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In search of expression bottlenecks in recombinant CHO cell lines--a case study.

Abstract：

:The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Reinhart D,Sommeregger W,Debreczeny M,Gludovacz E,Kunert R

doi

10.1007/s00253-014-5584-z

subject

Has Abstract

pub_date

2014-07-01 00:00:00

pages

5959-65

issue

eissn

0175-7598

issn

1432-0614

journal_volume

pub_type

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In search of expression bottlenecks in recombinant CHO cell lines--a case study.