Trypsin is a commonly used dissociation reagent
Trypsin use in cell culture
Trypsin is a cell dissociation reagent that is used remove adherent cells from a culture surface. The most commonly available trypsin is derived from porcine pancreases. Trypsin 1X solutions can range from 0.025% to 0.5%. EDTA (trypsin EDTA) can improve the efficiency of the cell dissociation process.
The differences in the range of effective concentrations are due to varying trypsin purity, incubation time, dissociation buffer composition, and the cell type. Incubating cells with too high a trypsin concentration for too long a time period can damage and kill the cells. The use of soybean trypsin inhibitor (STI), or centrifugation, or both, is commonly used to inhibit the activity of trypsin prior to re-plating when cells are grown in serum-free media.
Challenges and Risks from the Use of Animal-Derived Trypsin
The use of porcine derived trypsin presents some challenges and risks in biomanufacturing. There have been several well-publicized contamination events caused by bovine/porcine agents. These include facility contamination at Genzyme by calicivirus 2117 that forced a costly facility shutdown and deprived patients of products, multiple contaminations of fermenters with reovirus, and the detection of porcine circovirus (PCV1) nucleic acids in live rotavirus vaccines.
The U.S Government, the EMA, and the WHO have recommended quality control standards when trypsin is used for the manufacture of human biological medicinal products. These standards include testing for the presence of advantageous agents and for pathogen inactivation. The FDA, EMA, and WHO support the use of non-animal derived dissociation reagents such as recombinant trypsin or other recombinant proteases.
The use of newer, recombinant proteases avoid many of the limitations and concerns of animal-derived trypsin in cell culture and in other applications. These alternatives include TrypLE™ (Life Technologies, fungal protease), rTrypsin (Novozymes; fungal protease) and recombinant porcine trypsin (Roche, recombinant porcine).
Other trypsin Information
Trypsin is a ~ 23 KDa serine protease that catalyzes the breakdown of peptide bonds containing lysine or arginine residues. Cell culture grade trypsin is produced via extract of pancreases, varies in purity, and contains varying degrees of chymotrypsin. Crystalized trypsin is of higher purity; however, animal-derived crystalized trypsin typically contains remaining chymotrypsin activity. Treatment of animal-derived trypsin with TCPK removes residual chymotrypsin activity, which is required for the production of some virus, such as influenza, and for the tryptic digests of protein.
Recombinant human albumin – Speeds up cell doubling times and enhances cell growth, maximizing antibody productivity.
Recombinant human transferrin – Animal Free and defined source of transferrin for optimal cell growth and productivity.
Recombinant insulin and transferrin cell culture media supplement formulated with selenium and ethanolamine for improved cell growth and productivity. Animal component free.
- Bethencourt V. (2009). Virus stalls Genzyme plant. Nature Biotechnology. 27:681.
- European Medicines Agency (Feb. 2014). Guideline on the use of porcine trypsin used in the manufacture of human biological medicinal products. EMA/CHMP/BWP/814397/2011
- Kunitz, M. (1945). Crystallization of a Trypsin Inhibitor from Soybean. Science. 101:668-669.
- Marcus-Sekura, et al. (2011) “Evaluation of the Human Host Range of Bovine and Porcine Viruses That May Contaminate Bovine Serum and Porcine Trypsin Used in the Manufacture of Biological Products.” Biologicals. 39(6). 359–369.
- Tokiwa, T., et al. (1979). Mechanism of cell dissociation with trypsin and EDTA. Acta. Medica. Okayama 33:1-4.
- Travis, J. (1967). The specificity of porcine trypsin. Biochemical and biophysical research communications 29:294-297.
- World Health Organization (2010). WHO recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks.
- Wu, Y. V., and H. A. Scheraga. (1962). Studies of soybean trypsin inhibitor. I. Physicochemical properties. Biochemistry. 1:698-705.
- U.S. Government (2012). 9 CFR 113.53 Requirements for ingredients of animal origin used for production of biologics.