ArticleMicrobial cell factories2022
Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli.
Article in Microbial cell factories, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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1 citing paper in PubMed, 1 citations in OpenAlex.
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9 authors at 3 institutions in 3 countries.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundEscherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don't require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody's disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing.
resultsIn this study, we found that the reduction in antibody's disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc C
conclusionsDuring E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.
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