SBIR-STTR Award

Increased production of protein from senescent cells. Monoclonal antibodies (MAbs) are emerging as robust therapeutic options in the fight against cancer and autoimmune diseases. The estimated market in 2004 for therapeutic MAbs is more than $7 billion from 16 FDA approved biopharmaceutical products. Kilogram quantities of MAbs are produced on in large-scale bioreactors from traditional hybridoma cultures or genetically engineered mammalian cells. Bioprocessing of MAbs is a prohibitively expensive endeavor, costing nearly $10,000 per gram. There is a tremendous need for technologies that can make bioprocessing of MAbs easier and reduce production costs to make mAb therapies more affordable. Our company has developed a novel technology that produces premature senescence of cells in culture resulting in a 30-fold enhancement in the production of secreted proteins. The major goal of the proposed Phase I project is to translate this senescence technology into commercially relevant hybridoma cells. Specifically, plasmid or retroviral vectors with tetracycline-inducible promoters driving expression of cyclin-dependent kinase inhibitors are introduced into hybridoma cells. Following antibiotic selection, premature-senescence is tested in populations of induced-transformed cells, as measured by relative cell growth, DNA cell cycle analysis, and expression of senescence-associated biomarkers. Senescence-competent hybridoma cell lines are isolated by limiting dilution and tested individually for enhanced production of immunoglobulin. Future Phase I project goals are to transfer senescent-competent cell lines developed in this Phase I project into commercial-scale bioreactors to assess mAb production on a large scale. We intend to license this technology to biopharmaceutical companies and contract manufacturing laboratories to reduce their cost in bioprocessing of therapeutic proteins from mammalian cells.

Thesaurus Terms:
biotherapeutic agent, cell senescence, chemical synthesis, drug design /synthesis /production, monoclonal antibody, protein aging, biomarker, combinatorial chemistry, cyclin dependent kinase, enzyme inhibitor, plasmid, tetracycline Retroviridae, SDS polyacrylamide gel electrophoresis, enzyme linked immunosorbent assay, polymerase chain reaction, transfection /expression vector

Most commercial therapeutic proteins, such as monoclonal antibodies (Mabs), are produced from traditional hybridoma cultures or genetically engineered Chinese hamster ovary (CHO) cells using large scale : bioreactors. Bioprocessing of therapeutic proteins is exceedingly expensive because of the high costs of , building and maintaining FDA-approved facilities, complex serum-free media, and talented labor. There is a tremendous market need for methods that can enhance protein production that ideally complement the current methods of production. The overall goal of the proposed project is to evaluate the effectiveness of senescence-enhanced protein production (RP Shift) in commercially relevant bioreactor setting. In the Phase I portion .of this project, Mab production was enhanced nearly 20-fold from RP Shift-competent hybridoma cells in small flasks. The Phase II portion of this project addresses the effectiveness of the RP Shift in commercially relevant bioreactors, the most commonly used being batch and flow-through systems. The aims of this Phase II proposal are to monitor commercially relevant endpoints from RP Shift-competent CH450 and MH70 hybridoma cells that were engineered in the Phase I portion of the project. Specifically, 1) enhanced productivity of Mab (in pg/cell/day monitored daily), 2) increased culture lifetime measured in days), and 3) the stability and integrity of the Mab produced will be measured. First cell lines with increased productivity of at least 2-fold during RP Shift in small volume test systems will be isolated. Of these RP Shift competent cell lines, the best two will be transferred to commercial scale bioreactors and examined for enhanced protein production and increased bioreactor lifetimes. It is expected that enhancements of at least 2-fold in large-scale bioreactors will attract interest from biopharmaceutical manufacturers. During Phase III, the RP Shift technology and ready-to-go RP Shift-competent cell lines will be licensed to biopharmaceutical manufacturers in an effort to reduce costs for them and ultimately the general public.

Thesaurus Terms:
bioreactor, cell senescence, protein biosynthesis, technology /technique development, tissue /cell culture CHO cell, biotherapeutic agent, cell line, hybridoma, monoclonal antibody biotechnology