Tat, J.; Lay, F. D.; Stevens, J.; Lewis, N. E.

Chinese hamster ovary (CHO) cells are the dominant host for therapeutic protein production, yet intra- and inter-clonal heterogeneity in manufacturing phenotypes, and the underlying metabolic and secretory circuitry, remain poorly defined at single-cell resolution. Here, we apply secretion encoded single-cell sequencing (SEC-seq) to simultaneously measure transcriptomes and secreted IgG in single-cells from a parental production cell line and five CHO clones, each varying in cell-specific productivity. IgG mRNA and recombinant protein secretion are only moderately correlated across single cells, indicating that transcription alone does not explain intra-clonal secretion heterogeneity. By integrating SEC-seq with single-cell metabolic and secretory task scoring, we find that CHO cells accommodating recombinant protein expression burden have more active translation-associated pathways and suppressed energy-intensive endogenous secreted protein processing. Three high-secreting clones converge on this translation-focused state but differ in their subpopulation composition and energy/redox programs coupled to IgG output: one highly productive clone shows a low-growth, glycolytic, NAD/one-carbon-associated and UPR-activated program; a second shows increased oxidative phosphorylation and fatty-acid {beta}-oxidation, and a third shows higher lipid-uptake with modest central carbon metabolism. Genes such as Aldoa, Ndufab1, Acsl5, Mthfd2 showed clone-specific correlations with IgG, linking glycolysis, mitochondrial respiration, fatty-acid metabolism, and redox to secretion. Together, these results demonstrate that SEC-seq can resolve IgG-coupled metabolic-secretory wiring within and between CHO clones, providing a framework to identify subpopulation and circuit features to engineer or select for improved recombinant protein production.