Over‐expression of Hsp70 in BHK‐21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion

Abstract

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

“…For cell lines with high oxygen requirements, 2 mm is suggested, meanwhile 5 mm is proposed for cells with low oxygen necessities (Freshney 2010). In our study, it was demonstrated that BHK-21 cell line can grow satisfactoryly in static cultures at 5 mm medium height, the maximum viable cell concentration were comparable with others previously reported using agitated systems (Handa-Corrigan et al 1989;Cruz et al 2002;Moreira et al 1995;Ishaque et al 2007). Increase of liquid column height is not recommend, this is based on unnecessary culture medium consumption (around 50 % for 10 mm liquid column height) and increasing cost of inoculation procedure.…”

“…The non-influence of inoculum volume-working volume percentage (10-30 %) on maximum viable cell concentration was useful for reducing handling, cell loss and contaminations risks associated to centrifugation operation in lab scale. Maximum viable cell concentration ([4 9 10 6 -cell mL -1 ) in optimal conditions was higher than usual values of this kinetic parameter for BHK-21 cell lines cultured in batch mode (1-3.2 9 10 6 cell mL -1 ) (Handa-Corrigan et al 1989;Cruz et al 2002;Moreira et al 1995;Ishaque et al 2007). Glutamine was exhausted at the end of the exponential growth phase (72 h post inoculation).…”

Efficacy and safety of BAY 81‐8973, a full‐length recombinant factor VIII: results from the LEOPOLD I trial

“…BAY 81-8973 (Kovaltry â ; Bayer, Berkeley, CA, USA) is a full-length, unmodified recombinant human FVIII being developed for treatment of haemophilia A. BAY 81-8973 has the same primary amino acid sequence as sucrose-formulated recombinant FVIII (rFVIII-FS) and is manufactured using additional advanced technologies [4,5], including production without addition of human-or animal-derived raw materials to the cell culture, purification or formulation processes, a new cell bank that coexpresses the human chaperone protein heat shock protein 70 (which facilitates proper folding of proteins and enhances cell survival), and a 20-nm nanofiltration step to remove small non-enveloped viruses and potential protein aggregates. These overall advances in manufacturing have resulted in a recombinant FVIII (rFVIII) product of high and consistent purity, with low levels of high molecular weight impurities.…”

BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

“…The cell line was developed through introduction of the gene for human heat shock protein 70 (HSP70) [5,12] into the cell line used for manufacture of rFVIII-FS. HSP70 is a chaperone protein that may increase FVIII expression by facilitating proper protein folding and improves cell survival by inhibiting apoptosis (ie, programmed cell death) [5,13,14].…”

“…Comparison of BHK-21 cells transfected with HSP70 (rBHK-21-HSP70) with host clone cells expressing only rFVIII (rBHK-21-host) demonstrated that rBHK-21-HSP70 cells displayed improved rFVIII productivity and resisted apoptosis [14]. Specifically, FVIII expression into culture media was at least 2-fold greater for rBHK-21-HSP70 cells compared with rBHK-21-host cells [14].…”