Long-term Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in a Fibrous-Bed Bioreactor

Abstract

The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of approximately 8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 x 10(8)/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of approximately 0.5 g/l, with reactor productivity of approximately 7 mg/h.l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to approximately 0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of approximately 0.3 g/l with less than 8% drift. At the end of the study, it was found that approximately 25% of the cells were strongly attached to the fiber surfaces and the other approximately 75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of approximately 90%, compared to approximately 75% for cells weakly immobilized and only approximately 1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

“…The increase in the number of cell subcultures performed over a time, or cell passages over a time (number of cell passages), could favor the growth of unproductive cell cultures, reduce productivity and reduce the mAb protective potential against infections (Xin and Cutler 2006;Zhu and Yang 2004).…”

Technological progresses in monoclonal antibody production systems

“…Although large-scale production of mAbs by mammalian cells is usually done in suspension culture, continuous efforts are being made to develop high-density adherent cell culture systems. This is a consequence of the adherent growth being the natural state for mammalian cells, 4 with problems related to suspension culture, such as sensitivity to hydrodynamic shear forces 59,60 and time-consuming processes of adaptation, 61,62 not being an issue in the adherent cultures.…”

“…66 Other immobilization systems include small beads of different materials 67 (agarose, 68 gelatin and alginate 67,69 ), gel particles (collagen 70 ), membranes, [71][72][73] and fibers. [74][75][76][77] All these systems are ideal for perfusion culture, 11 which increases cell concentration in the reactor (10 7 -10 8 cells/mL) and extends the productive lifetime of cells, 60,74 resulting in increased levels of mAb productivity when compared with suspension culture. 60,74 Furthermore, immobilized cell reactors have other advantages such as simplifying downstream processing because of cell retention, 78 which provides cell-free products, 60,74 creating a favorable microenvironment for cell growth and mAb production.…”

“…[74][75][76][77] All these systems are ideal for perfusion culture, 11 which increases cell concentration in the reactor (10 7 -10 8 cells/mL) and extends the productive lifetime of cells, 60,74 resulting in increased levels of mAb productivity when compared with suspension culture. 60,74 Furthermore, immobilized cell reactors have other advantages such as simplifying downstream processing because of cell retention, 78 which provides cell-free products, 60,74 creating a favorable microenvironment for cell growth and mAb production. 60,78 However, these bioreactors still have limitations that result in a problematic long-term use and scale-up.…”

“…60,74 Furthermore, immobilized cell reactors have other advantages such as simplifying downstream processing because of cell retention, 78 which provides cell-free products, 60,74 creating a favorable microenvironment for cell growth and mAb production. 60,78 However, these bioreactors still have limitations that result in a problematic long-term use and scale-up. For example, in bioreactors that use beads, destruction of the beads can occur with cell leaking and washout, resulting in declined mAb production over time.…”