title: Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.
abstract: The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.
authors.affiliation: Cell Sciences and Technology, Amgen Inc., 1201 Amgen Court West, Seattle, Washington. zhimeidu@gmail.com.
authors.forename: Zhimei
authors.initials: Z
authors.lastname: Du
authors.forename: David
authors.initials: D
authors.lastname: Treiber
authors.forename: John D
authors.initials: JD
authors.lastname: McCarter
authors.forename: Dina
authors.initials: D
authors.lastname: Fomina-Yadlin
authors.forename: Ramsey A
authors.initials: RA
authors.lastname: Saleem
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: McCoy
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tharmala
authors.initials: T
authors.lastname: Tharmalingam
authors.forename: Matthew
authors.initials: M
authors.lastname: Leith
authors.forename: Brian D
authors.initials: BD
authors.lastname: Follstad
authors.forename: Brad
authors.initials: B
authors.lastname: Dell
authors.forename: Brent
authors.initials: B
authors.lastname: Grisim
authors.forename: Craig
authors.initials: C
authors.lastname: Zupke
authors.forename: Carole
authors.initials: C
authors.lastname: Heath
authors.forename: Arvia E
authors.initials: AE
authors.lastname: Morris
authors.forename: Pranhitha
authors.initials: P
authors.lastname: Reddy
mesh_terms: D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D018844:Cyclin-Dependent Kinases
keywords: glycosylation; product quality; recombinant antibody production; specific productivity