Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors–separators

Abstract

Growth of a hybridoma culture, along with production of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules. The molecular weight cutoffs of the membranes were 70,000, 50,000, and 100,000 daltons. The hybridoma cell line, designated 65/26, produced IgG (2b/kappa) directed at mouse thymus cell surface antigen, TL.1. Cell growth occurred in the shell space of the reactor, using supplemented RPMI 1640 (20% fetal bovine serum) supplied from a separate reservoir vessel through the hollow fiber lumen. The reservoir contained 125 mL media, which was changed every 4 days. Concentrations of immunoglobulin were determined by an enzyme immunoassay (using protein A and alkaline phosphatase-labeled antibody conjugate). For the 10K, 50K, and 100K hollow fiber membrane modules, the maximum IgG concentrations detected in the 2.5-mL shell space were 47.5-80, 510, and 740 mug/mL, respectively. In the 125-mL reservoir for the 100K hollow fiber membrane module, the IgG concentration was measured at 260 mug/mL These values compare with an IgG concentration of 1 mug/mL when grown in a standard tissue culture flask and 3.2-7.6 mug/mL when grown in 100 ml media in a spinner flask. In addition, 10K and 50K hollow fiber membrane modules were run in a mode that decreased the fetal bovine serum supplement with time. Differences between these systems suggest that it is possible to obtain high IgG accumulation rates, both during and after the exponential growth phase of the hybridoma population.

Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production

“…Purity of the antibody product has been facilitated by gradual serum reduction in the culture medium which has been achieved in hollow fiber systems without significant inhibitory effect on production (42,45,46,50,51).…”

“…Use of microporous hollow fiber bioreactors with large fiber pore sizes, between 0.2 and 0.5 p, have been described (26,32,51,52). In comparison to ultrafiltration membranes, greater antibody production has been achieved with these microporous membranes (26,52), however the product is not retained within the small volume of the extracapillary space and must be concentrated from the large volume of perfusing media.…”

“…It should be noted that of the hollow fiber bioreactor publications cited, only 2 described commercially available systems similar to the small scale systems that were used in the current study (45,46 Each of the five hybridoma cell lines was grown in a group of 20 mice to provide data on monoclonal antibody production in murine ascites. Additionally, three of these cells lines, 2B11, 3C9, and RMK, were also grown in each of three different hollow fiber bioreactor systems to provide comparative production data from these in vitro systems.…”

Two-Stage Chemostat Studies of Hybridoma Growth, Nutrient Utilisation, and Monoclonal Antibody Production

“…The few reports that are available on the relationship between the cell cycle and monoclonal antibody synthesis suggest that the relationship is cell line dependent. Alshuler et al (1986) reported the detection of intracellular antibody throughout the cell cycle in mouse hybridomas while Al-Rubeai et al (1989) claimed production was G l phase-specific. Suzuki and Ollis (1989) proposed a cell cycle model based on experimental evidence which showed that antibody production was restricted to the late G l and S phases.…”

A Self-Feeding Roller Bottle for Continuous Cell Culture

“…Although several novel continuous bioreactors have been developed for the culture of suspended mammalian cells (1)(2)(3)(4)(5)(6)(7)(8)(9), conventional roller bottles still provide the simplest means for culturing suspended mammalian cells that are shearsensitive but still require some means of agitation for cell suspension and oxygen and nutrient dispersion. Although each novel bioreactor has its own advantages, such as improvements in cell density, viability percentages, monoclonal antibody secretion rates, etc., these systems are generally not as mechanically simple or as easily scaleable as are roller bottles, require special user training, and require capital investment.…”