Monoclonal antibodies (Mabs) have been widely used in the diagnostics, separation and purification of proteins since their discovery in 1975 and recent application has been extended to therapeutic areas. Mabs are commercially produced by using homogeneous suspension cultures or immobilized cell cultures. But commercial culture systems may cause some problems in large scale applications. In this study, continuous cultures of high density hybridoma cells secreting Mabs against hCG were investigated for the production of Mabs.
In suspension cultures, culture characteristics such as specific rates of cell growth, Mab production, and glucose consumption were studied. In the case of hybridoma Alps 25-3, 0.065 $h^{-1}$, 1.0μg Mab/$10^6$ cells·h, and 0.1 mg glucose/$10^6$ cells·h were obtained, respectively. In comparison with batch or continuous suspension cultures, two or three times higher cell densities and Mab concentrations were obtained by using cell settler. But long term continuous culture failed, because the media conditions within cell settler got out of control.
In membrane cell recycle system, fragility of hybridoma led to cell death by disruption of cellular membrane, when they were recirculated by pumping systems. For successful operations, mild pumping system should be developed to recirculate suspended cells.
In immobilized cell culture systems, cells were immobilized and cultivated within dual hollow fiber, Ca-alginate capsule and cylindrical depth filter. In dual hollow fiber bioreactor (DHFBR), hybridomas could be successfully cultured in high density of ca. $1.87\times 10^8$ cells/mL for two months. The Mab productivity reached to 205 mg/L day. The mathematical analysis of oxygen transfer in DHFBR showed that oxygen transfer to immobilized cell matrix was improved much compared with conventional hollow fiber bioreactor.
Encapsulation of hybridomas using only Ca-alginate gel seemed to be simpler and more economical than the Lim's encapsulation process. High density cells of $1.5\times 10^8$ cells/mL in capsules resulted in high Mab productivity of 652.8 mg/L day. But long term continuous culture could not be carried out in Ca-alginate capsule system because supply of nutrients and removal of wastes were limited as grown cells packed the entire intra-capsule space, that was common to encapsulation systems. The analysis of oxygen transfer in the Ca-alginate capsules showed that oxygen transfer was seriously limited.
A newly developed depth filter perfusion system (DFPS) based on the immobilization of hybridoma cells within cylindrical depth filter matrix yielded a high Mab productivity of 774 mg/L day. The cells could be easily immobilized, and high density continuous culture could be successfully carried out in DFPS.
In the study of growth regulation of hybridomas, the cell growth was retarded by using growth inhibitors (hydroxy urea and thymidine), but culture time of hybridomas would not be elongated in batch stationary cultures. Inoculum cell density was found to be an important parameter in determining the viability of hybridomas cultivated in serum-deficient medium.
