IMPROVED METHODS FOR ENHANCING ANTIBODY PRODUCTIVITY IN MAMMALIAN CELL CULTURE AND MINIMIZING AGGREGATION DURING DOWNSTREAM, FORMULATION PROCESSES AND STABLE ANTIBODY FORMULATIONS OBTAINED THEREOF

• US 20200131251A1
• Filed: 12/20/2017
• Published: 04/30/2020
• Est. Priority Date: 12/23/2016
• Status: Active Application

• Current Assignee: Serum Institute of India Pvt. Ltd. (Poonawalla Group of Cos.)
• Original Assignee: Serum Institute of India Pvt. Ltd. (Poonawalla Group of Cos.)
• Inventors: MHALASAKANT, Dhere Rajeev, SHANKAR, Pisal Sambhaji, REDDY, Peddi Reddy Srinivas, CHAHAR, Singh Digamber, RAVINDRA, Yeolekar Leena, SINGH, Chouhan Pankaj, DATTATRAY, Avalaskar Nikhil
• Application Number:
  US16/472,673
• Publication Number:
  US 20200131251A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 39/39525 Purification
• CPC Class Codes: A61K 39/39591 Stabilisation, fragmentation
• CPC Class Codes: A61K 9/19 lyophilised , i.e. freeze-dried, solutions or dispersions lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 16/1003 Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
• CPC Class Codes: C07K 16/1081 Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention describes an efficient platform for antibody manufacturing and formulation that provides i) cell culture process with improved feeding strategy resulting in high antibody titer between 2 gm/L to 5 gm/L; ii) improved purification process showing optimal percentage recovery, high purity monomer content, minimum aggregation/particulate formation, minimum impurity levels; and iii) high concentration stable liquid formulation with optimal osmolality and low viscosity across different temperature excursions and devoid of aggregation. The preferred antibodies include IgG1 monoclonal antibody specific to the Dengue virus epitope in domain III of the E protein and IgG1 monoclonal antibody specific to the rabies virus surface G glycoprotein.

Claims

1. A method of manufacturing a pharmaceutical antigen binding protein with high yield and minimum aggregation, comprising:

a) culturing large scale mammalian cells that express antigen binding protein in a cell culture production medium, wherein the method effectively maintains the cell count and results in a yield of least 2 gm/L;

b) purification of antigen binding protein from harvested supernatant obtained in step (a), wherein the method results in recovery of at least 80% and purity of at last 99%; and

c) obtaining a stable antigen binding protein formulation, wherein Osmolality of the stable formulation is in a range of 300-400 mOsm/Kg and viscosity of the stable formulation is less than 2.5 mPa-S.

2. The method of claim 1 comprising culturing large scale mammalian cells that express antibody in a cell culture production medium;

wherein the culturing step includes use of a basal medium, use of a concentrated basal medium as a feed solution, and use of feed solutions along-with a definite feeding strategy, resulting in enhanced cell growth, maintaining lower concentrations of lactate and ammonia, and effectively maintaining the cell count thereby increasing cell longevity and high yield.

3. The method of claim 2, wherein the cell culture production medium comprises of at least one medium selected from the group comprising Cell Vento 220 (Merck), ACTIPRO (HyClone/GE), and Gibco™

Dynamism™

Medium (Thermo Fisher).

4. The method of claim 1, wherein the cell culture production medium is supplemented with one or more other nutrients, at least once during the method.

5. The method of claim 1, wherein the cell culture production medium is supplemented on a schedule comprising supplementation that is continuous, daily, every other day, every two days, or any combination thereof.

6. The method of claim 4, wherein the cell culture production medium is supplemented with a feed solution comprising at least one medium selected from the group comprising Glucose, Cell Boost™

5 Supplement (Hyclone), EX-CELL 293 (Sigma Aldrich), Cell Boost 7a and 7b supplements (Hyclone), 3×

Actipro medium, Cell Vento 220 (3×

medium), EX-CELLO Advanced™

CHO Feed 1, EfficientFeed™

A, EfficientFeed™

B, and EfficientFeed™

C, and any combination thereof.

7. The method of claim 1;

wherein the cell count is in the range of 10×

10⁶-20×

10⁶ cells/ml.

