Abstract
In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.
Highlights
Improving recombinant therapeutic protein manufacturing using mammalian Chinese hamster ovary (CHO) cell hosts is a major challenge for biopharmaceutical companies
CHO cells were cotransfected with expression vectors encoding an “easy‐to‐express” (ETE) trastuzumab or a “difficult‐to‐ express” (DTE) infliximab or Enbrel therapeutic protein, together with the vitamin B5 transporter SLC5A6 or with an antibiotic resistance gene as a control
We showed that overexpressing ACTC1 actin gene, known to be involved in cardiac muscle alpha‐actin synthesis, acts to improve ETE and DTE therapeutic protein expression and secretion by CHO cells
Summary
Improving recombinant therapeutic protein manufacturing using mammalian Chinese hamster ovary (CHO) cell hosts is a major challenge for biopharmaceutical companies. We previously described how the deprivation of vitamin B5 in CHO cell culture medium, coupled to the cotransfection of an expression vector for a vitamin B5 transport protein with the gene of interest, allowed the identification of cell variants that are capable of expressing at very high levels recombinant therapeutic proteins (Pourcel et al, 2020). We wished to identify specific gene expression alterations that accompany efficient therapeutic protein expression, with the hope to construct CHO cell line derivatives that would be permanently more efficient for production. Among these alterations, two genes involved in cytoskeleton organization were significantly induced after vitamin B5 selection, namely actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP). Selection based on vitamin B5 deprivation was performed by culturing the cells cotransfected with the vitamin B5 transporter SLC5A6 expression vector in a chemically defined medium with a low concentration of vitamin B5 (B5‐deprived BalanCD CHO‐M Growth A supplemented with 2.5 nM vitamin B5), as described previously (Pourcel et al, 2020)
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