Three-Dimensional Culture for Monoclonal Antibody Production by Hybridoma Cells Immobilized in Macroporous Gel Particles

摘要

Cell proliferation and long-term production of monoclonal antibody IgG_(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7x9 mm~2 ). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM α-ketoglutarate or L-alanyl-glutamine (GlutaMAX~(TM)) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + α-ketoglutarate was similar to cells grown on Gluta-MAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10 cells mL~(-1)) and highest specific mAb production rate (3.9 μg mL~(-1) 10~(-4) viable cell day~(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reseryoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoctonul-antibody production.