Ultrapurified DsbA and DsbC and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-10073098-B2
Application number	US-201615061943-A
Country	US
Kind code	B2
Filing date	Mar 4, 2016
Priority date	Mar 6, 2015
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

The present invention provides methods for producing disulfide oxidoreductase A (DsbA) and disulfide oxidoreductase C (DsbC) polypeptides at very high levels of purity. Also provided are ultrapure DsbA and DsbC and methods of using same, e.g., for use in immunoassays to show removal of DsbA and DsbC from biologics produced in bacteria.

First claim

What is claimed is:

1. A method for quantifying disulfide oxidoreductase A (DsbA) in a sample, comprising detecting DsbA in the sample using a detection system and comparing the amount of DsbA detected in the sample with the detection of one or more concentrations of an ultrapure DsbA reference standard, wherein ultrapure DsbA comprises at least about 95% monomeric DsbA.

2. A method for analyzing a recombinant polypeptide sample for the presence of and/or quantity of DsbA, comprising detecting DsbA in the sample using an immunoassay and comparing the amount of DsbA detected in the sample with the detection of one or more concentrations of an ultrapure DsbA reference standard, wherein the immunoassay comprises antibodies that specifically bind ultrapure DsbA which are used as capture antibodies and/or detection antibodies, wherein ultrapure DsbA comprises at least about 95% monomeric DsbA.

3. The method of claim 2 , wherein the ultrapure DsbA reference standard is prepared by
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbA polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbA polypeptide to an anion exchange chromatography material,
d) eluting the DsbA polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbA polypeptide,
e) applying the anion exchange eluate comprising the DsbA polypeptide to a cation exchange chromatography material,
f) eluting the DsbA polypeptide from the cation exchange chromatography material to generate a cation exchange eluate comprising the purified DsbA polypeptide.

4. The method of claim 2 , wherein the antibodies that specifically bind ultrapure DsbA are polyclonal antibodies.

5. The method of claim 2 , wherein the antibodies that specifically bind ultrapure DsbA are generated by exposing an animal to the composition comprising a DsbA polypeptide prepared by
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbA polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbA polypeptide to an anion exchange chromatography material,
d) eluting the DsbA polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbA polypeptide,
e) applying the anion exchange eluate comprising the DsbA polypeptide to a cation exchange chromatography material,
f) eluting the DsbA polypeptide from the cation exchange chromatography material to generate a cation exchange eluate comprising the purified DsbA polypeptide.

6. The method of claim 2 , wherein the recombinant polypeptide is prepared in a host cell overexpressing DsbA.

7. The method of claim 2 , wherein the recombinant polypeptide preparation is a final purified product.

8. The method of claim 2 , wherein the recombinant polypeptide contained in the recombinant polypeptide sample is an antibody or an immunoadhesin.

9. An immunoassay method for detecting DsbA in a sample, wherein the sample is obtained from a recombinant polypeptide preparation or a host cell line, the method comprising:
(a) contacting a capture antibody that binds DsbA with the sample thereby generating a sample-capture antibody combination material;
(b) contacting a detection antibody that binds DsbA with the sample-capture antibody combination material;
(c) detecting the detection antibody bound to the sample-capture antibody combination material;
(d) quantifying the level of the detection antibody bound using a standard titration curve and calculating an amount of DsbA present in the sample based on the level of the detection antibody bound wherein the amount of DsbA present in the sample is determined by comparing the standard titration curve with a standard titration curve generated with an ultrapure DsbA composition, wherein the ultrapure DsbA composition comprises at least about 95% monomeric DsbA.

10. The method of claim 9 , wherein the ultrapure DsbA in the composition is prepared by
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbA polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbA polypeptide to an anion exchange chromatography material,
d) eluting the DsbA polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbA polypeptide,
e) applying the anion exchange eluate comprising the DsbA polypeptide to a cation exchange chromatography material,
f) eluting the DsbA polypeptide from the cation exchange chromatography material to generate a cation exchange eluate comprising the purified DsbA polypeptide.

11. The method of claim 9 , wherein the capture antibody and/or detection antibody specifically binds ultrapure DsbA, wherein ultrapure DsbA comprises at least about 95% monomeric DsbA.

12. The method of claim 11 , wherein the antibody that specifically binds ultrapure DsbA is a polyclonal antibody.

13. The method of claim 12 , wherein the polyclonal antibody is generated by exposing an animal to the composition comprising a DsbA polypeptide prepared by
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbA polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbA polypeptide to an anion exchange chromatography material,
d) eluting the DsbA polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbA polypeptide,
e) applying the anion exchange eluate comprising the DsbA polypeptide to a cation exchange chromatography material,
f) eluting the DsbA polypeptide from the cation exchange chromatography material to generate a cation exchange eluate comprising the purified DsbA polypeptide.

14. The method of claim 9 , wherein the host cell line overexpresses DsbA.

15. The method of claim 9 , wherein the recombinant polypeptide preparation is final purified product.

16. The method of claim 9 , wherein the recombinant polypeptide contained in the recombinant polypeptide preparation is an antibody or an immunoadhesin.

17. A quality assay for a pharmaceutical composition comprising a recombinant polypeptide prepared from a bacterial cell, the release quality assay comprising subjecting a sample of the pharmaceutical composition to the immunoassay method of claim 9 , wherein the amount of DsbA detected in the composition determines if the pharmaceutical composition is suitable for administration to an animal.

18. The quality assay of claim 17 , wherein an amount of DsbA in the pharmaceutical composition of less than about 10 ppm indicates that the pharmaceutical composition is suitable for administration to the animal.

19. The quality assay of claim 17 , wherein the bacterial cell is an E. coli cell.

20. The quality assay of claim 17 , wherein the E. coli cell overexpresses DsbA.

21. The quality assay of claim 17 , wherein the sample is cell lysate.

22. The quality assay of claim 17 , wherein the recombinant polypeptide preparation is final purified product.

23. The quality assay of claim 17 , wherein the antibody is a multispecific antibo

Assignees

• Genentech Inc

Inventors

• Wong Marc
• Yee Liliana T
• Lim Amy
• Fong Chris B

Classifications

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• C12N9/90 Primary

  Isomerases (5.) · CPC title

• G01N2333/90212

  acting on a sulfur group of donors (1.8) · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12Y503/04001

  Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme · CPC title