title: 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.
authors.affiliation: Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria, David.Reinhart@boku.ac.at.
authors.forename: David
authors.initials: D
authors.lastname: Reinhart
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sommeregger
authors.forename: Monika
authors.initials: M
authors.lastname: Debreczeny
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Gludovacz
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism*; D016466:CHO Cells; D003412:Cricetulus; D015870:Gene Expression; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins