Galactose-alpha-1, 3-galactose-containing n-glycans in glycoprotein products derived from CHO cells

• US 10,023,894 B2
• Filed: 08/21/2015
• Issued: 07/17/2018
• Est. Priority Date: 01/22/2009
• Status: Active Grant

• Current Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Original Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Inventors: Bosques, Carlos J., Murphy, Jennifer, Sarvaiya, Hetal, Washburn, Nathaniel, Liu, Cuihua, Xu, Xiao-Jin
• Primary Examiner(s): Gabel, Gailene
• Application Number:
  US14/832,784
• Publication Number:
  US 20160053292A1
• Time in Patent Office: 1,061 Days
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: G01N 2400/02 involving antibodies to sugar part of glycoproteins
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

Methods of producing recombinant therapeutic fusion proteins described herein can include culturing Chinese hamster ovary (CHO) cells under conditions suitable for expression of a recombinant therapeutic fusion protein including one or more glycans, where the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans; treating the one or more glycans of the recombinant glycoprotein with one or more exoglycosidases; measuring glycans containing terminal galactose-alpha-1-3-galactose residues on the fusion protein by nuclear magnetic resonance (NMR); and producing a recombinant therapeutic fusion protein in the CHO cells if a target level of terminal galactose-alpha-1-3-galactose residues is measured.

Claims

1. A method of producing a recombinant therapeutic fusion protein, comprising the steps of:

(a) culturing Chinese hamster ovary (CHO) cells under conditions suitable for expression of a recombinant therapeutic fusion protein comprising one or more glycans, wherein the CHO cells have not been genetically engineered to produce terminal alpha-galactosyl residues on glycans;

(b) treating the one or more glycans of the fusion protein with one or more exoglycosidases;

(c) measuring glycans containing terminal galactose-alpha-1-3-galactose residues on the fusion protein by nuclear magnetic resonance (NMR); and

(d) producing a recombinant therapeutic fusion protein in the CHO cells if a target level of terminal galactose-alpha-1-3-galactose residues is measured.

2. The method of claim 1, wherein the recombinant therapeutic fusion protein is abatacept.

3. The method of claim 1, wherein the CHO cells are in a cell culture.

4. The method of claim 1, wherein the measuring step is performed on any of:

the recombinant therapeutic fusion protein produced by the CHO cells, peptides obtained from the recombinant therapeutic fusion protein produced by the CHO cells, cell surface glycans of the CHO cells, glycan preparations obtained from the CHO cells, glycan preparations obtained from the recombinant therapeutic fusion protein produced by the CHO cells, and combinations thereof.

5. The method of claim 1, wherein the producing step further comprises isolating the recombinant therapeutic fusion protein from the CHO cells.

6. The method of claim 1, wherein the culturing step occurs in a bioreactor.

7. The method of claim 1, wherein the measuring step further comprises use of a technique selected from the group consisting of:

chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, and combinations thereof.

8. The method of claim 1, wherein the recombinant therapeutic fusion protein is a human recombinant therapeutic fusion protein and in step (d) the CHO cells have been transformed with a vector encoding the human recombinant therapeutic fusion protein.

9. The method of claim 1, wherein the CHO cells are from at least one of:

at least two different CHO strains, at least two different clonal cell populations, and at least two different samples from a cell culture in a manufacturing process strain for a fusion protein.

10. The method of claim 1, wherein the CHO cells are from a first CHO cell population and a second CHO cell population and the method further comprises comparing a level of glycans containing terminal galactose-alpha-1-3-galactose residues produced by the first CHO cell population with a level of glycans containing terminal galactose-alpha-1-3-galactose residues produced by the second CHO cell population.

11. The method of claim 1, further comprising comparing a level of glycans containing terminal galactose-alpha-1-3-galactose residues produced by the CHO cells to a reference sample.

12. The method of claim 1, further comprising recording a level of glycans containing terminal galactose-alpha-1-3-galactose residues in a print or computer-readable medium.

13. The method of claim 1, wherein the target level is a quality criterion for a pharmaceutical preparation.

14. The method of claim 1, wherein the target level is a range or value in a product specification.

15. The method of claim 1, wherein the target level is no more than 5% terminal galactose-alpha-1-3-galactose residues.

16. The method of claim 1, wherein the one or more exoglycosidases are selected from the group consisting of sialidase, galactosidase, hexosaminidase, mannosidase, fucosidase, and combinations thereof.

17. The method of claim 1, wherein the treating step comprises treating with one or more exoglycosidases for a time and under conditions suitable for the one or more exoglycosidases to cleave one or more terminal glycosidic bonds from a non-reducing end of the one or more glycans.