Therapeutic Immunology Group:
Large scale production of monoclonal antibodies
See also:
Request form for academics who would like TIG cell lines to make their own monoclonal antibodies.
Some notes about making and growing Y3 derived 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:
- high antibody concentrations that minimise and simplify purification
- no need to develop serum free lines/optimal medium combinations
- minimal serum usage
- simple to keep sterile and endotoxin free
- low capital and consumable costs
- relatively compact and convenient
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:-
- negotiate bulk purchase in 20L disposable bags: we use a special formulation of IMDM w/o HEPES from Gibco/Life Technologies (Cat No: 041/91344R).
- use serum at 5% only on cell (EC) side.
- use HEPES (the major cost component) free medium .
- use high glucose, rich medium (eg. IMDM) likely to give best yields for a range of different cell lines (with minor modifications can be made suitable for Glutamine Sythetase selection).
A home-made hollow fibre bioreactor system
(NB: Antibody product not suitable for clinical use)
Figure 1We 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:
- Run in warm room on trolleys (cost/space/convenience): however, it can also be run on a normal laboratory bench if the circulating medium is warmed by using a plastic coil (eg."surgical blood warmer") inserted in the IC circuit just prior to the cartridge that can also be insulated with "bubble wrap"
- Medium supplied commercially, on demand, in 20L bags with all connections in place/HEPES free (cost/QA/containment)
- Pump - high capacity [greater than 200ml/min] (improves gas/nutrient exchange and avoids air bubbles trapping in cartridge) - twin head possible (seeding second cartridge)
- Sample ports (both IC and EC) - 3 way taps with sample drawn through 0.2 micron filter (containment)
- Feed to EC (medium + 5% FCS + 5mM HEPES/HCl) and harvest from EC with sterile parenteral nutrition bags (containment)
- Harvest with 50ml push-pull syringe on 3 way tap (containment)
Hints and Tips
- Every hybridoma/cell line/clone behaves differently (production vs growth [dead cells], oxygen, glucose and glutamine requirement). Use rich medium to minimize.
- Clone and select for high producing, long term stable, and 'moderately sticky' cell lines.
- Production may depend on EC (cell side) FCS concentration (5% min), unless you have been able to specially adapt cells to serum free production.
- Can monitor harvest by native gel electrophoresis when >0.2 mg/ml (also cross-checks mAb identity).
- Monitor glucose with urine test strips - less easy to read than blood sticks but 1/10 the cost (Fresh medium 25mM [reads 28], change when between 7-14mM on stick). However, some cell lines (especially those derived from NS0) seem to use glutamine rather than glucose and these cannot be easily monitored this way. Such lines also tend to be low producers of lactic acid and so do not maintain their pH without help by addition of extra acid (5mM HCl daily up to a total of 20mM, until established).
- If running in air, charge cartridge with as many cells as possible (5-50x10**8) from roller culture, with bolus of FCS on EC (cell) side (20-40%) to "coat" fibres (also see previous paragraph). 7) Seed second cartridge in parallel with first to sustain long runs (feeder effect, especially in air). 8) Circulate IC (non-cell side) medium at greater than 200 ml/min to avoid bubbles and possible air lock. 9) Run antibiotic free - avoids low level bugs/mycoplasma and gives option of adding curatively if infection on IC side (LPS does not pass fibres).
- Adjust harvest time/volume to maintain high product concentration.
- For higher production and establishing more difficult lines use a cartridge made from triacetate membranes (eg Hy-Med Ltd Nipro FB210U) that although cost approx. 30 pounds each we have found to be more reliable for growing NS0 derived hybridomas or transfectants than the cheaper cartridges (eg Althin AltraNova 140)
Example of an actual Home-made Bioreactor Run
Cell line:
YTS 177.9.6.1 rat IgG2a anti-CD4 (DA spleen x Y3/Ag1.2.3 hybridoma)
NB. This is a reliably good producer by all methods (eg 100 micrograms/ml in roller culture)!
Cartridge:
Initial seeding with 2x10**8 cells only - this took 2 weeks to reach optimal production and we would normally use more cells.
Medium:
Total usage over 6 weeks: 40L (cost 80 pounds)
Harvest:
Total harvest over last 4 weeks: 2.15L (average of 77ml/day)
Production:
Total mAb after 50% SAS purification: 3.2 g (avge. of 114mg/day at greater than 1.5mg/ml)
N.B. final mAb greater than 90% pure [native gel].
Other costs:
Estimate 40 pounds per litre of harvest for cost of nutrition bags, FCS, filters, syringes etc.
Total cost:
80 pounds; consumables + 80 pounds; medium + 12 pounds; cartridge (AltraNova 140) = 172 pounds total (for 3.2 g)
ie. approx. 50 pounds per gm (excluding labour/purification/hidden and indirect costs).
"Developing short-term treatments for long-term outcomes"
Related topics:
Last Updated 27th January 2003 by Steve Cobbold