Title: Genetic engineering for the reduction of high mannose species in biopharmaceutical-producing CHO cells
Abstract:
In biopharmaceutical manufacturing, approximately 60-70% of all recombinant proteins are produced in mammalian cells, with Chinese Hamster Ovary (CHO) cells representing the preferred expression system. Clinical efficacy and safety can be significantly affected by the glycosylation profile of the product.
High mannose (HM) species are immature glycoforms that may reduce drug half-life and increase the risk of off-target effects, particularly in antibody-drug conjugates (ADCs). As HM levels are considered a critical quality attribute (CQA), clones exhibiting elevated HM are often discarded (potentially leading to the loss of high-producing clones) or manufacturing processes are modified through manganese supplementation to reduce HM content. Both strategies increase process complexity, timelines, and costs.
To enable glycan maturation and reduce HM species, we engineered 4 CHO cell lines to overexpress MGAT1 gene, which encodes for Alpha-1,3-Mannosyl-Glycoprotein 2-Beta-N-Acetylglucosaminyltransferase involved in the maturation of M5, the most represented HM forms.
An additional copy of MGAT1 gene was integrated into the CHO genome using two strategies: (i) one-step integrase-based insertion and (ii) a landing-pad approach. Construct integration and its stability were assessed by digital droplet PCR (ddPCR). MGAT1 overexpression was evaluated by RT-ddPCR and Western blotting.
Engineered lines were cultured in batch and fed-batch conditions to assess growth profiles, viability, and monoclonal antibody titer. HM species were quantified using lectin-based assay and LC-MS.
ddPCR confirmed single-copy integration of MGAT1 gene in all engineered lines. RT-ddPCR and Western blotting demonstrated robust overexpression at both transcript and protein level. Across batch and fed-batch cultures, MGAT1 integration and overexpression did not adversely affect cell growth, viability or titer. Lectin-based profiling and LC-MS analyses confirmed a reduction in released HM species in MGAT1-overexpressing lines compared with controls.
These findings demonstrated that MGAT1 overexpression is an effective cell-line engineering strategy to reduce HM species while maintaining viability and productivity. This approach can facilitate meeting glycosylation CQA targets and may support improved safety and efficacy of the product while reducing the need to discard high-producing clones and limiting reliance on manganese supplementation, ultimately saving time and costs.
