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Increased t-PA Yields Using Ultrafiltration of an Inhibitory Product from CHO Fed-Batch Culture

Authors

  • Jason E. Dowd,

    1. Biotechnology Laboratory, University of British Columbia, #237 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada
    2. Department of Chemical and Biological Engineering, University of British Columbia, 2216 Main Mall, Vancouver, BC V6T 1Z4, Canada
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  • K. Ezra Kwok,

    1. Department of Chemical and Biological Engineering, University of British Columbia, 2216 Main Mall, Vancouver, BC V6T 1Z4, Canada
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  • James M. Piret

    Corresponding author
    1. Biotechnology Laboratory, University of British Columbia, #237 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada
    2. Department of Chemical and Biological Engineering, University of British Columbia, 2216 Main Mall, Vancouver, BC V6T 1Z4, Canada
    • Biotechnology Laboratory, University of British Columbia, #237 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada. Tel: (604) 822–5835. Fax: (604) 822–2114
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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.

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