Pneumatic microplate mixing accelerates CHO single-cell cloning workflows and improves early clone expansion

Stable Chinese hamster ovary (CHO) cell line development depends on efficient recovery and expansion of monoclonal populations after single-cell cloning (SCC). Early-stage clones, however, are typically expanded under static microplate conditions before transfer to suspension culture, creating a physiological discontinuity that can delay adaptation and obscure differences in clone performance. Here, we evaluated C.NEST, a pneumatic microplate mixing system, as a strategy for improving early CHO-K1 clone expansion and facilitating transition to suspension culture. Benchmark studies showed that pneumatic microplate mixing improved culture performance compared with static culture and supported shake-flask-like growth. Operating-window studies in 24-well plates showed higher viable cell densities under pneumatic mixing while generally maintaining high viability, with the response depending on inoculation density and mixing intensity. In clone-level validation studies, pneumatic-mixing pre-culture was associated with faster subsequent suspension adaptation. In an integrated SCC workflow demonstration, pneumatic mixing shortened microplate expansion by 3 days and reached the fed-batch production stage 7 days earlier than the corresponding static workflow. Among the eight clones advanced to production, 96-well relative titer was positively correlated with final fed-batch titer under pneumatic mixing (Spearman’s ρ = 0.7857, P = 0.0279), while the static workflow showed a weak negative, non-significant correlation. Early pneumatic mixing in microplates improves clone expansion and facilitates transition from static SCC culture to suspension culture, thereby accelerating progression through CHO cell line development. Among the selected clones advanced to production, the observed correlation pattern was consistent with greater preservation of clone ranking under the tested pneumatic-mixing conditions.