Monoclonal Antibodies

“I have used FiberCell‘s hollow fiber chambers for many years to produce high titre monoclonal antibodies. They are simple to use and produce a highly concentrated product that is easily purified. An excellent alternative to ascites or flask production.”
– David Winkler, Bethesda, Md.

 

“Thanks for the great product!  I am actually more impressed with the Bioreactor now using the CDM-HD than I was using FBS. CDM-HD is cheaper than FBS, no purification required (Protein A or Melon gel), and the yields are about the same. “
– Dr. Erin Bromage, US Veterinary Immune Reagents Network

casestudy

Figure 1: Data courtesy of: Dr. Erin Bromage c/o the US Veterinary Immune Reagents Network

A hollow fiber bioreactor from FiberCell Systems is the ideal method for the in vitro production of 25mg on up to gram quantities of a monoclonal antibody. A technologically advanced high-gross-filtration rate hydrophilic polysulfone fiber with a molecular weight cut off  (MWCO) of 20kd is specified.  Hybridomas secrete an inhibitory factor called TGF-Beta that in its active form has a molecular weight of 27kd. The MWCO of the hollow fibers permits this inhibitory cytokine to diffuse away while the secreted antibody and cells remain in the small volume of the extra-capillary space. The gross filtration rate of the polysulfone fiber is 10X higher than that of cellulosic fibers for more rapid exchange of nutrients and waste products. Cell densities of 108 or higher are attained facilitating the reduction of serum, the adaptation to commercially available serum free formulations and the use of FiberCell Systems’ CDM-HD.

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

Figure 2: Hybridoma cells growing at high density in the C2011 cartridge using CDM-HD medium

CDM-HD is a chemically defined, protein free, animal component free CGMP compliant serum replacement optimized for hollow fiber and designed to take advantage of the unique cell culture conditions found within a hollow fiber bioreactor. Harvest volume is 20-40 mL and antibody concentration can range from .5 mg/mL to 5 mg/mL.  Since no animal host is required (such as mice or nude mice), chimeric (mouse/human) and non-murine antibodies such as rabbit, rat and hamster can be easily produced. We have demonstrated a reduction in apoptosis when cells are cultured in hollow fiber bioreactors. When used with CDM-HD, contaminants such as lipids, endotoxin, proteins, intracellular DNA and viruses are reduced or eliminated. This lack of contaminants can simplify or reduce the purifications steps required improving overall yields as well.

The scale-up of antibody production has just been made easier.  The C5011 cartridge can be managed in the same manner as the C2011 but with 2X greater productivity and twice the antibody concentration.  The C5011 is the same cartridge as the C2011 but with more than twice the oxygenation capacity.

Data shared by a FiberCell customer:

Catridge C5011
Cell Line SP2 Hybridoma
Medium H-SFM
Medium Consumed Per Day 1 Liter
Total Medium Consumed 66 Liters (11 weeks production)
Total Antibody Produced 1.7 grams after purification
Total Harvest Volume 450 mL
Average Concentration (after purification) 3.77 mg/mL

IMAG0054-mabP5291220-mabProduction of multiple gram amounts of a monoclonal antibody can be easily attained using the C5011 cartridge.

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 FiberCellFlask 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.

This 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 as 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.

 

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 3: 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. 200 ml of harvested supernatant was equivalent to 10 liters of culture supernatant from the other systems again reflecting the 50 X higher concentration of antibody generated in the FiberCell® Systems bioreactor cartridge.

Figure 4: 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. 200 ml of harvested supernatant was equivalent to 10 liters of culture supernatant from the other systems again reflecting the 50 X higher concentration of antibody generated in the FiberCell® Systems bioreactor cartridge.

 

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Figure 5: RAb M5114 Production results from the Fibercell Hollow Fiber System. Cartridge used was the C2011 20 kd MWCO polysulfone fiber and medium used was DMEM with 10% CDM-HD. Protocol followed was per the FiberCell Systems manual. The M5/114.15.2 monoclonal antibody reacts with the mouse major histocompatibility complex class II, both I-A and I-E subregion encoded glycoproteins. In the gel shown, purified M5114 was a positive control purified from Protein G column, lane 1. Lanes 2,3, and 4 are harvests from the cartridge on day 7, 9 and 12. No purification has been performed.  Note how clean the harvests are and free from extraneous proteins. Lane 5 is 10X concentrated supernatant from a T-flask using DMEM and 10% CDM-HD. Lane 6 is from a T flask using DMEM and 10% serum.  3 ug of control was loaded, and the supernatant from HFB was loaded with 2ul of each. The supernatant from T-flasks were 10x concentrated and i loaded 2 ul of each.  Based on the gel, it is estimated that the concentration of supernatant from HFB was about 2.5 mg/ml. 140 ml of supernatant was harvested to date for a total about 350 mg of non-purified antibody.  Both SDS-PAGE gel and gel filtration analysis showed that the antibody from the hollow fiber bioreactor was very clean and properly folded. The cartridge was allowed to run for several weeks after this initial data was collected, producing additional antibody. Data courtesy of Liying Lu, University of Massachusetts, Worcester, Worcester, Massachusetts.

Suggested ordering for Monoclonal Antibody Cell Culture

Bioreactor

P3202 The FiberCell® Systems Duet more info

Cartidges

Catalog No. Size Surface Area Fiber Type Packing Density ECS Vol MWCO 50% Max. Cell#
C2011 Medium 3000 cm2 high flux PS 50% 20 mL 20 kd 109 more info
C5011 Medium 3000 cm2 high flux PS 50% 20 mL 20 kd 2 x 1010 more info

Reservoir Caps

A1005 33 mm Reservoir Cap more info
A1006 38 mm Reservoir Cap more info

Serum Replacement

CDM-HD more info

 

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