Yoshiharu Takazawa

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.

High cell density perfusion culture of mouse-human hybridomas

SummaryFive mouse-human hybridomas, H2, H3, V1, V2 and V6 cells secreting anti-virus human monoclonal immunoglobulin G (IgG) were cultured in serum-free media at high density in a settling perfusion culture vessel with an inner cell sedimentation zone. The H2, H3 and V6 cells reached a density of 107 cells/ml in 0.5% (w/v) BSA-ITES-eRDF (see Materials and methods). The H2 cells reached only 6.8 × 106 cells/ml in the absence of bovine serum albumin (BSA), but the addition of 0.2% (w/v) Pluronic F68 increased the maximum cell density to 1.1 × 107 cells/ml, which was the same level as in BSA including medium. On the other hand, Pluronic F68 showed no stimulative effect on the growth of H2 cells in static culture. Pluronic F68 also increased the maximum cell density of V2 cells from 4.6 × 106 cells/ml to 6.9 × 106 cells/ml even in the presence of 0.5% (w/v) BSA.

Production of human-mouse chimeric antibody by high cell density perfusion culture.

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×107 cells/ml and produced chimeric antibody to a maximum value of 60 μg/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×107 cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 μg/ml.

Transferrin recycling perfusion culture of hybridoma cells

A perfusion culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg ml−1 in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2≈3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the culture supernatant.

Enhancement of γ -Carboxylation of Recombinant Activated Protein C by Cell Fusion

Suspension adapted low-aggregate BHK expressing activated Protein C (APC) was fused with human embryonal kidney cell line 293 to make a hybrid that have both low aggregation property derived from BHK and high γ-Carboxylation activity derived from 293. A direct expression plasmid for recombinant APC containing dhfr gene was constructed and introduced into low-aggregate BHK, and amplified with MTX. The best clone of the gene amplified BHK was fused with 293 transfected with pSV2neo, and one clone of the hybrid was investigated, γ -Carboxylated APC productivity of the hybrid in static culture was sixty to seventy percent higher than that of BHK, and stable at least for three months. In the case of suspension perfusion culture, it was about two times higher than that of BHK. The hybrid cells did not form large cell aggregates unlike 293. The appearance of cells in suspension culture was much closer to low-aggregate BHK than 293. These results show that hybridization is one of the useful methods to improve post-translational modification of recombinant proteins.