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Prevention of disulfide bond reduction during recombinant production of polypeptides

a technology of disulfide bond and recombinant protein, which is applied in the direction of peptides, drug compositions, immunological disorders, etc., can solve the problems that the production of recombinant proteins is still not without difficulties, and achieve the effect of preventing the reduction of a disulfide bond

Inactive Publication Date: 2009-02-26
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The instant invention generally relates to a method for preventing reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell, comprising supplementing the pre-harvest or harvested culture fluid of the recombinant host cell with an inhibitor of thioredoxin or a thioredoxin-like protein.

Problems solved by technology

Although this process has been the subject of much study and improvements over the past several decades, the production of recombinant proteins is still not without difficulties.

Method used

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  • Prevention of disulfide bond reduction during recombinant production of polypeptides
  • Prevention of disulfide bond reduction during recombinant production of polypeptides
  • Prevention of disulfide bond reduction during recombinant production of polypeptides

Examples

Experimental program
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example 1

Description of Materials and Methods

[0281]The following materials and methods were used in Examples 2-8 below.

[0282]Materials

[0283]Materials and devices used in the experiments described in the experimental examples include: stainless steel vials (mini-tanks, Flow Components, Dublin, Calif.; short (50 cc) and tall (55 cc)); dialysis tubing (Spectra / Por, 6-8000 MWCO, cat. #132645), 0.22 μm filter (Millipore Millipak Gamma Gold cat. #MPGL04 GH2); phosphate buffered saline (PBS, EMD, cat. #6506); ethylenediaminetetraacetic acid (EDTA, Sigma, cat. #E4884); α-nicotinamide adenine dinucleotide phosphate (NADPH, Calbiochem, cat. #481973); dehydroepiandrosterone (DHEA, TCI, cat. #D0044); cupric sulfate (Sigma, cat. #C8027), glucose-6-phosphate (G6P, Calbiochem, cat. #346764); aurothioglucose (ATG, USP, cat. #1045508); aurothiomalate (ATM, Alfa Aesar, cat. #39740); reduced glutathione (GSH, J. T. Baker, cat. #M770-01); monobromobimane (mBB, Fluka, cat. #69898); histidine (J. T. Baker, cat. #...

example 2

Dialysis Experiment

[0317]A dialysis experiment was designed and carried out to determine if the reduction of ocrelizumab was caused by small reducing molecules or macromolecules (e.g., enzymes). In this dialysis experiment, purified intact ocrelizumab was placed in a dialysis bag with a molecular weight cut off (MWCO) of 7000 and incubated the dialysis bag in HCCF containing ocrelizumab in a stainless steel mini-tank. As shown in FIGS. 1 and 2, the ocrelizumab inside the bag was not reduced after the incubation period (FIG. 1), whereas the ocrelizumab outside the bag in the HCCF was significantly reduced soon after the incubation started. This was evidenced by the loss of intact ocrelizumab (˜150 kDa) and the formation of ocrelizumab fragments (various combinations of heavy and light chains) (FIG. 2). The mass spectrometry analysis of the ocrelizumab in the protein A elution pools from the reduced manufacturing runs indicated that those observed fragments were formed by reduction of...

example 3

Reduction of Ocrelizumab (rhuMAb 2H7, Variant A) by Trx / TrxR In Vitro

[0319]The Trx system was tested for its ability to reduce ocrelizumab in vitro by incubating intact ocrelizumab with Trx, TrxR, and NADPH. The Bioanalyzer results indicate that ocrelizumab was reduced in vitro by the Trx system (FIG. 5). The rate of reduction in this in vitro system appears to be slower than that in the HCCF (for example when compared to the reduction shown in FIG. 2). This is likely due to lower concentrations of the enzymes (Trx and Trx-R) and / or the buffer system used in the in vitro reaction because reaction rate of Trx system is dependent on both the enzyme concentrations and buffer systems.

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Abstract

The invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a non-provisional application filed under 37 CFR 1.53(b)(1), claiming priority under 35 USC 119(e) to provisional application No. 60 / 948,677 filed Jul. 9, 2007, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.BACKGROUND OF THE INVENTION[0003]In the biotechnology industry, pharmaceutical applications require a variety of proteins produced using recombinant DNA techniques. Generally, recombinant proteins are produced by cell culture, using either eukaryotic cells, such as mammalian cells, or prokaryotic cells, such as bacterial cells...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/99C12N5/06
CPCA61K39/39591C07K2317/24C07K16/2887C07K1/14C07K14/00A61P37/02A61P5/00C12N1/38C07K1/22C07K16/28C07K16/2863C07K16/32C12N5/0018C07K2317/14C07K2317/31
Inventor KAO, YUNG-HSIANGLAIRD, MICHAEL W.SCHMIDT, MELODY TREXLERWONG, RITA L.HEWITT, DANIEL P.
Owner GENENTECH INC
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