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Inhibition of glutamine‐dependent autophagy increases t‐PA production in CHO Cell fed‐batch processes
Author(s) -
Jardon Mario A.,
Sattha Beheroze,
Braasch Katrin,
Leung Amy O.,
Côté Hélène C.F.,
Butler Michael,
Gorski Sharon M.,
Piret James M.
Publication year - 2012
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.24393
Subject(s) - autophagy , chinese hamster ovary cell , glutamine , recombinant dna , cell culture , microbiology and biotechnology , biochemistry , fed batch culture , glycosylation , chemistry , biology , apoptosis , amino acid , gene , receptor , genetics , fermentation
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. Biotechnol. Bioeng. 2012; 109:1228–1238. © 2011 Wiley Periodicals, Inc.