Single-Cell Mitochondrial DNA Analysis of Recombinant Chinese Hamster Ovary Cells Reveals Widespread Heteroplasmy

Abstract

Recent bulk analysis of Chinese hamster ovary (CHO) cell mitochondrial DNA revealed widespread heteroplasmy across cell lines and even within clones of the same parental host. To address this, we applied our previously developed single-cell mtDNA sequencing (scmtDNAseq) method to 84 single CHO cells. We identified widespread intercellular heteroplasmy across the CHO cell population and predicted possible phenotypic impacts. 3/11 (27%) of the most variable mutations were only identified by scmtDNAseq, indicating greater resolution when compared to bulk cell analysis. Single-cell RNAseq (scRNAseq) was also performed at the same time point and, compared to scmtDNAseq, significant differences in intercellular heteroplasmy were observed. Using an inducible mAb expression system demonstrated that short-term additional biosynthetic burden of exogenous protein production had little impact on intercellular heteroplasmy. We additionally monitored bulk heteroplasmy over 38 days, reflecting the typical timespan from vial thaw to production vessel in a Biopharmaceutical upstream cell culture process. We observed minimal change in heteroplasmy, finding no evidence that a mAb-producing CHO cell line develops impactful changes in heteroplasmy over that timeframe. This would suggest that for our cell line, the heteroplasmy profile established on Day 1 should be maintained throughout a full fed-batch bioprocess run.