Monoclonal Antibody Production Using a New Supermacroporous Cryogel Bioreactor

Abstract

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.

Cryogels for Biotechnological Applications

“…Small gel plugs with modified surfaces for use as efficient cell culture media have been used in continuous processing for production of urokinase (Figs. 4 and 5) [52] and monoclonal antibodies [43,53]. In this latter study, cryogel that was surface modified (also within the pores) with gelatin was produced within plastic housings C that was crosslinked with 10 μL glutaraldehyde.…”

Technological progresses in monoclonal antibody production systems

“…3 This small-scale cell culture provides sufficient amounts for research and diagnostic purposes. However, the market needs for therapeutic products require large-scale manufacturing capacity, 3 with demands on mAbs of 100-1,000 kg/year, which can only be achieved in large bioreactors and by efficient processes that produce several kg/day. 8,9 Systems for small-scale production Small-scale culture systems usually have a simple design and a low level of instrumentation and control.…”

“…They constitute more than 30% of total biopharmaceutical production 1,2 and are the largest class of proteins currently under clinical trials. 3 Several mAbs have been approved for use in therapeutic applications, as summarized in Table 1 (for a more extensive and detailed list that includes mAbs approved for diagnostic purposes as well as mAbs under development, consult the Immunogenetics (IMGT) webpage http://imgt.cines.fr/textes/IMGTrepertoire/ GenesClinical/monoclonalantibodies/#Approval_antibodies).…”

Efficacy of supermacroporous poly(ethylene glycol)–gelatin cryogel matrix for soft tissue engineering applications

“…Different conventional scaffolding technologies [3] have been used but cryogelation technique has an advantage over other fabrication methods and provides a suitable engineered 3-D surface known as cryogels similar to the native extracellular matrix. These cryogel matrices posses ideal characteristics like large pores, high porosity, high mechanical strength and scale-up opportunities seem to offer a promising alternative towards the treatment of many diseases [4][5][6][7][8][9][10][11][12]. The aim of this study was to design 3-D cryogels by cryogelation technique which shows its potentiality for different soft tissue engineering applications such as neural, cardiac and skin.…”