Recombinant Trypsin, Animal Origin Free

Transcription

1 Recombinant Trypsin, Animal Origin Free PRODUCT INFORMATION: BioGenomics r-trypsin powder is ready to use, animal origin free optimized for cell culture applications. It is derived by r-dna technology. BioGenomics r-trypsin is a pure enzyme, which helps maximize the yield of functionally viable cells from culture vessels, while preventing the toxic effect induced by other non-desirable proteases. In addition, BioGenomics r-trypsin, being a recombinant product, completely eliminates the risk of viruses, or other potential adventitious agents found in animal derived products. It is most commonly used for dissociation and disaggregation of adherent cells. When the powder Trypsin is used in the solution form, a chelating agent like Ethylenediaminetetraacetic acid (EDTA) is often added to enhance the enzymatic activity of Trypsin solution. EDTA acts by neutralizing Calcium and Magnesium ions which increases the cell adherence onto the surfaces. FEATURES AND BENEFITS 1. Product Quality: BioGenomics r-trypsin is manufactured under stringent GMP environment. The robust manufacturing process ensures the final quality of the product as per the regulatory requirements. Robust process controls ensures high quality standards and consistency and eliminates batch to batch variability. 2. Non-animal origin: Derived as a Recombinant product from E coli, BioGenomics r- Trypsin eliminates the risk of viruses, TSE/BSE or other potential adventitious agents. 3. Enzyme inhibition: Soybean Trypsin inhibitor and other inhibitors have similar inhibitory activity for BioGenomics r-trypsin solution as they do with native Trypsin. 4. High purity: BioGenomics r-trypsin provides increased specificity and eliminates contaminating activities found in lower purity animal origin (Bovine/Porcine) enzymes.

2 5. Convenience: BioGenomics r-trypsin is formulated at the optimal concentration can that be readily converted to solution for use in dissociation of adherent cells. 6. Shelf life: BioGenomics r-trypsin has a proven stability period of 24 months when stored at -20 C. Gel picture showing comparative SDS-PAGE profile of Biogenomics recombinant trypsin with that of reference standard SDS-PAGE analysis of recombinant Biogenomics Trypsin performed under reducing conditions followed by Coomassie staining. Lane 1: Molecular weight marker; Lane 2: Recombinant Biogenomics Trypsin; Lane 3: Reference standard. TRYPSINIZATION Trypsinization is usually done with buffer composition of BioGenomics r-trypsin at a desired concentration with addition of chelating agents like EDTA which is stored at 2-8 C.This solution is stable for 24 months under the storage conditions of 2-8 C.

3 Standard Protocol for Trypsinization: 1. BioGenomics r-trypsin solution should be thawed at room temperature before its use. The container should be opened at room temperature in an aseptic area, ideally under a laminar air flow hood. 2. This r-trypsin solution can now be used in cell culture trypsinization process. 3. The excess cell culture medium should be removed by aspiration. The cell culture monolayer is then rinsed with a Ca 2+ and Mg 2+ free salt solution, such as Dulbecco s Phosphate Buffered Saline, or Hanks Balanced Salt Solution. 4. The salt solution is further replaced with BioGenomics r-trypsin. This process should be carried out by aspirating the salt solution out, followed by a refill of BioGenomics r- Trypsin solution into the vessels. It needs to be ensured that the r-trypsin completely covers the monolayer of the cells. 5. Incubate the vessel at 37 C, until the cells start detaching from the surface of the culture vessel. The progress of trypsinization shall be checked intermittently at regular time points like 1, 2 or 5 minutes or even more time points based on the cell line being used under the inverted microscope. To ensure proper detachment of the cells, the culture vessel shall be gently tapped. 6. When all the adherent cells become suspended and the cell morphology appears rounded under the microscope, it shall mark the successful completion of the trypsinization process and prove its efficiency. 7. Upon the completion of the trypsinization process, the cell culture suspension shall be diluted with media containing an appropriate concentration of serum. Suspended cells tend to form clumps and these shall be broken up by gently pipetting the cell culture medium. This cell culture suspension shall now be transferred to a centrifuge tube for further processing. 8. Centrifugation of the cell culture suspension shall be carried out at 100 x g for 5 to 10 minutes. At this stage, the Trypsin activity shall be inhibited by addition of Soybean Trypsin inhibitor. The centrifugation step shall be carried out multiple times to get rid of trypsin solution by re-suspending the cell pellet with fresh cell culture medium.

