Recombinant Proteins
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"I have been using FiberCell™ cartridges for the production recombinant proteins in CHO cells for five years. I routinely produce a half-gram of protein in two months in a volume of less than five liters. This type of production would not be possible using conventional methods. FiberCell™ Hollow-fiber systems have made an invaluable contribution to my research."
--Dr. James Arthos -- Bethesda , Md.
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Current methods for the production of secreted biologicals such as recombinant proteins, cytokines and conditioned medium involve the use of inefficient in-vitro culture systems such as roller bottles or large numbers of tissue culture flasks. FiberCell™ hollow fiber culture systems facilitate the easy culture of large numbers of cells (1-2 X 1010) replacing roller bottles or hundreds of flasks while concentrating the secreted protein 100X or more.
Secreted proteins can be 100 times more concentrated vs. tissue culture supernatant. |
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- Reduced serum requirements facilitates purification
- Small harvest volume for easy handling
- No splitting of cells required, cartridge maintenance just 15 minutes a day
- Cultures can be maintained for several months of production
- Optimal cell culture conditions can result in improved protein assembly and folding
Comparison (See chart below) of a purified recombinant human IgM produced from CHO cells grown in either a 150cm2 tissue culture flask (upper panel) or a FiberCell™ polysulphone cartridge (lower panel). Gel filtration chromatography reveals incomplete polymerization of the IgM produced in the flask. Both a highly polymerized IgM (peak A.) and non-polymerized (peak B.) are observed. In contrast, the same protein produced in a FiberCell™ cartridge is expressed almost entirely in highly polymerized form (peak C.) 478 mg of purified protein were produced in 2 months in a volume of less than 5 liters using catalog #2018.
SDS-Page gel of successive harvests from the ECS of a recombinant protein secreted by a 293 cell line. Initial medium is DMEM with 10% FBS (Monday). Medium is changed from DMEM-FBS to Hyclone serum free medium for 293 cells. Note the diminution of serum proteins, especially BSA in the harvest from the ECS while the protein of interest remains constant or increases as the adaptation continues. Protein of interest is higher in concentration on Mondays as it has accumulated over the weekend to a higher level.
Production of a hexeramized IgG consisting of 6 IgG1 subunits held together by 3 IgA tails in a CHO cell line. When grown in flask culture (top trace) about 40% of the protein is expressed as an incompletely folded monomeric sub-unit. When the same cells are culture in the FiberCell™ hollow fiber bioreactor module more than 90% of the protein is expressed as a properly folded hexamer. This is due to the superior cell culture conditions inside the hollow fiber cartridge. Mediums used was DMEM with 2% FBS. 475 mgs of purified protein was produced in FiberCell™ catalog number C2018 in a period of 8 weeks in a collected volume of less than 5 liters.
Production of a recombinant IgG in 293 cells using serum-free medium:
The medium 20kd MWCO FiberCell™ cartridge (catalog number C2011) was used to produce 276 mgs of protein in a total volume of 900 mls in 2 months using serum-free medium. The cartridge was run over the weekends with a one liter bottle of serum-free medium. This accounts for the extremely high levels of lactate and protein concentration seen on peak days. At it's highest point lactate was 3.8 mgs/ml, glucose less than 100 mgs/ml and pH below 5.9. Highest protein concentration was 900µg/ml. Although a certain amount of cell viability was lost the replacement of the medium with a fresh bottle resulted in continued cell growth and protein production. When grown in the FiberCell™ hollow fiber bioreactor cells most cell types demonstrate high resistance to lactate resulting in easier management of cell culture medium changes.
Adaptation of cell cultures from GMEM to serum free media
Figure legend: Proteins from cell culture supernatants containing whole IgG preparations of 2F5 antibody were subjected to SDS-PAGE gel electrophoresis and visualized via staining with Microwave Blue (Protiga). Lane 1 marker (in kDa from highest to lowest) = 225, 150, 100, 75, 50, 35, 25, 15, 10, 5. Lane 2 = Protein produced in GMEM media containing 10% FBS. Lane 3 = blank. Lane 4 = (begins migration to serum free media) Protein produced in GMEM media containing 4% FBS. Lane 5 = Protein produced in GMEM media containing 3% FBS. Lane 6 = Protein produced in GMEM media containing 2% FBS. Lane 7 = Protein produced in ¾ GMEM media containing 2% FBS and ¼ serum free media. Lane 8 = Protein produced in ¼ GMEM media containing 2% FBS and ¾ serum free media. Lane 9 = Protein produced in Serum free media. Lane 10 = Protein produced in Serum free media.
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