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Optimization of fed‐batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivation
Author(s) -
Senger Ryan S.,
Karim M. Nazmul
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21428
Subject(s) - asparagine , glutamine , glycosylation , metabolite , biochemistry , amino acid , chemistry , leucine , alanine , product inhibition , fed batch culture , recombinant dna , chromatography , biology , non competitive inhibition , fermentation , enzyme , gene
Optimization of fed‐batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue‐type activator (r‐tPA) protein. Dynamic inactivation models were written to describe particular r‐tPA glycoform inactivation in the presence and absence of free‐glucose. A glucose‐independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r‐tPA at N184. Inactivation rate constants of the glucose‐dependent mechanism were not affected by glycosylation at N184. Fed‐batch optimization was performed for r‐tPA production by CHO cell culture in a stirred‐tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free‐glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L −1 glucose and 1.18 g L −1 of amino acids. Optimization was also performed in absence of metabolite control using fixed feed‐flow rates initiate during the exponential growth phase. Fixed feed‐flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation. Biotechnol. Bioeng. 2007;98: 378–390. © 2007 Wiley Periodicals, Inc.