Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles

Abstract

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 (7 x 9 mm2). 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™) 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 × 106 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 reservoir 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 monoclonal antibody production. © 2008 American Institute of Chemical Engineers.