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(12) Patent Application Publication (10) Pub. No.: US 2007/0037192 A1 Ziomek Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2007/0037192 A1 Ziomek Et Al US 20070037192A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0037192 A1 Ziomek et al. (43) Pub. Date: Feb. 15, 2007 (54) METHOD OF PURIFYING RECOMBINANT Related U.S. Application Data HUMAN ANTITHROMBIN TO ENHANCE THE VIRAL AND PRON SAFETY PROFILE (60) Provisional application No. 60/702,194, filed on Jul. 25, 2005. (75) Inventors: Carol A. Ziomek, Milford, MA (US); Christopher Hendry, Medway, MA Publication Classification (US) (51) Int. Cl. Correspondence Address: CI2O I/68 (2006.01) WOLF GREENFIELD & SACKS, PC CI2P 2/06 (2006.01) FEDERAL RESERVE PLAZA (52) U.S. Cl. ............................................... 435/6; 435/69.1 6OO ATLANTIC AVENUE BOSTON, MA 02210-2206 (US) (57) ABSTRACT (73) Assignee: GTC Biotherapeutics, Inc., Framing- Y ham, MA Methods of purifying antithrombin from a variety of source materials including from the milk of transgenic mammals to (21) Appl. No.: 11/493,124 enhance its safety profile vis-a-vis the removal and/or the inactivation of contaminants. Contaminants would include (22) Filed: Jul. 25, 2006 particulate matter, viruses, and/or prions. FLOWCHART OF AN EMBODIMENT OF THE CURRENT INVENTION Initial transfection of mammalian cell line with transgene of interest for the bifunctional protein Selection ir cell-lines Nuclear Transfer & Embryo Transfer procedure or Microinjection Procedures* Birth of hemises animal(s) Characterization of hemises transgenic animal(s) Biopsy of transgenic animal tO generate cell population Expansion of biopsied beingu cell-line in culture Selection of homozygous cells with inised concentration of selective agents Pick surviving cell colonies Characterizing surviving els (FISH, Southern blot) Using homozygous cell ins in Embryo Transfer Production of a homoguin for desired transgene Accelerated production of herd hogs for desired transgene(s) Production of desired biopharmaceutical/Production of genetically desirable livestock or non-human mammals *NT Procedures would include earlier step of transfection of cell lines, cell line expansion and selection. *Microinjection Procedure would include microinjection of appropriate Zygote Patent Application Publication Feb. 15, 2007 Sheet 1 of 19 US 2007/0037192 A1 FLOWCHART OF AN EMBODIMENT OF THE CURRENT INVENTION Initial transfection of mammalian cell line with transgene of interest for the bifunctional protein Selection ir cell-lines Nuclear Transfer & Embryo Transfer procedure or Microinjection Procedures Birth of hemicou animal(s) Characterization of hemious transgenic animal(s) Biopsy of transgenic anim tO generate cell population Expansion of biopsied hiogu cell-line in culture Selection of homozygous cells with inised concentration of selective agents Pick surviving cell colonies Characterizing Surviving els (FISH, Southern blot) Using homozygous cell line in Embryo Transfer Production of a homoguin for desired transgene Accelerated production of herd hiogu for desired transgene(s) l Production of desired biopharmaceutical/Production of genetically desirable livestock or non-human mammals *NT Procedures would include earlier step of transfection of cell lines, cell line expansion and selection. * Microinjection Procedure would include microinjection of appropriate Zygote Figure 1 Patent Application Publication Feb. 15, 2007 Sheet 2 of 19 US 2007/0037192 A1 Figure 2 Antithrombin Arg393-Ser 394 Cys 247-430 Patent Application Publication Feb. 15, 2007 Sheet 3 of 19 US 2007/0037192 A1 Figure 3 ATVector Construction Goat beta Casein AT cDNA Patent Application Publication Feb. 15, 2007 Sheet 4 of 19 US 2007/0037192 A1 Figure 4 Making a haT Transgenic Goat Select 8 mate founder Goat beta casein induce DNA isolate lactation fertilized Transfer into recipient female "Milk rhAT Microinject s production herd A Wector Measure into embryo AT hAT . r expression cDNA hat expression Test offspring Wector for transgene ATIk Patent Application Publication Feb. 15, 2007 Sheet 5 of 19 US 2007/0037192 A1 Figure 5 rhAT Purification Process a 5. Pall DV-20ill Anion Hydrophobic 80C Heparin Viral exchange interaction 300gm for column filter Column Column 55% Yield 72 hr. Fill/lyo 6 Caseinmiceles NA8Lactose >99.9% pureby Mineral Sats Milk proteins Milk proteins contaminating Bacteria Witars protein JSA Hortones Wiruses Milk proteins Patent Application Publication Feb. 15, 2007 Sheet 6 of 19 US 2007/0037192 A1 Figure 6 Purity Profile for AT Process & Final Product Silver stained gel ShifS27S. S. Styluss 8 s 1 2 3 4 5 --- 4. 