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Compositions and methods for detecting and quantifying host cell protein in cell lines and recombinant polypeptide products

A technology for recombinant polypeptides and antibody detection, applied in biochemical equipment and methods, detection of programmed cell death, chemical instruments and methods, etc., can solve the problem of low sequence homology

Active Publication Date: 2016-06-29
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although microorganisms have similar enzymatic activity, the proteins responsible for this activity are distinct, and sequence homology between microbial and mammalian PLBL2 enzymes is low

Method used

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  • Compositions and methods for detecting and quantifying host cell protein in cell lines and recombinant polypeptide products
  • Compositions and methods for detecting and quantifying host cell protein in cell lines and recombinant polypeptide products
  • Compositions and methods for detecting and quantifying host cell protein in cell lines and recombinant polypeptide products

Examples

Experimental program
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Embodiment

[0220] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0221] As used in the Examples below and elsewhere herein, "PLB2" and "PLBL2" and "PLBD2" are used interchangeably and refer to the enzyme "phospholipase B-like 2" or its synonym "phospholipase B-like 2" domain-like 2".

[0222] Example 1 - General method

[0223] Monoclonal antibody raw material

[0224] The mAb starting material used in all examples was selected from industrial, pilot or small scale cell culture batches from Genentech (South San Francisco, CA, U.S.A.). After a period of cell culture fermentation, the cells are isolated and, in some cases, purified and clarified by protein A chromatography and one or more additional chromatography steps and filtration steps as indicated in the Examples below. liquid (harvested cell culture fluid, HCCF). HCCF or in-process mixtures...

Embodiment 2

[0235] Example 2 - Generation of antibodies that bind hamster PLBL2

[0236] The identification of PLBL2 as an impurity in antibody preparations prompted us to synthesize the gene and subsequently express and purify hamster PLBL2. The literature on PLBL2 describes it as a lysosomal enzyme with a molecular weight of approximately 66 kD (F. Deuschl et al., FEBS Lett 580:5747-5752 (2006)). Like other lysosomal enzymes, this protein contains multiple post-translational modifications with mannose-6-phosphate and is initially synthesized as a preproenzyme. During processing, the leader sequence was clipped and proteolytic cleavage occurred, resulting in three bands of the protein on SDS-PAGE gel: intact PLBL2 (MW66kD), N-terminal domain (28kD) and C-terminal domain (40kD). Shearing occurs at acidic pH levels. Although the N-domain and C-domain separate on SDS-PAGE, these fragments may not separate under native conditions, as we have observed that strong solvents (eg, urea, guanid...

Embodiment 3

[0247] Example 3 - Mouse Monoclonal Anti-Hamster PLBL2 ELISA Assay

[0248] An ELISA assay was developed to detect and quantify the CHOP impurity PLBL2 in recombinant polypeptide samples such as recombinant antibody or immunoadhesin preparations. The procedure is as follows: Coat the mouse monoclonal antibody 19C10 at a concentration of 0.5 μg / mL in carbonate buffer (0.05 M sodium carbonate, pH 9.6) onto a half-area 96-well microtiter plate, at 2-8° C. overnight. After coating, block buffer (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% 300 [Sigma-Aldrich]; also known as assay diluent) to block the plate to prevent non-specific adhesion of proteins. In assay diluent (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% 300 [Sigma-Aldrich]) and then loaded in duplicate into separate wells and incubated at room temperature (22-27°C) for 2 hours. PLBL2 (if present in the sample) will bind to the coating (also ...

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Abstract

Monoclonal and polyclonal antibodies that bind hamster phospholipase B-like 2 are provided. Also provided are methods for detecting and quantifying hamster phospholipase B-like 2, for example, in recombinant polypeptide preparations, as well as kits for carrying out such methods. Methods of screening or selecting host cell lines or recombinant polypeptide-expressing cell lines that express low levels of hamster phospholipase B-like 2 are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 61 / 991,228, filed May 9, 2014, and U.S. Provisional Application No. 61 / 877,503, filed September 13, 2013, both of which are hereby incorporated by reference in their entirety into this article. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been submitted via EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 28, 2014, is named P5701R1WO_PCTSequenceListing.txt and is 28,965 bytes in size. technical field [0005] Monoclonal and polyclonal antibodies that bind hamster phospholipase B-like 2 are provided. Also provided are methods for detecting and quantifying hamster phospholipase B-like 2, eg, hamster phospholipase B-like 2 in recombinant polypeptide preparations, and kits for performing such methods. Also provided are methods of screening or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/44C12N5/07C12P21/08
CPCG01N2333/918G01N2500/10C07K16/40C07K2317/33G01N33/573C07K2317/565G01N33/577G01N2333/92
Inventor F·古纳万Y-C·肖D·C·可拉维茨M·S·林M·范德兰R·维吉I·H·尤克J·朱-希莫尼
Owner F HOFFMANN LA ROCHE & CO AG
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