title: 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.
authors.affiliation: Research and Development, Process Sciences, Bayer Healthcare, Biological Products Division, 800 Dwight Way, Berkeley, California 94701, USA.
authors.forename: Adiba
authors.initials: A
authors.lastname: Ishaque
authors.forename: John
authors.initials: J
authors.lastname: Thrift
authors.forename: John E
authors.initials: JE
authors.lastname: Murphy
authors.forename: Konstantin
authors.initials: K
authors.lastname: Konstantinov
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D018840:HSP70 Heat-Shock Proteins; C078147:F8 protein, human; D005169:Factor VIII