Engineering TPT1-mediated apoptosis resistance for improved mammalian cell biomanufacturing.

Cell death during late-stage culture remains a major limitation in mammalian manufacturing processes for antibody therapeutics, constraining yield and process robustness. Here, we identified the predominant apoptotic signalling axis associated with culture decline in Chinese hamster ovary (CHO) cells and engineering against it. Recombinant CHO lines were engineered to overexpress BCL-2 (intrinsic pathway and benchmark control), CFLAR (death receptor pathway regulator), or TPT1 (a multifunctional stress-response protein). Apoptosis profiling across fed-batch cultures indicated that viability loss is predominantly associated with intrinsic pathway activation, characterised by increased cleavage of caspase-9, caspase-7 and caspase-3, with minimal activation of caspase-8. In batch and fed-batch studies, CFLAR and TPT1 improved late-stage viability and extended culture lifespan relative to the control, with TPT1 providing the most consistent benefit and outperforming BCL-2 in overall process performance. Under apoptosis challenges, including chemical induction, pro-apoptotic BAK overexpression and caspase 3 activation, TPT1-expressing cells maintained higher viability, decreased apoptosis and attenuated caspase 3 activation. Finally, TPT1 overexpression was transferred to industrially relevant CHO DG44 production platforms expressing monoclonal and bispecific antibodies and improved culture longevity and titres in both formats, without altering cell-specific productivity and N-glycan profile. TPT1 also presented similar behaviour to a BAK/BAX double knockout on viability and exceeded it on growth and titre, and combining the two gave no further gain in unfed batch culture. TPT1 overexpression therefore offers a single-cassette route to longer culture and high volumetric output inCHO-based bioprocesses.