Integrated dual carbon source and TCA intermediate supplementation in fed-batch fermentation of CHO cells for reduced lactate and ammonia accumulation and improved IgG1 monoclonal antibody titre.

Chinese hamster ovary (CHO) cells are the workhorse of biopharmaceutical manufacturing, yet lactate and ammonia accumulation remain persistent bottlenecks that compromise cell viability, productivity, and product quality. To address this, we present an integrated feeding strategy systematically combining Ala-Gln, TCA intermediates, and glucose-galactose dual carbon feed evaluated head-to-head across four defined fed-batch conditions for CHO cultures producing an IgG1 monoclonal antibody (mAb). Conventional L-glutamine was replaced with L-alanyl-glutamine (Ala-Gln) to curb spontaneous ammonia release, while a customized "efficient feed mix" enriched with 2× TCA cycle intermediates and branched-chain amino acids combined with a glucose-galactose co-feed (2 mM glucose + 30 mM galactose) was introduced upon glucose depletion. Four fed-batch conditions were evaluated: a glutamine control (FB-01), Ala-Gln alone (FB-02), Ala-Gln with TCA intermediates (FB-03), and the complete integrated strategy (FB-04). FB-04 delivered an > 83% reduction in peak lactate (4.0 mM) and 80% reduction in ammonia (< 2.0 mM), sustained cell viability above 85% through day 11, and achieved a 93% higher mAb titre (1.72 g/L) with peak Viable count density (VCD) of 39 × 10⁶ cells/mL. Glycosylation profiles (19.0% galactosylation) remained aligned with innovator benchmarks. This study presents the first direct head-to-head benchmarking of Ala-Gln substitution, TCA intermediate supplementation, and galactose co-feeding in a single CHO fed-batch platform, demonstrating their combined metabolic and glycosylation benefits against a glutamine control.