title: Optimization of fed-batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivation.
abstract: 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.
authors: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
authors: Ryan S
authors: RS
authors: Senger
authors: M Nazmul
authors: MN
authors: Karim
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical; D006224:Cricetinae; D003412:Cricetulus; D004345:Drug Industry; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D046509:Tissue Culture Techniques / Q000295:instrumentation / Q000379:methods*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D005947:Glucose