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Increased t‐PA Yields Using Ultrafiltration of an Inhibitory Product from CHO Fed‐Batch Culture
Author(s) -
Dowd Jason E.,
Kwok K. Ezra,
Piret James M.
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp000079b
Subject(s) - ultrafiltration (renal) , product (mathematics) , chemistry , fed batch culture , chromatography , inhibitory postsynaptic potential , by product , food science , biology , biochemistry , mathematics , endocrinology , fermentation , geometry
Abstract Fed‐batch operation for the production of t‐PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2‐ to 2.5‐fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5‐fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed‐batch results. The concentrate addition rate was based on a constant 4 pmol/cell·day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t‐PA concentrations compared to batch. The fed‐batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4‐fold increase in viable cell index yielded a 4‐fold increase in antibody concentration. The half‐life of t‐PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t‐PA threshold. Instead, the CHO results were explained by a reduction in t‐PA production at higher extracellular t‐PA concentrations that limited the fed‐batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t‐PA mRNA levels revealed no response to increasing extracellular t‐PA concentrations upon exogenous additions. Instead, intracellular t‐PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t‐PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t‐PA fed‐batch production was increased over 2‐fold, up to a yield of 80 mg/L.