Carole A. Heath

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.

Comparison of a quadroma and its parent hybridomas in fed batch culture.

A quadroma (#22 x 63), formed by the fusion of two hybridomas, and its parent hybridomas (#22 and FMC 63) were each grown in fed batch cultures in order to examine the change in antibody productivity over time of the quadroma compared to its parent hybridomas. The growth rate, glucose uptake rate and lactate production rate of the quadroma were found to be intermediate between those of its parent cells of origin. The specific antibody productivity and internal antibody content of the quadroma followed the same decreasing trends over time as those seen in both parent hybridomas. Losses in specific antibody production rate and antibody content, however, occurred at a faster rate for the quadroma than for either of its parent hybridomas. Although the growth of a non-producing subpopulation is presumed to account for the drop in antibody production, there was no direct correlation between the percentage of high antibody containing cells, as determined by flow cytometry, and the specific antibody production rate.

New Developments in Membrane Bioreactors

Although membrane bioreactors have been extensively used in the biotechnology industry for small-scale culturing of mammalian cells and to a lesser extent plant and insect cells, they remain for the most part, unused in large scale bioprocessing. We address the main reasons for this and review recent developments and opportunities in the design and application of membrane bioreactors.

Biotechnology Processes: Membrane Materials, Modules and Process Design

Pressure-driven membrane processes are being increasingly integrated into existing reaction, isolation, and recovery schemes for the production of valuable biological molecules. In some cases they are replacing traditional unit processes. Membrane systems are being used for both upstream and downstream processing taking advantage of their permselectivity, high surface area per unit volume, and their potential for controlling the level of contact and/or mixing between two separate phases. Advances in both membrane materials and module design and operation have led to better performance, i.e., control of concentration polarization and membrane fouling. After reviewing how membranes can be integrated into bioprocesses and presenting some recent advances in membrane materials, module design and fluid mechanics, we review established and emerging membrane separation processes. Several examples are used to emphasize the synergism between biological processes and synthetic membranes.