Therapeutic Immunology Group:
Large scale production of monoclonal antibodies
A note about scale:
The methods described here are most suited to the 1-10g production range of a normal rat or mouse hybridoma. We would recommend the following methods as a rough guide for different scales of operation:
Why the Therapeutic Immunology Group need LARGE quantities of antibodies
We are studying transplantation tolerance induction and treatment of autoimmunity with CD4, CD8 and other T-cell specific monoclonal antibodies in vivo. Experimental studies use 1-10mg/experiment while pre-clinical studies use 100-1000mg/course. Therefore we need to produce 10-40 g each of 6-8 different monoclonal antibodies per year (preferably free of endotoxin), and it is this that has led us to develop our own methods for large scale production. In addition, a number of genetically engineered humanized mAbs and structural variants are required from transfected cells in sufficient quantitiy for in vitro functional and structural studies, in some cases leading to pre-clinical studies. Therefore, in total we require 1-10 g of each of a number of mAb variants to allow selection of those which can be moved to pilot scale production in the Therapeutic Antibody Centre (TAC) for clinical trials.
For laboratory experimental purposes we do not need the high levels of quality assurance required by the TAC, and we do not wish to have to optimise growth conditions for a whole range of different cell lines. We therefore chose a hollow fibre based method that has the following advantages:
Total experimental antibody production target is 150 g per year, but total budget allowed for consumables is 30,000 pounds/year. Therefore we must keep costs below an average of: 200 pounds per gm.
Strategies to minimize mAb production costs
Ascitic fluid must be phased out, but we need antibody produced at high concentration/small volume to minimize handling and purification costs.
Equipment must have low cost consumables: we use hollow fibre based on cheap kidney dialysis cartridge.
Run system in air in available 37deg C warm room to cut costs of gassed incubators, and for convenience (we also use trolleys carrying both the bioreactor and medium bags so that sterile connections etc can be made in a hood).
Select high producing cell lines/clones where possible, but definitely reclone until all clones are positive for antibody production over 3 month period to avoid outgrowth of negative variants.
Major cost component is medium:-
A home-made hollow fibre bioreactor system(NB: Antibody product not suitable for clinical use)
We have developed a home made bioreactor system that is efficent and easy to run in a normal laboratory environment. A simplified diagram of the system is shown in Figure 1. The hollow fibre bioreactor itself is a kidney dialysis cartridge with essentially two circuits for liquid flow - the intracapilliary (IC) circuit, in the lower half of the diagram, is used to rapidly circulate serum free medium from the 20L reservoir, via the peristaltic pump. After passing through the cartridge, where the nutrients and gases are exchanged with the extracapilliary (EC) space (that contains the growing cells), it is returned to the reservoir via a length of gas permeable silicon rubber tubing that allow re-oxygenation and release of carbon dioxide. The cells grow on the outside of the capilliaries, where they are fed by medium containing 5% FCS (or a suitable FCS replacement) from a 1L parenteral nutrition bag (as used for hospital drips). The antibody secreted by the cells and any dead cells are removed daily by using the simple push-pull syringe arrangement to pump the harvest into the collecting bag, and at the same time replenishing the feeding medium. Harvests of 50-100ml per day per cartridge are possible once the cells are established, and yields are usually from 0.5 to 5 mg/ml depending on the cell line.
For simple methods of purifying endotoxin free antibodies click here
Cartridges on trolleys in a warm room
Particular points of the system that are novel and we think most valuable are:
Hints and Tips
Example of an actual Home-made Bioreactor Run
"Developing short-term treatments for long-term outcomes"
Last Updated 27th January 2003 by Steve Cobbold