title: CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.
abstract: The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey.
authors.forename: Duncan
authors.initials: D
authors.lastname: McVey
authors.affiliation: Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Aronov
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Rizzi
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey.
authors.forename: Alexis
authors.initials: A
authors.lastname: Cowan
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey.
authors.forename: Charo
authors.initials: C
authors.lastname: Scott
authors.affiliation: Discovery Toxicology, Bristol Myers Squibb Company, Pennington, New Jersey.
authors.forename: John
authors.initials: J
authors.lastname: Megill
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey.
authors.forename: Reb
authors.initials: R
authors.lastname: Russell
authors.affiliation: Division of Global Manufacturing and Supply, Bristol Myers Squibb Company, Bloomsbury, New Jersey. boazt@ekmd.huji.ac.il.|Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. boazt@ekmd.huji.ac.il.
authors.forename: Boaz
authors.initials: B
authors.lastname: Tirosh
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors; D016466:CHO Cells; D064113:CRISPR-Cas Systems / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D055786:Gene Knockout Techniques / Q000379:methods*; D000076222:Mechanistic Target of Rapamycin Complex 1; D060847:Metabolic Engineering / Q000379:methods*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D051059:PTEN Phosphohydrolase / Q000235:genetics; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D000906:Antibodies; D046912:Multiprotein Complexes; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D051059:PTEN Phosphohydrolase
keywords: CHO cells; CRISPR/Cas9; mTOR; productivity