title: Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production.
abstract: Industrial therapeutic protein production has been greatly improved through fed-batch development. In this study, improvement to the productivity of a tissue-plasminogen activator (t-PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed-batch feed and the reduction of ammonia generation by glutamine replacement. The t-PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed-batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed-batch operation increased the generation of by-products such as lactate and ammonia that can adversely affect the fed-batch performance. To reduce the ammonia production, a glutamine-containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two-step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t-PA titer to over 420 mg/L without compromising the t-PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed-batch strategy that could be applied to the production of other recombinant therapeutic proteins.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: Do Yun
authors.initials: DY
authors.lastname: Kim
authors.forename: Muhammad Arshad
authors.initials: MA
authors.lastname: Chaudhry
authors.forename: Malcolm L
authors.initials: ML
authors.lastname: Kennard
authors.forename: Mario A
authors.initials: MA
authors.lastname: Jardon
authors.forename: Katrin
authors.initials: K
authors.lastname: Braasch
authors.forename: Ben
authors.initials: B
authors.lastname: Dionne
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
authors.forename: James M
authors.initials: JM
authors.lastname: Piret
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D005973:Glutamine; D000641:Ammonia; D010959:Tissue Plasminogen Activator