8. The method of claim 1, wherein the cell culture production medium has an Osmolality in a range of 250-500 mOsm/Kg;

a pH in a range of 6.5-7.5;

dissolved oxygen is maintained in a range of 10-60%;

Cell culture temperature is in a range of 30% C to 38°

C.;

Glucose concentration is maintained below 7%;

harvesting the cell culture when viability is decreased to 80%;

wherein cell culture conditions are maintained in a manner such that Lactate concentration is not more than 5 g/L; and

Ammonia concentration is not more than 5 mMol/L.

11. The method of claim 1, wherein the antigen binding protein is selected from the group comprising of humanized antibody, chimeric antibody, human antibody, bispecific antibody, multivalent antibody, multi-specific antibody, antigen binding protein fragments, polyclonal, monoclonal, diabodies, nanobodies, monovalent, bispecific, heteroconjugate, multispecific, autoantibodies, single chain antibodies, Fab fragments, F(ab)′

2, fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments and combinations thereof.

12. The method of claim 1, wherein the cell culture Production medium comprises a cell line selected from the group consisting of Chinese Hamster Ovary (CHO) cells, GS-CHO, CHOK1SV GS-KO, CHO DUX-B11, CHO-K1, BSC-1, NSO myeloma cells, CV-1 in Origin carrying SV40 (COS) cells, COS-1, COS-7, P3X3Ag8.653, SP2 cells, human embryonic kidney (HEK 293) cells, baby hamster kidney (BHK 21) cells, African green monkey kidney VERO-76 cells, HELA cells, human lung cells (W138), Retinal cells, and human hepatoma line (Hep G2). VERO, BHK, MDCK, W138 cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, HsS78Bst cells, PER.C6, SP2/0-Ag14, a myeloma cell line, a hybridoma cell line, human lung cells (W138), Retinal cells, human hepatoma line (Hep G2), CHO-K1 (ATCC CCL-61), DGI 44, SH87 cellCHO-DXB11, CHO designated ECACC 85050302, CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), CHOK1sv, CHO-S, dihydrofolate reductase negative CHO cells (CHO/−

DHFR), and dp12.CHO cells;

monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651);

human embryonic kidney cells;

baby hamster kidney cells (BHK, ATCC CCL-10);

CAP cell, AGE1.HN cell, monkey kidney cells (CV 1, ATCC CCL-70);

African green monkey kidney cells (VERO-76, ATCC CRL-1587;

VERO, ATCC CCL-81);

mouse sertoli cells (TM4);

human cervical carcinoma cells (HELA, ATCC CCL-2);

canine kidney cells (MDCK, ATCC CCL-34);

human lung cells (W138, ATCC CCL-75);

human hepatoma cells (HEP-G2, HB

     8065);

mouse mammary tumor cells (MMT 060562, ATCC CCL-51);

buffalo rat liver cells (BRL 3A, ATCC CRL-1442);

TR1 cells;

MCR 5 cells; and

FS4 cells and hybridoma cells.

13. The method of claim 12, wherein the cell line is CHO-K1 SV GS-KO.

14. The method of claim 12, wherein the cell line is GS-CHO.

15. The method of claim 1, the cells are cultivated in a batch, fed batch-, continuous mode, or perfusion mod.

16. The method of claim 1, wherein the salt concentration of the buffers used in purification is in the range of 30 mM-500 mM.

18. The method of claim 1, wherein the purification step comprises one or more of Protein A affinity chromatography, cation exchange chromatography, and anion exchange chromatography.

19. The method of claim 1, wherein the purification step comprises affinity chromatography, Low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, and Tangential flow filtration/Ultrafiltration in a sequential manner.

20. The method of claim 1, wherein the purification step comprises affinity chromatography, Low pH viral inactivation, anion exchange chromatography, cation exchange chromatography, nanofiltration, and Tangential flow filtration/Ultrafiltration in a sequential manner.

21. The method of claim 1, wherein the purification step occurs in an affinity chromatography matrix selected from Protein A, Protein G and Protein L.

22. The method of claim 1, further comprising an additional chromatography step selected from the group comprising one or more of Hydrophobic interaction chromatography, Hydrophobic charge induction chromatography, Ceramic hydroxyapatite chromatography, Multimodal chromatography (Capto MMC and Capto Adhere), Membrane chromatography (Q membranes including Intercept™

(Millipore), Mustang®

(Pall Corporation) and Sartobind™

(Sartorius)).

23. The method of claim 21, wherein the protein A chromatography matrix comprises one or more resins selected from the group comprising Eshmuno A, KanCapA™

, MabSelect SuRe™

, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros®

MabCapture A, Amsphere™

Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus.

