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

Patent metadata

Field	Value
Publication number	US-10690671-B2
Application number	US-201816044360-A
Country	US
Kind code	B2
Filing date	Jul 24, 2018
Priority date	Mar 6, 2015
Publication date	Jun 23, 2020
Grant date	Jun 23, 2020

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 C (Dsb C) in a sample, comprising detecting DsbC in the sample using a detection system and comparing the amount of DsbC detected in the sample with the detection of one or more concentrations of an ultrapure DsbC reference standard, wherein ultrapure DsbC comprises at least about 95% monomeric DsbC.

2. The method of claim 1 , wherein the ultrapure DsbC reference standard is prepared by the method comprising
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbC polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbC polypeptide to an anion exchange chromatography material,
d) eluting the DsbC polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbC polypeptide,
e) applying the anion exchange eluate comprising the DsbC polypeptide to a hydrophobic interaction chromatography (HIC) material,
f) eluting the DsbC polypeptide from the HIC material to generate a HIC eluate,
g) applying the HIC eluate comprising the DsbC polypeptide to a size exclusion chromatography,
h) collecting fractions from the size exclusion chromatography comprising the purified DsbC polypeptide.

3. The method of claim 1 , wherein the detection system is an immunoassay.

4. The method of claim 3 , wherein the immunoassay comprises antibodies that specifically binds ultrapure DsbC.

5. A method for analyzing a recombinant polypeptide sample for the presence of and/or quantity of DsbC, comprising detecting DsbC in the sample using an immunoassay and comparing the amount of DsbC detected in the sample with the detection of one or more concentrations of an ultrapure DsbC reference standard, wherein the ultrapure DsbC reference standard comprises at least about 95% monomeric DsbC polypeptide.

6. The method of claim 5 , wherein the ultrapure DsbC reference standard is prepared by the method comprising
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbC polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbC polypeptide to an anion exchange chromatography material,
d) eluting the DsbC polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbC polypeptide,
e) applying the anion exchange eluate comprising the DsbC polypeptide to a hydrophobic interaction chromatography (HIC) material,
f) eluting the DsbC polypeptide from the HIC material to generate a HIC eluate,
g) applying the HIC eluate comprising the DsbC polypeptide to a size exclusion chromatography,
h) collecting fractions from the size exclusion chromatography comprising the purified DsbC polypeptide.

7. The method of claim 5 , wherein the immunoassay comprises antibodies that specifically bind ultrapure DsbC.

8. The method of claim 7 , wherein the antibodies that specifically bind ultrapure DsbC are polyclonal antibodies.

9. The method of claim 7 , wherein the antibodies that specifically bind DsbC are prepared by exposing an animal to the composition comprising a DsbC 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 DsbC polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbC polypeptide to an anion exchange chromatography material,
d) eluting the DsbC polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbC polypeptide,
e) applying the anion exchange eluate comprising the DsbC polypeptide to a hydrophobic interaction chromatography (HIC) material,
f) eluting the DsbC polypeptide from the HIC material to generate a HIC eluate,
g) applying the HIC eluate comprising the DsbC polypeptide to a size exclusion chromatography,
h) collecting fractions from the size exclusion chromatography comprising the purified DsbC polypeptide.

10. The method of claim 5 , wherein the recombinant polypeptide is prepared in a host cell overexpressing DsbC.

11. The method of claim 5 , wherein the recombinant polypeptide preparation is a final purified product.

12. The method of claim 5 , wherein the recombinant polypeptide contained in the recombinant polypeptide sample is an antibody or an immunoadhesin.

13. An immunoassay method for detecting DsbC 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 DsbC with the sample thereby generating a sample-capture antibody combination material;
(b) contacting a detection antibody that binds DsbC with the sample-capture antibody combination material; and
(c) detecting the 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 DsbC present in the sample based on the level of the detection antibody bound wherein the amount of DsbC present in the sample is determined by comparing the standard titration curve with a standard titration curve generated with an ultrapure DsbC composition, wherein the ultrapure DsbC composition comprises at least about 95% monomeric DsbC.

14. The method of claim 13 , wherein the ultrapure DsbC in the composition is prepared by the method comprising
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbC polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbC polypeptide to an anion exchange chromatography material,
d) eluting the DsbC polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbC polypeptide,
e) applying the anion exchange eluate comprising the DsbC polypeptide to a hydrophobic interaction chromatography (HIC) material,
f) eluting the DsbC polypeptide from the HIC material to generate a HIC eluate,
g) applying the HIC eluate comprising the DsbC polypeptide to a size exclusion chromatography,
h) collecting fractions from the size exclusion chromatography comprising the purified DsbC polypeptide.

15. The method of claim 13 , wherein the capture antibody and/or detection antibody specifically binds ultrapure DsbC, wherein the ultrapure DsbC composition comprises at least about 95% monomeric DsbC polypeptide.

16. The method of claim 15 , wherein the antibody that specifically binds ultrapure DsbC is a polyclonal antibody.

17. The method of claim 13 , wherein the polyclonal antibody is generated according to the method comprising
a) adding polyethyleneimine (PEI) to a final concentration of about 0.01% to about 1.0% to a cell lysate comprising the DsbC polypeptide,
b) clarifying the cell lysate by centrifugation,
c) applying the clarified cell lysate comprising the DsbC polypeptide to an anion exchange chromatography material,
d) eluting the DsbC polypeptide from the anion exchange chromatography material to generate an anion exchange eluate comprising the DsbC polypeptide,
e) applying the anion exchange eluate comprising the DsbC polypeptide to a hydrophobic interaction chromatography (HIC) material,
f) eluting the DsbC polypeptide from the HIC ma

Assignees

• Genentech Inc

Inventors

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

Classifications

• G01N2333/99

  Isomerases (5.) · CPC title

• G01N2333/90212

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

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• C12Y503/04001

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

• C12Y108/04002

  Protein-disulfide reductase (glutathione) (1.8.4.2), i.e. BdbC or BdbD · CPC title