Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein

摘要: Shear stress in suspension culture was investigated as a possible manipulative parameter for the control of glycosylation recombinant tissue-type plasminogen activator protein (r-tPA) produced by Chinese hamster ovary (CHO) cell culture, grown protein-free media. Resulting fractions partially glycosylated, Type II, and fully I, r-tPA were monitored direct function shear characteristics environment. The shear-induced response CHO to levels low stress, where exponential growth not obtained, higher which resulted extensive death, examined through manipulation bioreactor stirring velocity. Both apparent intrinsic growth, metabolite consumption, byproduct production, glycosylation, from variable site-occupancy standpoint, throughout. Kinetic analyses revealed shear-stress-induced alteration cellular homeostasis resulting nonlinear dependency metabolic yield coefficients an lysis kinetic constant on stress. Damaging used investigate dependence death lysis, well effects rate culture. models also developed basis state compared traditional models. Total production maximized under moderate conditions, viable density However, II fraction glycoform ratio damaging Analyses biomass coupled with plug-flow reactor model glycan addition endoplasmic reticulum (ER) propose overall mechanism decreased increasing Decreased residence time ER result increased synthesis related protection mechanisms is proposed limit contact site Asn184 membrane-bound oligosaccharyltransferase enzyme ER.