Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest

A vertebrate cell and cell technology, applied in the field of new vertebrate cells and methods for recombinant expression of polypeptides of interest, can solve problems such as expensive and time-consuming adaptation

Pending Publication Date: 2016-12-21
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this adaptation is time-consuming and expensive

Method used

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  • Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest
  • Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest
  • Novel vertebrate cells and methods for recombinantly expressing a polypeptide of interest

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0101] According to one embodiment, the method comprises

[0102] (a) cultivating the vertebrate host cell described in the first aspect under certain conditions that allow expression and secretion of the polypeptide of interest;

[0103] (b) isolating the polypeptide of interest from the cell culture medium; and

[0104] (c) Optional processing of the isolated polypeptide of interest.

[0105] Therefore, as a preferred embodiment of the method described in the third aspect, a method for recombinantly producing a polypeptide of interest is provided, comprising

[0106] (a) cultivating the vertebrate host cell described in the first aspect under conditions that allow expression and secretion of the polypeptide of interest into the cell culture medium;

[0107] (b) isolating the polypeptide of interest from the cell culture medium; and

[0108] (c) Optional processing of the isolated polypeptide of interest.

[0109] The host cells can be cultured under serum-free conditions...

Embodiment 1

[0167] II. Example 1: Cleavage of different polypeptides of interest when expression of different proteases is silenced by RNA interference (RNAi)

[0168] First, the proteolytic cleavage sites of different recombinant proteins prone to cleavage were analyzed by different techniques, including CE-SDS analysis, mass spectrometry and computer simulation analysis (data not shown). This analysis, in conjunction with other assays employing different protease inhibitors, revealed that the majority of the tested proteins tended to be cleaved by trypsin-like proteases, a subfamily of serine proteases. The RNAi experiments of Example 1 were set up to demonstrate that interstitial protease is the key protease responsible for cleavage and that silencing of the interstitial protease gene can significantly reduce cleavage, whereas silencing of other genes encoding different proteases does not reduce cleavage of the polypeptide of interest. siRNAs were designed against the following target mR...

Embodiment 2

[0190] III. Example 2: Interstitial protease gene knockout (KO) in CHO cells results in reduced cleavage

[0191] A. KO with TALEN technology

[0192] Nine interstitial protease (MT-SP1) knockout cell clones were generated based on CHO-K1 derived cells, using TALEN (transcription activator-like effector nuclease) technology. For the knockout, exon 2 of the interstitial protease was targeted to the region preceding the coding region of the transmembrane domain. Exon 2 was chosen because it covers different alternative splicing variants. Incorporating a frameshift mutation in exon 2 has the advantage that the truncated protein is shorter and intracellular, and moreover, is unstable and nontoxic to cells.

[0193] 2.A.1. Design / preparation and application of interstitial protease exon 2 specific TALEN

[0194] Sequencing of interstitial protease exon 2 and flanking introns in a CHO-K1-derived parental cell line (see figure 2 , SEQ ID NO: 31).

[0195] Design of 2 truncated ...

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Abstract

The present application pertains inter alia to an isolated vertebrate cell suitable for recombinant expression of a polypeptide of interest, wherein the vertebrate cell is altered to impair the function of the endogenous protease matriptase and wherein said cell comprises at least one heterologous polynucleotide encoding a polypeptide of interest and wherein the polypeptide of interest is secreted by the cell. It was found that using respective vertebrate cells for producing a recombinant polypeptide of interest significantly reduces clipping of the polypeptide of interest that is secreted into the cell culture medium. Also provided are improved production and screening methods.

Description

technical field [0001] The present disclosure relates to the field of recombinant expression technology. It relates to altered vertebrate cells and their use in recombinant expression methods, among others. When the recombinantly expressed polypeptide of interest in the modified cells is subsequently secreted into the cell culture medium, the cleavage of the recombinantly expressed polypeptide of interest in the cell culture medium is significantly reduced or even completely prevented. Background of the invention [0002] As biomedicine becomes more and more important to today's medicine, the biomedicine market continues to grow rapidly. Currently, more and more biopharmaceuticals are produced in vertebrate cells, such as mammals in particular. Biopharmaceuticals include, but are not limited to, antibodies, antibody fragments, ADCs (antibody-drug conjugates), Nanobodies, Fc-fusion proteins, growth factors, hormones, cytokines, enzymes and other therapeutic polypeptides. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90C12P21/00
CPCC12N9/50C12N15/907C12P21/00C12N9/6424C12Y304/21109C12N5/00C12N5/0602C12N15/66C12N2510/02G01N15/14G01N2015/0065G01N2015/1006
Inventor H·劳克斯S·罗曼德U·博登多夫
Owner NOVARTIS AG
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