ULTRAPURIFIED DsbA AND DsbC AND METHODS OF MAKING AND USING THE SAME
AssigneeGenentech, Inc.
InventorsMarc WONG, Liliana T. YEE, Amy LIM, Chris B. FONG
Life of this patent
Estimating the term…
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.
Claims78
A method for purifying a DsbA polypeptide from a cell lysate comprising the DsbA polypeptide, 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 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.
2-33. (canceled)
35-50. (canceled)
52-55. (canceled)
A method for purifying antibodies that specifically bind DsbA, comprising contacting a composition comprising anti-DsbA antibodies to chromatography material comprising ultrapure DsbA attached to a support material, washing the chromatography material to remove unbound compounds, and eluting the anti-DsbA antibodies.
57-61. (canceled)
63-66. (canceled)
A method for quantifying 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.
68-71. (canceled)
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 conjugated to a detection agent.
(canceled)
77-80. (canceled)
(canceled)
84-87. (canceled)
90-91. (canceled)
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;
- and
- (c) detecting the detection antibody bound to the sample-capture antibody combination material.
(canceled)
(canceled)
103-106. (canceled)
The method of claim 106, wherein the host cell line overexpresses DsbA.
108-109. (canceled)
112-113. (canceled)
(canceled)
(canceled)
A method for purifying a DsbC polypeptide from a cell lysate comprising the DsbC polypeptide, 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.
125-157. (canceled)
159-173. (canceled)
175-178. (canceled)
A method or purifying antibodies that specifically bind DsbC, comprising contacting a composition comprising anti-DsbC antibodies to chromatography material comprising ultrapure DsbC attached to a support material, washing the chromatography material to remove unbound compounds, and eluting the anti-DsbC antibodies.
180-184. (canceled)
(canceled)
188-189. (canceled)
A method for quantifying DsbC 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.
191-194. (canceled)
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.
196-214. (canceled)
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.
216-236. (canceled)
238-246. (canceled)
248-249. (canceled)
251-252. (canceled)
254-255. (canceled)
Dependencies are inferred from the claim text.