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 -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,

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