Improving Antibody Production in Stably Transfected CHO Cells by CRISPR-Cas9-Mediated Inactivation of Genes Identified in a Large-Scale Screen with Chinese Hamster-Specific siRNAs.

The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.

Generation of cell lines for monoclonal antibody production.

Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes.

Rapid protein production using CHO stable transfection pools.

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post-transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence-activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three-fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin-opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines.