title: Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.
abstract: DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative.
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, BP Process Science, 88397 Biberach an der Riss, Germany. lore.florin@boehringer-ingelheim.com
authors.forename: Lore
authors.initials: L
authors.lastname: Florin
authors.forename: Carolin
authors.initials: C
authors.lastname: Lipske
authors.forename: Eric
authors.initials: E
authors.lastname: Becker
authors.forename: Hitto
authors.initials: H
authors.lastname: Kaufmann
mesh_terms: D000818:Animals; D001702:Biopharmaceutics / Q000379:methods*; D016466:CHO Cells / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000737:chemistry*; D017931:DNA Primers / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D055786:Gene Knockout Techniques; D019271:Hypoxanthine / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency* / Q000235:genetics; D013936:Thymidine / Q000494:pharmacology*
chemical_list: D016895:Culture Media, Serum-Free; D017931:DNA Primers; D019271:Hypoxanthine; D013762:Tetrahydrofolate Dehydrogenase; D013936:Thymidine