Compositions and methods for producing a composition
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.
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 sialic acid to CTLA4-Ig molecules of at least about 8;
(b) an average molar ratio of N-acetyl neuraminic acid (NANA) to CTLA4-Ig molecules of from about 8 to about 12;
(c) an average molar ratio of N-glycolyl neuramininic acid (NGNA) to CTLA4-Ig molecules less than or equal to 1.5;
(d) an average molar ratio of N-Acetylgalactosamine (GalNAc) to CTLA4-Ig molecules from about 1.7 to about 3.6;
(e) an average molar ratio of N-Acetylglucosamine (GlcNAc) to CTLA4-Ig molecules from about 15 to about 35;
(f) an average molar ratio of galactose to CTLA4-Ig molecules from about 8.0 to about 17;
(g) an average molar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3;
(h) an average molar ratio of mannose to CTLA4-Ig molecules from about 7.7 to about 22;
and
(i) less than or equal to 5.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection, and
wherein the CTLA4-Ig molecules comprise one or more CTLA4-Ig polypeptides having the amino acid sequence set forth in SEQ ID NO: 2, 5, 6, 7, 8, 9, or 10.
2. The CTLA4-Ig composition of claim 1 , wherein the concentration of MCP-1 in the CTLA4-Ig composition is less than about 5 ppm.
3. The CTLA4-Ig composition of claim 1 , wherein the CTLA4-Ig composition comprises at least 95% of dimeric CTLA-Ig forms.
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 1 , wherein the CTLA4-Ig molecules are produced by a population of recombinant CHO cells.
10. The CTLA4-Ig composition of claim 9 , wherein at least 75% of the CHO cells comprise at least 30 copies of a CTLA4-Ig expression cassette comprising a nucleic acid sequence encoding a CTLA4-Ig polypeptide of SEQ ID NO:2.
11. The CTLA4-Ig composition of claim 10 , wherein the 30 or more copies are integrated at a single site in the genome of the CHO cell.
12. The CTLA4-Ig composition of claim 9 , wherein the CHO cells are clonal.