Carbohydrate content of CTLA4 molecules

Patent metadata

Field	Value
Publication number	US-10508144-B2
Application number	US-8678606-A
Country	US
Kind code	B2
Filing date	Dec 19, 2006
Priority date	Dec 20, 2005
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

The invention provides for mammalian cells capable of producing recombinant CTLA4-Ig and variants thereof. The invention also provides for compositions comprising CTLA4-Ig and formulations thereof. The invention further provides for methods for mass-producing CTLA4-Ig from mammalian cells capable of producing this recombinant protein, and for purifying the CTLA4-Ig.

First claim

What is claimed:

1. A composition comprising cytotoxic T lymphocyte antigen 4-Ig (CTLA4-Ig) molecules, wherein the CTLA4-Ig composition comprises
(a) an average molar ratio of N-acetyl neuraminic acid (NANA) to CTLA4-Ig molecules of from about 8 to about 12, and
(b) less than or equal to 5.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection,
wherein the CTLA4-Ig molecules are isolated from a soluble fraction of a liquid culture medium comprising an initial population of CTLA4-Ig molecules recombinantly produced by mammalian cells, wherein
(1) the CTLA4-Ig molecules of the initial population have one or more sialic acid residues;
(2) the number of sialic acid residues per CTLA4-Ig molecule varies within the initial population;
(3) the initial population comprises CTLA4-Ig dimers and high molecular weight aggregates;
(4) the liquid culture medium contains Monocyte Chemotactic Protein-1 (MCP-1); and,
(5) the CTLA4-Ig molecules comprise one or more polypeptides having the amino acid sequence set forth in SEQ ID NO: 2, 5, 6, 7, 8, 9, or 10,

wherein the CTLA4-Ig composition is obtainable by a method comprising:
(i) obtaining the soluble fraction of the liquid culture medium, wherein the liquid culture medium comprises mammalian cells that produce the initial population of CTLA4-Ig molecules; then
(ii) subjecting the soluble fraction of the liquid culture medium to affinity chromatography to reduce MCP-1 content; then
(iii) subjecting the composition of step (ii) to anion chromatography to separate the CTLA4-Ig molecules into two or more fractions, wherein at least one fraction has a greater molar ratio of sialic acid to CTLA4-Ig molecules compared to at least one other fraction; and then
(iv) subjecting the composition of step (iii) to hydrophobic interaction chromatography to separate CTLA4-Ig dimers from CTLA4-Ig high molecular weight aggregates.

2. The CTLA4-Ig composition of claim 1 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

3. The CTLA4-Ig composition of claim 1 , wherein the concentration of MCP-1 in the CTLA4-Ig composition after the method is less than about 5 ppm.

4. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises less than or equal to 4.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

5. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises less than or equal to 3.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

6. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises less than or equal to 2.5 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

7. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises less than or equal to 2.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

8. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10.

9. The CTLA4-Ig composition of claim 8 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

10. The CTLA4-Ig composition of claim 8 , wherein the concentration of MCP-1 in the CTLA4-Ig composition after the method is less than about 5 ppm.

11. The CTLA4-Ig composition of claim 8 , wherein the CTLA4-Ig composition comprises less than or equal to 4.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

12. The CTLA4-Ig composition of claim 8 , wherein the CTLA4-Ig composition comprises less than or equal to 3.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

13. The CTLA4-Ig composition of claim 8 , wherein the CTLA4-Ig composition comprises less than or equal to 2.5 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

14. The CTLA4-Ig composition of claim 8 , wherein the CTLA4-Ig composition comprises less than or equal to 2.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

Assignees

• Leister Kirk J
• Schaefer Eugene J
• Bates Ronald Charles
• Bramhall Elizabeth A
• Didio David Michael
• Donaldson Robert
• Flesher Alan R
• Haggerty Helen Gray
• Kirkley David Henry
• Tabor John Malcolm
• Tay Lee K
• Thammana Pallaiah
• Velayudhan Ajoy
• Smolin David Edward
• Russell Reb J
• Vanden Boom Thomas James
• Brownell Dean Woodrow
• Schrimsher Jeffrey
• Whitehead Joyce Patricia
• Bristol Myers Squibb Co

Inventors

• Leister Kirk J
• Schaefer Eugene J
• Bates Ronald Charles
• Bramhall Elizabeth A
• Didio David Michael
• Donaldson Robert
• Flesher Alan R
• Haggerty Helen Gray
• Kirkley David Henry
• Tabor John Malcolm
• Tay Lee K
• Thammana Pallaiah
• Velayudhan Ajoy
• Smolin David Edward
• Russell Reb J
• Vanden Boom Thomas James
• Brownell Dean Woodrow
• Schrimsher Jeffrey
• Whitehead Joyce Patricia

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P37/00

  Drugs for immunological or allergic disorders · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title