Arg Pro Phe Wall Wall Ala Trp Asp Wall Pro Thir Glin Asp Gly 35 4 O 45
Pro Arg His Met Pro Lell Asp Pro Asp Met Lys Ala Phe Asp SO 55 6 O
Wall Glin Ala Ser Pro Asn Glu Gly Phe Wall ASn Glin Asn Ile Thir Ile 65 70
Phe Arg Asp Arg Lell Gly Met Pro His Phe Asn Ser Wall Gly 85 90 95
Arg Ser Wall His Gly Gly Wall Pro Glin Asn Gly Ser Lell Trp Wall His 105 11 O
Lell Glu Met Luell Lys Gly His Wall Glu His Ile Arg Thir Glin Glu 115 12 O 125
Pro Ala Gly Luell Ala Wall Ile Asp Trp Glu Asp Trp Arg Pro Wall Trp 13 O 135 14 O
Wall Arg Asn Trp Glin Asp Asp Wall Arg Arg Lell Ser Arg Glin 145 150 155 160
Lell Wall Ala Ser His His Pro Asp Trp Pro Pro Glu Arg Ile Wall Lys 1.65 17O 17s
Glu Ala Glin Tyr Glu Phe Glu Phe Ala Ala Arg Glin Phe Met Luell Glu 18O 185 19 O
Thir Luell Arg Phe Wall Ala Phe Arg Pro Arg His Lell Trp Gly Phe 195
Luell Phe Pro Asp Tyr Asn His Asp Wall Glin Asn Trp Glu 21 O 215 22O
Thir Thir Gly Arg Cys Pro Asp Wall Glu Wall Ser Arg Asn Asp Glin 225 23 O 235 24 O
Lell Ser Trp Luell Trp Ala Glu Ser Thir Ala Luell Phe Pro Ser Wall Tyr 245 250 255
Lell Glu Glu Thir Lell Ala Ser Ser Thir His Gly Arg Asn Phe Wall Ser 26 O 265 27 O
Phe Arg Wall Glin Glu Ala Lell Arg Wall Ala Asp Wall His His Ala Asn 285
His Ala Luell Pro Wall Wall Phe Thir Arg Pro Thir Tyr Ser Arg Gly 29 O 295 3 OO
Lell Thir Gly Luell Ser Glu Met Asp Luell Ile Ser Thir Ile Gly Glu Ser 3. OS 310 315 32O US 9,084,743 B2 175 176 - Continued
Ala Ala Luell Gly Ala Ala Gly Wall Ile Luell Trp Gly Asp Ala Gly Phe 3.25 330 335
Thir Thir Ser Asn Glu Thir Arg Arg Luell Lys Asp Tyr Luell Thr Arg 34 O 345 35. O
Ser Luell Wall Pro Tyr Wall Wall Asn Wall Ser Trp Ala Ala Glin Tyr Cys 355 360 365
Ser Trp Ala Glin His Gly His Gly Arg Cys Wall Arg Arg Asp Pro 37 O 375
Asn Ala His Thir Phe Lell His Luell Ser Ala Ser Ser Phe Arg Lieu Wall 385 390 395 4 OO
Pro Ser His Ala Pro Asp Glu Pro Arg Luell Arg Pro Glu Gly Glu Lieu. 4 OS 415
Ser Trp Ala Asp Arg Asn His Luell Glin Thir His Phe Arg Cys Gln Cys 425 43 O
Luell Gly Trp Gly Gly Glu Glin Glin Trp Asp Arg Arg Arg Ala 435 44 O 445
Ala Gly Gly Ala Ser Gly Ala Trp Ala Gly Ser His Lell Thir Gly Lieu. 450 45.5 460
Lell Ala Wall Ala Wall Lell Ala Phe Thir Trp Thir Ser 465 470 47s
SEO ID NO 27 LENGTH: 114 TYPE : PRT ORGANISM: Ovis airies FEATURE: OTHER INFORMATION: PH2O partial sequence
< 4 OOs SEQUENCE: 27
Lieu. Tyr Val Arg Asn Arg Wall Arg Glu Ala Ile Arg Lell Ser Lys Ile 1. 5 15
Ala Ser Wall Glu Ser Pro Lell Pro Wall Phe Wall His Arg Pro Wall 25
Phe Thir Asp Gly Ser Ser Thir Tyr Luell Ser Glin Gly Asp Luell Wall Asn 35 4 O 45
