Generation and monitoring of cell lines producing humanized antibodies

Antibody humanization has eliminated or reduced the human antimouse antibod y response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hW12 (anti-idi otype to the anti-carcinoembryonic antigen antibody MN-14). All humanized a ntibodies demonstrated immunoreactivities comparable to their parent counte rparts. Previously, we reported the generation of high productivity cell li nes for hMN-14 and hLL2 using the amplifiable vector pdHL2, Through amplifi cation, selection, and cloning procedures, cell lines capable of large scal e production were established, and further enhancement of production was ac hieved by a fed-perfusion bioreactor process. Using a similar end improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the conc entration of methotrexate in the culture media. A reliable IgG determinatio n method is essential to monitor amplification, especially at the final clo ning stage, for the selection of the subclones with the highest productivit y. We found that measurement of humanized IgG concentration in culture medi a supplemented with more than 1 mu M methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and re producible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures, The assay is simple and sensitive, and it ma kes simultaneous determinations of relative antibody production from indivi dual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell Lines ca n be monitored in an efficient manner.