Biopharmaceutical manufacturing plays a critical role in global healthcare systems. Methods and techniques for protein expression in upstream bioprocesses can have the highest impact on product quality and safety and, ultimately, on delivering effective means of treating and curing life threatening diseases. In addition to novel products, development and regulatory approval of biosimilars require precise matching of the quality attributes between lots of reference innovator drug and those of the biosimilar candidate. With continuing pressure to reduce the cost of these lifesaving medicines, increased bioprocess yields are also key to reducing cost of manufacturing and allowing for lower prices at the pharmacy. With these goals in mind, a novel biomanufacturing platform was developed that harnesses the commercially established murine NS0 myeloma host cell in new ways to create stable, highly productive manufacturing cell lines. Cholesterol metabolic markers were screened to identify the optimal enzyme, Hsd17b7, to enable stable cell line selection and eliminate the need for cholesterol addition to the bioprocess. Enhancement of the expression vector promoters, isolation and cloning of highly efficient hybridoma-derived heavy and light chain signal peptides and optimization of coding sequences increased monoclonal antibody expression by 10-fold. The development of novel multiplex selection strategies that combines antibiotic selection with cholesterol and glutamine metabolic selection both sequentially and in parallel allowed for the rapid and consistent generation of commercial grade cell lines with greater than 1 g/L yield potential. The platform was demonstrated to be highly valuable in developing the most complex of next generation biologics and commercially significant for the production of biosimilar monoclonal antibodies whose reference drugs are currently manufactured in murine host cells.