This informed the design of a screening study in 500 mL shake flasks varying the concentrations of asparagine, cysteine, and tyrosine (experimental design not shown in this study). Cell culture intermediate retains obtained from a commercial production 300L Bioreactor were analyzed for amino acid concentrations. Enzyme activity levels for rHuPH20 were quantified using a micro-turbidity method, and amino acid concentrations were quantified by Ultra Performance Liquid Chromatography (UPLC) analysis. lactate) were quantified using a biochemical analyzer. viable cell density) were measured using an automated cell counter, while cell metabolites (e.g. The objective of this work was to demonstrate the effectiveness of spent media amino acid analysis to optimize amino acid supplementation of the cell culture feed media to improve the cellular growth and metabolic profile as well as increase productivity in a cell culture process.
Subsequent amino acid supplementation studies using a mini-bioreactor system at a fixed concentration for the 3 depleted amino acids led to an overall increase in the bioreactor IVCD by approximately 68% and enzyme titer by 90%, while decreasing cell specific lactate production by 45%. Using a Design of Experiment (DOE) approach, optimal concentrations of each of the 3 amino acids were assessed to increase cell growth and enzyme titer, while reducing waste product production (eg, lactate). This analysis revealed that 3 amino acids in particular (asparagine, cysteine, and tyrosine) were either depleted or reduced during the early stages of the 14 day process. To further improve bioreactor performance, as measured by an increase in Integral Viable Cell Density (IVCD) and productivity (enzyme titer), spent media analysis was performed to assess individual amino acid consumption rates on a commercial rHuPH20 production bioreactor. The current manufacturing process uses a chemically defined basal media and complex feeds consisting of carbohydrates, amino acids, lipids, growth factors, vitamins, etc. Recombinant human hyaluronidase enzyme (rHuPH20) is expressed using a Chinese Hamster Ovary (CHO) cell line in a fed-batch bioreactor process. Purpose:An important focus of upstream BioProcess Development is to improve product yields and minimize cost of goods while maintaining product quality.
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