25. The method of claim 18, wherein viral inactivation of an eluate from the protein A affinity chromatography is accomplished by holding the eluate at a pH of 3.3-3.5 for a period of 50-100 minutes.

26. The method of claim 18, wherein the cation exchange chromatography is conducted using a resin selected from the group comprising one or more of sulfonate based group (e.g., MonoS, MiniS, Source 15S and 30S, SP SEPHAROSE®

Fast Flow, SP SEPHAROSE®

High Performance from GE Healthcare, TOYOPEARL®

SP-650S and SP-650M from Tosoh, MACRO-PREP®

High S from BioRad, Ceramic HyperD S, TRISACRYL®

M and LS SP and Spherodex LS SP from Pall Technologies);

a sulfoethyl based group (e.g., FRACTOGEL®

SE, from EMD, POROS®

S-10 and S-20 from Applied Biosystems);

a sulphopropyl based group (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, POROS®

HS-20, HS 50, and POROS®

XS from Life Technologies);

a sulfoisobutyl based group (e.g., FRACTOGEL®

EMD S03 “

from EMD);

a sulfoxy ethyl based group (e.g., SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g., CM SEPHAROSE®

Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., MACRO-PREP®

CM from BioRad, Ceramic HyperD CM, TRISACRYL®

M CM, TRISACRYL®

LS CM, from Pall Technologies, Matrex CELLUFINE®

C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL®

CM-650S, CM-650M and CM-650C from Tosoh);

sulfonic and carboxylic acid based groups (e.g., BAKERBOND®

Carboxy-Sulfon from J.T. Baker);

a carboxylic acid based group (e.g., WP CBX from J.T Baker, DOWEX®

MAC-3 from Dow Liquid Separations, AMBERLITE®

Weak Cation Exchangers, DOWEX®

Weak Cation Exchanger, and DIAION®

Weak Cation Exchangers from Sigma-Aldrich and FRACTOGEL®

EMD COO-from EMD);

a sulfonic acid based group (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX®

Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J.T. Baker, SARTOBIND®

S membrane from Sartorius, AMBERLITE®

Strong Cation Exchangers, DOWEX®

Strong Cation and DIAION®

Strong Cation Exchanger from Sigma-Aldrich); and

a orthophosphate based group (e.g., PI 1 from Whatman).

28. The method of claim 18, the anion exchange chromatography is conducted using a resin selected from the group comprising one or more of DEAE cellulose, POROS®

PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND®

Q from Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE®

Fast Flow, Q SEPHAROSE (GE), Q SEPHAROSE®

High Performance, QAE SEPHADEX® and

FAST Q SEPHAROSE®

(GE Healthcare), WP PEI, WP DEAM, WP QUAT from J.T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., U Osphere Q, MACRO-PREP DEAE and MACRO-PREP®

High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL®

M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL®

LS, QMA SPHEROSIL®

M and MUSTANG®

Q from Pall Technologies, DOWEX®

Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX®

MONOSPHER E 77, weak base anion from Dow Liquid Separations, INTERCEPT®

Q membrane, Matrex CELLUFINE®

A200, A500, Q500, and Q800, from Millipore, FRACTOGEL®

EMD TMAE, FRACTOGEL®

EMD DEAE and FRACTOGEL®

EMD DMAE from EMD, AMBERLITE®

weak strong anion exchangers type I and II, DOWEX®

weak and strong anion exchangers type I and II, DIAION®

weak and strong anion exchangers type I and II, DUOLITE®

from Sigma-Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL®

SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q from Whatman.

30. The method of claim 18, wherein the anion exchange chromatography is in “

flow through and wash mode”

or “

bind and elute mode.”

31. The method of claim 18, wherein removal of viral particles is accomplished by nanofiltration using a virus retentive filter selected from the group comprising one or more of Viresolve PRO (Merck), Planova 20N (Asahi Kasei), Bio EXL PALL PEGASUS PRIME, PEGASUS SV4 (Pall Life Sciences), and Virosart (Sartorius), Virosart CPV filter from Sartorius, Virosolve from Millipore, Ultipor DV20 or DV50 from Pall, Planova 20N and 50N, and BioEx from Asahi.

32. The method of claim 18, wherein the antigen binding protein is further concentrated using Tangential Flow Filtration (TFF).

33. The method of claim 32, wherein the TFF is carried out using a 30 kDa membrane selected from the group comprising one or more of Centramate T series PES membrane (Pall Corporation), Hydrosart (Sartorius), and Pelicon 3 (Merck).

