Inhibition of glutamine-dependent autophagy increases t-PA production in CHO Cell fed-batch processes

Authors

  • Mario A. Jardon,

    1. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4. Tel.: 604-822-5835, Fax: 604-822-2114
    2. Department of Chemical and Biological Engineering, UBC, 2360 East Mall, Vancouver, BC, Canada, V6T 1Z3
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  • Beheroze Sattha,

    1. Department of Pathology and Laboratory Medicine, UBC, G227-2211 Wesbrook Mall, Vancouver, BC, Canada, V6T 2B5
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  • Katrin Braasch,

    1. Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB, Canada, R3T 2N2
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  • Amy O. Leung,

    1. Michael Smith Genome Sciences Centre, BC Cancer Research Centre, 675 West 10 Ave, Vancouver, BC, V5Z 1L3
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  • Hélène C.F. Côté,

    1. Department of Pathology and Laboratory Medicine, UBC, G227-2211 Wesbrook Mall, Vancouver, BC, Canada, V6T 2B5
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  • Michael Butler,

    1. Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB, Canada, R3T 2N2
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  • Sharon M. Gorski,

    1. Michael Smith Genome Sciences Centre, BC Cancer Research Centre, 675 West 10 Ave, Vancouver, BC, V5Z 1L3
    2. Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6
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  • James M. Piret

    Corresponding author
    1. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4. Tel.: 604-822-5835, Fax: 604-822-2114
    2. Department of Chemical and Biological Engineering, UBC, 2360 East Mall, Vancouver, BC, Canada, V6T 1Z3
    • Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4. Tel.: 604-822-5835, Fax: 604-822-2114.
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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 107 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.

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