title: Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes.
abstract: Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.
authors: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
authors: Mario A
authors: MA
authors: Jardon
authors: Beheroze
authors: B
authors: Sattha
authors: Katrin
authors: K
authors: Braasch
authors: Amy O
authors: AO
authors: Leung
authors: Hélène C F
authors: HC
authors: Côté
authors: Michael
authors: M
authors: Butler
authors: Sharon M
authors: SM
authors: Gorski
authors: James M
authors: JM
authors: Piret
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000737:chemistry; D020869:Gene Expression Profiling; D005973:Glutamine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
chemical_list: D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005973:Glutamine; C025946:3-methyladenine; D010959:Tissue Plasminogen Activator; D000225:Adenine