MONOCLONAL ANTIBODIES

"I have used FiberCell™'s hollow fiber chambers for many years to produce high titre monoclonal antibodies. They are easy to use and produce a highly concentrated product that is easily purified. An excellent alternative to ascites or flask production."

--David Winkler -- Bethesda, Md.

The FiberCell Systems hollow fiber system is the method of choice for the in-vitro production of 100 mg to two grams of monoclonal antibody per month. A hydrophilic polysulfone fiber with a MWCO of 20 kd is used. This permits the inhibitory factor TGF beta to freely diffuse away while trapping the higher molecular weight antibody along with the hybridoma cells in the extra capillary space. The polysulfone fiber has a filtration rate that is 10 times higher than conventional hollow fibers permitting cell densities greater than 10⁸/mL and antibody concentrations in the range of 0.5 mg to 5 mg per ml. Harvest volume is only 20-40 mls and each cartridge will produce antibody for up to 6 months. Adaptation to serum free medium is facilitated or serum requirements can be reduced by as much as 5 fold. Since no animal host is required (such as mice or nude mice) chimeric (mouse/human) and non-murine antibodies can be easily produced. Contaminants such as lipids, endotoxin, proteins and viruses are reduced or eliminated. Purification is simplified and overall yields are improved.

• C2011 will produce between 10mg and 50mg of antibody every two days
• C5011 will produce between 35mg and 150mg of antibody every two days
• Low harvest volume of 15 to 30 mls
• Cartridges can be maintained for over 6 months of continuous production
• Endotoxin burden is 10X lower than ascites fluid, ideal for animal injections
• Easy adaptation to reduced serum or serum free medium
• Chimeric, humanized and non-murine antibodies can be easily produced

Hybridoma cells growing at high density in  the C2011 cartridge using CDM HD medium

To demonstrate the utility of CDM-HD, a direct comparison was performed between monoclonal antibody production from the same standard murine SP2 clone in a 20 liter wave bag containing 10 liters of medium, T175 tissue culture flasks (1.2 liters consumed, data normalized to 10 liters for comparison) and a FiberCell Systems C2011 medium 20kd MWCO hollow fiber bioreactor cartridge, set up according to the manufacturer's protocol and allowed to consume 10 liters of medium. Flask data and Wave bag data were generated using a commercially available serum free hybridoma medium (SFM), while FiberCell Systems data was generated using DMEM medium with 10% CDM-HD chemically defined serum replacement. Total protein was measured before and after Protein A purification and antibody quantitation performed on column eluent.

Figure one: Purified antibody and ratio of antibody to total protein for the three systems. Flask culture yielded almost as much antibody as the FiberCell Systems cartridge, however the antibody in the FiberCell Systems harvests is about 50X more concentrated. Note the large amount of extra protein from the wave bag, probably due to cell lysis. Non-antibody protein in the flask is from the serum free media, which is not protein free. FiberCell Systems culture when used with CDM-HD is free from exogenous protein and cell lysis.

Figure two: Antibody yield per 10 liters of medium consumed, total harvested antibody concentration prior to purification. Note the extremely high concentration of antibody in the Fibercell Systems hollow fiber bioreactor cartridge. 200mls of harvested supernatant was equivalent to 10 liters of culture supernatant from the other systems again reflecting the 50X higher concentration of antibody generated in the FiberCell Systems bioreactor cartridge.

These data represents a direct comparison between a Wave bioreactor bag, standard T175 tissue culture flasks and a medium sized FiberCell Systems C2011 hollow fiber bioreactor cartridge. The FiberCell Systems cartridge produced 6 times the amount of antibody than the Wave bag produced at 50 times higher concentration with no exogenous protein. The FiberCell Systems cartridge produced slightly more antibody than the T175 flasks but at a concentration 50X higher with no detectable exogenous protein. CDM-HD is optimized for higher density cell culture, and therefore the media employed in this comparison were not identical. The comparison demonstrates the power of the combination of hollow fiber bioreactors and a medium designed to take advantage of this higher cell density. It should also be noted that the Wave bag is a 1-2 week batch mode process, while the FiberCell Systems cartridge can be maintained producing antibody for more than 6 months if so desired, further increasing amount of antibody produced per reactor cycle or annum.

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