title: A fibrous-bed bioreactor for continuous production of monoclonal antibody by hybridoma.
abstract: A fibrous-bed bioreactor (FBB) has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibody (MAb). A non-woven polyester fibrous matrix was used to immobilize the cells to reach a high viable cell density of 3 x 10(8) cells cm(-3) packed bed, which gave a high volumetric MAb productivity of 1 g L(-1) day(-1) under continuous feed conditions with the medium containing 10% serum. Reducing the medium serum content to 1% increased MAb production to 6.5 g L(-1) day(-1) in a repeated batch FBB culture. MAb production was higher at higher dissolved oxygen (DO) levels in the range between 10% and 70% of air saturation, although DO did not significantly affect glucose metabolism and lactate production. The medium LDH (lactate dehydrogenase) level increased dramatically when the DO level was decreased from 30% to 10%, suggesting that a critical DO level of approximately 30% is necessary for maintaining the FBB culture for long-term operation. Compared with suspension cultures in T-flasks and spinner flasks, the FBB culture had a lower lactate yield from glucose (0.80 vs. 0.91 g g(-1), produced MAb at a higher concentration (up to 442 mg L(-1) vs. 83.5 mg L(-1), and was stable for continuous long-term operation (more than 1 month). The superior FBB performance was attributed to the highly porous fibrous matrix that enabled the efficient mass transfer, cell immobilization, and continued growth and regeneration that are critical to maintaining a high density of viable and productive cell populations. The cells immobilized in the fibrous matrix had high viability (>85%) even though many of them were in growth arrest (G1/G0 phase) as indicated by their smaller cell size (<10 microm). Scanning electron microscopic studies of the cell-matrix showed that the high density of cells formed large clumps within the interstitial spaces of the fibrous matrix. Their close contact and interaction with each other might have contributed to their ability to survive well under adverse conditions such as low DO and low serum content in the medium. It was also found that the cells present inside the fibrous matrix had a higher viability and lower apoptosis than those present in the liquid suspension, indicating that the fibrous matrix had selectively retained healthy, nonapoptotic cells and dislodged apoptotic and dead cells; this also might have contributed to the stability of the long-term culture. This work demonstrated that the FBB originally developed for microbial fermentation also gave excellent results in achieving high cell density, productivity, and product concentrations, and should have a good potential for industrial animal cell culture applications.
authors.affiliation: Department of Chemical Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210, USA. yang.15@osu.edu
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.forename: Chunnuan
authors.initials: C
authors.lastname: Chen
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification*; D017209:Apoptosis / Q000502:physiology; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D018914:Cells, Immobilized; D004867:Equipment Design; D006825:Hybridomas / Q000166:cytology* / Q000378:metabolism*; D008567:Membranes, Artificial; D008954:Models, Biological; D013673:Technology Assessment, Biomedical; D013678:Technology, Pharmaceutical / Q000295:instrumentation* / Q000379:methods / Q000639:trends
chemical_list: D000911:Antibodies, Monoclonal; D008567:Membranes, Artificial