A 96-Well Plate Assay for Relative Monoclonal Antibody Titers

LEVEL: INTERMEDIATE M onoclonal antibodies are used to treat a wide range of illnesses such as rheumatoid arthritis, Crohn’s Disease, and follicular non-Hodgkin lymphoma (1). The identification and use of certain growth medium additives and modified culture conditions during cell culture processes that produce MAbs may lower production costs—which could help lower the overall costs of those drugs. Additives such as dimethyl sulfoxide, sodium butyrate, and rapamycin have been reported to increase MAb titers by 30–120% in various cell culture systems (2–5). Changes in culture conditions such as temperature and osmolality are also shown to influence MAb titers to similar extents in certain cell culture systems. For example, increasing culture temperature from 33 °C to 39 °C more than doubled specific MAb production (from 0.07 to 0.15 × 10–9 mg/cell/h) in one case, whereas increasing osmolality of the medium (from 286 mOsm/kg to 398 mOsm/kg) caused a 55% increase in specific MAb productivity for another (6, 7). Unfortunately, the effects of adding compounds or changing conditions on MAb productivity and titer are variable and unpredictable for any given cell culture system. One example is the reported 1.4-fold increase in production of a recombinant protein for a culture of Chinese hamster ovary (CHO) cells in the presence of 1% DMSO, conditions that suppressed MAb production in three hybridoma cell lines (8). In another work, just two of five hybridoma cell lines tested with retinyl acetate showed a significant increase in MAb titer (9). And switching from a serum-containing to a serum-free medium affects productivity unpredictably. In one case, the switch led to an increase in MAb titer from 3.5 μg/mL to 9.0 μg/mL (10). Highly variable and unpredictable cell line responses complicate selection of enhancers and optimization of cell culture conditions. Thus, a methodology is needed for quickly and efficiently assessing how reported enhancers and/or modified culture conditions will influence the MAb titer in a particular cell line or newly created clone under investigation. Presumably there are more compounds and conditions that could serve to increase MAb titers, and quickly identifying them would be useful. Our objective was to demonstrate a high-throughput capability for identifying statistically significant differences in 72-hour MAb titers for test and control conditions. To this end, we developed a 96-well plate assay for relative MAb titers that consists of a 72-hour batch culture followed by a MAb ELISA. Our assay is demonstrated for control hybridoma cultures and those provided with select concentrations of rapamycin (5), sodium butyrate (3, 4), dimethyl sulfoxide (2, 8), and sodium chloride.