4 9. Checking the cell viability and cell counting after centrifugation shall be done and the cell pellet shall be further sub cultured into fresh culture vessel with cell culture medium as per the standard operating procedure of the end user. Note: 1. Different cell lines require different times for the process of dissociation from the surface. Major factors affecting the process duration depends on the cell type, cell density, potency of Trypsin, serum concentration in growth medium and time since last subculture. Excessive exposure of the cell culture with respect to Trypsin concentration and reaction time shall be avoided to prevent cellular damage. 2. Whenever there is a use of serum in cell culture medium it is important to use Soybean Trypsin inhibitor in 1:1 ratio to neutralize the overt action of Trypsin. Extensive studies have been carried out on BioGenomics r-trypsin with various cell lines to study the effect of trypsinization. The result of our studies on different cell lines is as highlighted below: Cell Line Vero MDCK CHO K1 MRC5 Optimum Dissociation Time minutes minutes 1 2 minutes 4 5 minutes SUMMARY BioGenomics has conducted extensive, comparative cell dissociation studies with multiple cell lines for serum free and serum supplemented adherent cell cultures. The study has been carried out specifically focusing on Trypsin of animal origin in comparison with BioGenomics r-trypsin. BioGenomics stands to endorse the enhanced performance of its product based on the studies conducted. BioGenomics r-trypsin has given exceptional results vis-à-vis cell viability greater than 95% under all our typical experimental conditions of 37 C for 10 minutes for CHO cell line, in addition to other widely used mammalian cell lines. BioGenomics has generated extensive data on different cell lines like Vero, CHO, and MRC5 etc. Kinetics of dissociation has always been better for BioGenomics r- Trypsin under our experimental conditions. The viability of dissociated and subsequently harvested cells have always been greater than 90% (Figure: 2).

5 Figure: 1: The below graph shows the comparison of BioGenomics r-trypsin with Animal Origin Trypsin available in the market Comparison of Activity of Animal Origin Trypsin and BioGenomics r-trypsin 4.7 Cell Number (Cells/ml) X 10^ Animal Origin Trypsin (0.125%) BG-rTrypsin (0.125%) Animal Origin Trypsin (0.25%) BG-rTrypsin (0.25%) Adherent cultures of multiple cell lines were used to evaluate the utility of BioGenomics r- Trypsin for use in cell culture. In this experiment, BioGenomics r-trypsin and animal origin trypsin powders were used for trypsinization experiment. The cell viability exceeded 90% in comparison to Animal Origin trypsin. (Figure: 2). Figure: 2: The graph shows comparison of the viability of cells of various different cell lines after dissociation with BioGenomics r-trypsin and Animal Origin Trypsin. MDCK cell line Viability of Dissociated cells CHO K1 Cell line Vero cell line Trypsin formulation % Viability BioGenomics r-trypsin is proven for its stability. Trypsinization efficiency of BioGenomics r- Trypsin remains unchanged over a period of 24 months. (Figure: 3)

6 Figure: 3: Graph showing the Trypsinization efficiency of BioGenomics r-trypsin on various different cell lines. Two Years Trypsinization Data For BioGenomics r-trypsin 20 Formulation 16 Time (minutes) Vero Cell Line CHO K1 Cell Line MDCK Cell Line Months STORAGE & STABILITY BioGenomics r-trypsin is supplied as lyophilized powder which is stable for 24 months at C. The solution made out of lyophilized r-trypsin is also proven to be stable for 24 months at C. BioGenomics r-trypsin in its solution form remains stable for a period of 6 months at ambient temperature when stored under under sterile conditions. RECOMBINANT TRYPSIN: KEY QUALITY PARAMETERS : APPEARANCE SOLUBILITY ACTIVITY PER MILLIGRAM OF SPECTROSCOPIC PROTEIN(1mg/Ml solution of recombinant trypsin has absorbance of 1.47 units at 280nm) ACTIVITY PER MILLIGRAM OF TOTAL White or almost white powder Soluble in N HCl and 20Mm Tris, Ph 8.0 at a concentration of 5mg/Ml.(For enzyme preparation for Insulin production and cell culture) An Amount equivalent to USP Trypsin units, soluble in 10 Ml of water and in 10 ml of saline TS Not less than 2500 USP units/mg Not less than 2500 USP units/mg

7 POWDER SDS-PAGE IDENTIFICATION BY SDS-PAGE IMPURITIES WITH MOLECULAR MASSES HIGHER THAN THAT OF TRYPSIN BY SDS-PAGE RATIO OF TOTAL SPECTROSCOPIC PROTEIN CONTENT PER MILLIGRAM OF POWDER The electrophoretic mobility of the sample should correspond to the electrophoretic mobility of reference standard. Not more than 5 % Not less than 0.5 RESIDUE ON IGNITION Not more than 2.5% LOSS ON DRYING Not more than 5% MICROBIOLOGY TEST a. Escherichia coli b. Staphylococcus aureus c. Pseudomonas aeruginosa d. Salmonella REFERENCES: 1. Trypsin USP monograph NF 2. On the mechanism of action of proteolytic inhibitors: IV. Effect of 8M urea on the stability of trypsin in trypsin-lnhibitor complexes". Archives of Biochemistry and Biophysics Voet & Voet (1995). Biochemisty (2nd ed.). John Wiley & Sons. pp Complete disassociation of adult pancreas into viable single ce lls through cold trypsin-edta digestion. Journal of Zhejiang University - Science B (Biomedicine & Biotechnology) 5. Facile Trypsin Immobilization in Polymeric Membranes for Rapid, Efficient Protein Digestion. Anal Chem December 15; 82(24): doi: /ac101857j 6. Culture of Animal Cell - A Manual of Basic Technique by R. Ian Freshney 7. Cell Dissociation with trypsin - Sigma Aldrich 8. Dissociation of cell monolayers using trypsin solutions - Corning cellgro. 9. Trypan Blue exclusion test of cell viability - Warren Strober; Current Protocols in Immunology.