1 2 3 4 MW Milk Heparin ANX HIC MW rhAT rhAT hpAT MW rhAT rhAT hpAT markers etuate eluate eluate markers standard markers standard Patent Application Publication Feb. 15, 2007 Sheet 7 of 19 US 2007/0037192 A1 Figure 7: Chromatograms (A280 nm) showing the Methyl HyperD elution profiles of goat and human AT. Stack races Patent Application Publication Feb. 15, 2007 Sheet 8 of 19 US 2007/0037192 A1 Figure 8 Methionine Oxidation in Thrombate, Kybernin and rhAT KYXSox A Thrombate C Kybernin Recombinant ATIII O 5 Retention Time (min Patent Application Publication Feb. 15, 2007 Sheet 9 of 19 US 2007/0037192 A1 Figure 9 Non-Reducing Peptide Map of Thrombate and rhaT Thrombate A T C y s 8 - 1 2 8 C y s 21 - 9 5 C y s 247 - 43 0 A b s O r b a Jillt RhAT : A e C w is 8 - 1 28 C y s 247 - 4 3 O 2 y s - C y s 21 - 9 5 1 5 n l al --------------------------|- 2 O 4 O 6 O 8 O O O r im e ( m in ) ifssos Patent Application Publication Feb. 15, 2007 Sheet 10 of 19 US 2007/0037192 A1 Figures 10a & 10b Heparin Affinity Before (Left) and After (Right) Glycosidase Treatment 10a 10b 60 S00 - hpATIII w tgATIII 40 300 200 100 8 O 2 4. 6 Retention Time (min) SOC '' - hpATIII tgATIII 300 200 00 8 O 2 4. 6 Retention Time (min) Patent Application Publication Feb. 15, 2007 Sheet 11 of 19 US 2007/0037192 A1 Figure 11 rhAT III Immunogenicity Analysis • Primary Screening Assay - Patient Immune Response ELISA - Qualitative, not quantitative assay - Background threshold established by evaluation of 200 normal human sera, 98% of the population has OD below 0.10 - Any patient serum sample reading over 0.10 was above the normal range and screened with confirmatory assays • Confirmatory Assay - Western Blot Assay - Any patient serum sample giving a band within the size range of AT III would be considered positive • Confirmatory Assay - Immunoprecipitation Patent Application Publication Feb. 15, 2007 Sheet 12 of 19 US 2007/0037192 A1 Figure 12 Antithrombin's Role in Modulation of Coagulation (Roemisch et al, 2002) Modulation of coagulation Enant-ages- V -- 7 S ---------------------st verseese Patent Application Publication Feb. 15, 2007 Sheet 13 of 19 US 2007/0037192 A1 Figure 13 Anti-inflammatory Properties of AT (Roemisch et al 2002) ---. s Anti-inflammatory4. properties of AT ill. sa s - NaA testaursSco -- Sassisters/ -verera-a-we-armammar cogisticasurings -is - it...let SY-...-a, theliumvirast - rs k M M k . Patent Application Publication Feb. 15, 2007 Sheet 14 of 19 US 2007/0037192 A1 Figure 14 Pharmacodynamics of rhaT in Rats with Klebsiella pneumoniae Induced Sepsis. (Dickneite et al., THROMB HAEMOST. 1993 Feb 1;69(2):98-102.) Kaplan-Meier Estimates (Irrespective of Dose) t hp(n=40) AT III 0.8 a -0 - gAT III l (n=40) Elapsed Time (Days) Patent Application Publication Feb. 15, 2007 Sheet 15 of 19 US 2007/0037192 A1 Figure 15 Transgenic a MF Microfiltration Mik Men brane Diafiltration 2 Ultrafiltration UF Menbane 3 Aseptic Filtration Clarified Bulk Concentrate Patent Application Publication Feb. 15, 2007 Sheet 16 of 19 US 2007/0037192 A1 Figure 16 The equipment was assembled as follows: Figure 16a (Microfiltration Unit): Path 'A' Sample Point Sample Point Path 'B' eB Figure 16b (Dual TFF System): Shaded arrows represent sample points. Patent Application Publication Feb. 15, 2007 Sheet 17 of 19 US 2007/0037192 A1 Figure 17 UFPerneate Aseptic Filter Clarified Bulk GTC Process Equipment Schematic. Concentrate Peristaltic Pamp Patent Application Publication Feb. 15, 2007 Sheet 18 of 19 US 2007/0037192 A1 Figure 18 TFF Donal Process Nass Balance Starting Feed r 82.34% Yied WF Retentate Patent Application Publication Feb. 15, 2007 Sheet 19 of 19 US 2007/0037192 A1 Figure 19 ATryn?& Manufacturing Process Flow Diagram Sterilized, Type I. 20 mm, rubber closures (siliconized) sings with Nitrogen EP monograph 3.2.9 blanket Overlay Container Coding 0.22 um Filters Pre and Post integrity Tested Drug Substance Surge Wessel Coutiner Ery eat Oven waseptic connection Capping SC for his sters issi: Hold 3 2-8°C Target Fili: 0.5 mL per via Freezing: Shelf Temp -35°C, Depyrogenated. Type I, (10.- 0.8 ml) ld S.5s Crimped 20 mm 20 cc glass vials with 1 Drying: Ramp 45°C, Shelf aluminum bridge Storage 2 to 8°C 20 mm opening. Temp-20°C, Hold 17 Hrs; seals and plastic Complies with EP Ramp 15°Ch. Shelf Temp 0°C, flip-off caps Monograph 3.2.1 hold 5hrs; Wacuum CO mTorr. 2 Drying: Ramp 30°Ch. Shelf Temp 25°C, Hold 6hrs; vacuuin 00 mTorr, GD -> In process Testing US 2007/0037192 A1 Feb. 15, 2007 METHOD OF PURIFYING RECOMBINANT acquired AT deficiencies, including prophylaxis for preven HUMAN ANTITHROMBIN TO ENHANCE THE tion of DIC in hereditary deficient (HD) patients in high risk VIRAL AND PRON SAFETY PROFILE situations such as Surgery or delivery. Additionally, rh AT may prove beneficial for various acquired AT deficiency FIELD OF THE INVENTION states including but not limited to: DIC, burns, heparin 0001. The present invention relates to the transgenic resistance, neurocognitive deficit due to CABG Surgery and production of recombinant human proteins which are bio SepS1S. logically active and can be used to treat hereditary and 0008. In one aspect the invention provides a method for acquired AT deficiencies and associated pathologies.
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