Ser Wall Gly Glu Ile Wall Ala Luell Gly Ala Ser Gly Ile Ile Met Trp SO 55 6 O
Gly Ser Luell Asn Lell Ser Lell Thir Met Glin Ser Met Asn Lieu. Gly 65 70 8O
Asn Tyr Luell Asn Thir Thir Lell Asn Pro Tyr Ile Ile Asn Wall Thir Lieu. 85 90 95
Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Val Cys 1OO 105 11 O
Ile Arg
SEQ ID NO 28 LENGTH: 414 TYPE : PRT ORGANISM: Pongo pygmaeus FEATURE: OTHER INFORMATION: hyaluronidase 3
<4 OOs, SEQUENCE: 28 Met Thir Thr Arg Lieu. Gly Pro Ala Lieu Val Lieu. Gly Val Ala Lieu. Cys 1. 5 15
Lieu. Gly Cys Gly Glin Pro Leu Pro Glin Val Pro Glu Arg Pro Phe Ser 25 US 9,084,743 B2 177 178 - Continued
Wall Luell Trp Asn Wall Pro Ser Ala His Cys Ser Arg Phe Gly Wall 35 4 O 45
His Luell Pro Luell Asn Ala Lell Gly Ile Ile Ala Asn Arg Gly Glin His SO 55 6 O
Phe His Gly Glin Asn Met Thir Ile Phe Lys Asn Glin Luell Gly Luell 65 70
Pro Phe Gly Pro Gly Thir Ala His Asn Gly Gly Ile Pro 85 90 95
Glin Ala Luell Pro Lell Asp Arg His Luell Ala Luell Ala Ala Tyr Glin Ile 105 11 O
His His Ser Luell Arg Pro Gly Phe Ala Gly Pro Ala Wall Luell Asp Trp 115 12 O 125
Glu Glu Trp Pro Lell Trp Ala Gly Asn Trp Gly Arg Arg Arg Ala 13 O 135 14 O
Tyr Glin Ala Ala Ser Trp Ala Trp Ala Glin Glin Wall Phe Pro Asp Luell 145 150 155 160
Asp Pro Glin Glu Glin Lell Ala Tyr Thir Gly Phe Glu Glin Ala 1.65 17O 17s
Ala Arg Ala Luell Met Glu Asp Thir Luell Arg Wall Ala Glin Ala Luell Arg 18O 185 19 O
Pro His Gly Luell Trp Gly Phe Tyr His Pro Ala Cys Gly Asn Gly 195
Trp His Ser Met Ala Ser Asn Thir Gly Arg Cys His Ala Ala Thir 21 O 215 22O
Lieu Ala Arg Asn Thr Lieu His Trp Lieu Trp Ala Ala Ser Ser Ala 225 23 O 235 24 O
Lell Phe Pro Ser Ile Lell Pro Pro Arg Luell Pro Pro Ala His His 245 250 255
Glin Ala Phe Wall Arg His Arg Luell Glu Glu Ala Phe Arg Wall Ala Luell 26 O 265 27 O
Wall Gly His Luell Pro Wall Lell Ala Wall Arg Lell Thir His Arg Arg 27s 285
Ser Gly Arg Phe Lell Ser Glin Asp Asp Luell Wall Glin Thir Ile Gly Wall 29 O 295 3 OO
Ser Ala Ala Luell Gly Ala Ala Gly Wall Wall Luell Trp Gly Asp Luell Ser 3. OS 310 315
Lell Ser Ser Ser Glu Glu Glu Trp His Luell His Asp Luell Wall 3.25 330 335
Asp Thir Luell Gly Pro Gly Ile Asn Wall Thir Arg Ala Ala Met Ala 34 O 345 35. O
Ser His Glin Arg His Gly His Gly Arg Ala Arg Arg Asp 355 360 365
Pro Gly Glin Met Glu Ala Phe Luell His Luell Trp Pro Asp Gly Ser Luell 37 O 375 38O
Gly Asp Trp Ser Phe Ser His Tyr Trp Gly Trp Ala Gly 385 390 395 4 OO
Pro Thir Glin Glu Pro Arg Luell Gly Pro Glu Ala Wall 4 OS 41O
SEQ ID NO 29 LENGTH: 510 TYPE : PRT ORGANISM: Macaca fascicularis FEATURE: OTHER INFORMATION: PH2O