35. The method of claim 1, wherein the stable antigen binding protein formulation contains no greater than 2% aggregates.

36. The method of claim 1, wherein the stable antigen binding protein formulation comprises of least one antigen binding protein, at least one Stabilizer, one Buffer, at least one Tonicity agent, and at least one surfactant.

50. The method of claim 1, wherein the stable antigen binding protein formulation comprises about 1 mg/ml to about 100 mg/ml of the antigen binding protein.

53. The method of claim 1, wherein the concentration of the antigen binding protein monomer in the stable antigen binding protein formulation is greater than 99%;

residual CHO DNA is not more than 2 pg/mg of antigen binding protein, more particularly not more than 0.1 pg/mg of antigen binding protein;

residual CHO protein is not more than 100 ng/mg of antigen binding protein, more particularly not more than 10 ng/mg of antigen binding protein;

residual Protein-A is not more than 10 ng/mg of antigen binding protein, more particularly not more than 1.5 ng/mg of antigen binding protein;

Endotoxin is not more than 0.1 EU/mg of antigen binding protein;

viral clearance LRV for MuLV is atleast at least 20 fold 15 log 10, reduction factor and for MMV is atleast at least 12 log 10, reduction factor 10 fold.

54. A pharmaceutical formulation prepared according to the method of claim 1.

55. The pharmaceutical formulation of claim 54 comprising an antigen binding protein, a buffering agent, a tonicity agent, a surfactant and a stabilizing agent.

9-10. -10. (canceled)

17. (canceled)

24. (canceled)

27. (canceled)

29. (canceled)

34. (canceled)

37-49. -49. (canceled)

51-52. -52. (canceled)

56. (canceled)

57-68. -68. (canceled)

69. A pharmaceutical formulation comprising

a. 1-100 mg/ml of at least one antigen binding protein;

b. 20-40 mM of Histidine;

c. 50-100 mM of Arginine;

d. 0.002-0.02% of Polysorbate 80 (w/v);

e. 50-150 mM NaCl; and

f. not more than 2.5% Sucrose w/v;

wherein pH of the formulation is 6.5±

0.5;

wherein the Osmolality of the formulation is 300-450 mOsmol/kg and viscosity is less than 2.5 mPa-S and said formulation is stable at 2-8 deg C. for at least 9 months, at 25 deg C. for at least 1 month, at 40 deg C. for at least 40 days, and at 50 deg C. for at least 2 days.

70. The pharmaceutical formulation of claim 69, wherein the formulation comprises 2-80 mg/ml of a least one antigen binding protein;

25 mM of Histidine;

75 mM of Arginine;

101 mM NaCl;

0.02% Polysorbate 80 (w/v); and

0.5% Sucrose w/v;

wherein pH of the formulation is 6.5±

0.5, and wherein the osmolality of the formulation is about 380 mOsmol/kg.

75. The pharmaceutical formulation according to claim 69, wherein said antigen binding protein is a monoclonal antibody that binds to a dengue virus.

76. The pharmaceutical formulation according to claim 69, wherein said antigen binding protein is a monoclonal antibody that binds to a rabies virus.

77. The pharmaceutical formulation according to claim 70, wherein said antigen binding protein is a Dengue monoclonal antibody.

78. The pharmaceutical formulation according to claim 77, comprising 25 mg/ml of the Dengue monoclonal antibody.

79. The pharmaceutical formulation according to claim 77, comprising 50 mg/ml of the Dengue monoclonal antibody.

80. The pharmaceutical formulation according to claim 70, wherein said antigen binding protein is a Rabies monoclonal antibody.

81. The pharmaceutical formulation according to claim 80, comprising 25 mg/ml of the Rabies monoclonal antibody.

82. The pharmaceutical formulation according to claim 80, comprising 50 mg/ml of the Rabies monoclonal antibody.

83. The pharmaceutical formulation according to claim 70, wherein the formulation is a liquid formulation.

84. The pharmaceutical formulation according to claim 70, wherein the formulation is a lyophilized formulation.

87. The pharmaceutical formulation according to claim 70, wherein said antigen binding protein is an anti-dengue antibody or an anti-rabies antibody that can be administered alone or in combination with other agents, other prophylactics or therapeutic modalities.

71-74. -74. (canceled)

85-86. -86. (canceled)

88-89. -89. (canceled)