GAL ALPHA 1-3GAL-CONTAINING N-GLYCANS IN GLYCOPROTEIN PRODUCTS DERIVED FROM CHO CELLS

• US 20110045496A1
• Filed: 01/22/2009
• Published: 02/24/2011
• 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, Liu, Cuihua, Murphy, Jennifer, Xu, Xiao-Jin, Sarvaiya, Hetal, Washburn, Nathaniel
• Granted Patent: US 8,586,356 B2
• Time in Patent Office: Days
• US Class Current: 435/7.21
• 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 lectins from plants G01N2333/42, lectins from mammals G01N2333/4724
• 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

The present invention provides methods of evaluating CHO cells.

Claims

1. A method for evaluating a Chinese Hamster Ovary (CHO) cell population, the method comprising:

(a) providing one or more CHO cells from the population; and

(b) measuring glycans containing terminal galactose-alpha-1-3-galactose residues produced by said cells, wherein the CHO cells have not been genetically engineered to express an alpha-galactosyl transferase coding sequence.

2. The method of claim 1, wherein the measuring step comprises any of:

(a) isolating glycoproteins produced from the CHO cells and measuring the glycans containing terminal galactose-alpha-1-3-galactose residues on the glycoproteins,

(b) isolating a specific glycoprotein composition produced from the CHO cells and measuring the glycans containing terminal galactose-alpha-1-3-galactose residues from the isolated glycoprotein composition,

(c) obtaining a glycan preparation from a glycoprotein preparation or isolated glycoprotein produced from the CHO cells and measuring the glycans containing terminal galactose-alpha-1-3-galactose residues in the glycan preparation,

(d) cleaving monosaccharides from glycans present on a glycoprotein produced from the CHO cells or from glycans on the surface of the one or more CHO cells, and detecting the terminal released alpha-galactose residues from the cleaved monosaccharides,

(e) providing at least one peptide from a glycoprotein preparation produced from the CHO cells, and measuring the glycans containing terminal galactose-alpha-1-3-galactose residues on the at least one peptide, and

(f) measuring glycans containing terminal galactose-alpha-1-3-galactose residues from glycans on the cell surface of the one or more CHO cells.

3. The method of claim 1, wherein the CHO cell population is a clonal cell population.

4. The method of claim 1, further comprising a step of comparing the level measured in step (b) to a reference level or specification.

5. The method of claim 1, wherein measuring comprises use of a method for identifying or quantifying glycans containing terminal galactose-alpha-1-3-galactose residues selected from the group consisting of:

chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods, nuclear magnetic resonance (NMR) methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, use of a detection molecule, and combinations thereof.

6. The method of claim 1, wherein the CHO cell population is a sample from a cell culture in a bioreactor for manufacturing a therapeutic glycoprotein.

7. The method of claim 1, wherein the providing and measuring steps are repeated at least once over time.

8. The method of claim 1, wherein the measuring step comprises using a detection molecule that binds to galactose-alpha-1-3-galactose epitopes.

9. The method of claim 8, wherein the detection molecule comprises a lectin that is able to bind to galactose-alpha-1-3-galactose epitopes.

10. The method of claim 8, wherein the detection molecule comprises an antibody against galactose-alpha-1-3-galactose epitopes.

11. The method of claim 8, wherein the detection molecule comprises or is bound to a molecule comprising a labelled moiety.

12. The method of claim 1, further comprising a step of recording the result of the measuring step in a print or computer-readable medium.

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

14. The method of claim 1, wherein the CHO cells have been transformed with a vector encoding a human therapeutic glycoprotein.

15. The method of claim 1, further comprising the step of quantifying the amount of terminal galactose-alpha-1-3-galactose epitopes or glycans containing the epitopes present on the CHO cells.

16-25. -25. (canceled)

26. A method for screening one or more Chinese Hamster Ovary (CHO) cells for the ability to produce glycoproteins comprising glycans containing -alpha-1-3-galactose epitopes, the method comprising:

(a) providing a plurality of CHO cell populations wherein none of the plurality have been genetically engineered to produce terminal alpha-galactosyl residues on glycans;

(b) culturing each of the plurality of CHO cell populations under conditions suitable for expression of a glycoprotein expression product;

(c) measuring glycans containing terminal galactose-alpha-1-3-galactose residues produced by each of the plurality of CHO cells, and

(d) selecting one or more of the plurality of CHO cell preparations based on the presence of a target level of terminal galactose-alpha-1-3-galactose residues produced by the selected CHO cell preparation, wherein the CHO cells have not been transfected with an alpha-galactosyl transferase coding sequence.

27. The method of claim 26, wherein each of the plurality of CHO cell populations is in a cell culture.

28. The method of claim 26, wherein the measuring is performed on any of:

an isolated glycoprotein expression product of the CHO cell preparations, peptides obtained from a glycoprotein expression product of the CHO cell preparations, cell surface glycans of the CHO cell preparations, glycan preparations obtained from the CHO cell preparations or from a glycoprotein expression product thereof, and combinations thereof.

32. The method of claim 26, wherein the measuring step includes the step of (i) isolating a glycoprotein expression product from each of the plurality of CHO cell populations, and (ii) measuring the terminal galactose-alpha-1-3-galactose residues on the glycoprotein expression product.

33. The method of claim 27, wherein the cell culture is in a bioreactor.

34. The method of claim 26, wherein measuring comprises use of a technique for identifying or quantifying glycans containing terminal galactose-alpha-1-3-galactose residues selected from the group consisting of:

chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods, nuclear magnetic resonance (NMR) methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, use of a detection molecule, and combinations thereof.

35. The method of claim 26, wherein at least one the plurality of CHO cell populations have been transformed with a vector encoding a human therapeutic glycoprotein.

36. The method of claim 26, wherein the plurality of CHO cell populations comprises at least one characteristic selected from the group consisting of:

at least two different CHO strains, at least two different clonal cell populations, and at least two different samples from a manufacturing process train for a therapeutic glycoprotein.

37. The method of claim 26, further comprising the step of culturing the selected CHO cell preparation to produce a therapeutic glycoprotein product.

29-31. -31. (canceled)

38. A method for evaluating a glycoprotein composition produced in a CHO cell host, comprising:

measuring the amount of terminal galactose-alpha-1-3-galactose present in a glycoprotein composition, wherein the glycoprotein composition was produced in CHO host cells, and wherein the CHO host cells were not genetically engineered to express an alpha-galactosyl transferase coding sequence.

39. The method of claim 38, further comprising recording the level of terminal galactose alpha-1-3-galactose present in the glycoprotein composition in a print or computer-readable medium.

40. The method of claim 38, further comprising comparing the measured level of terminal galactose-alpha-1-3-galactose present in the glycoprotein composition with a reference level.

41. The method of claim 40, wherein the reference level is a specification or quality criterion for a pharmaceutical preparation containing the glycoprotein composition.

42. The method of claim 40, wherein the reference level is no more than 5% terminal galactose-alpha-1-3-galactose on a glycan/total glycan basis.

43. The method of claim 38, wherein measuring comprises use of a method for identifying or quantifying glycans containing terminal galactose-alpha-1-3-galactose selected from the group consisting of chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods, nuclear magnetic resonance (NMR) methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, use of a detection molecule, and combinations thereof.

44-50. -50. (canceled)