Prajakta Dandekar

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.

FT-IR spectroscopy coupled with PCA enables rapid assessment of multiple monoclonal antibody critical quality attributes using a simple, low-complexity analytical workflow.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.