P-CHECK. Protease Detection Kit Manual and Instructions. distributed by. Contact Information

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1 P-CHECK Detection Kit Manual and Instructions distributed by Contact Information For any further information, questions or remarks please contact us. Jena Bioscience GmbH Loebstedter Strasse 71 D Jena Germany phone: fax:

2 1. Introduction 1. Introduction s are an enzyme class showing very diverse specificities and characteristics. Some have one specific substrate in vivo while some are much more promiscuous. This makes it very difficult to detect unknown protease contaminations, which are a major problem in production and storage of protein or peptide based pharmaceuticals and diagnostic assays. Most common assays for protease detection are based on the degradation of heavily fluorescencelabeled natural proteins such as casein, generating a fluorescence increase due to reduced quenching. These assays are quite sensitive and well established. However, there might be protease contaminations for which casein or other natural proteins are no suitable substrate and are therefore not detected. In contrast, the P-CHECK assay is based on a FRET pair (Mca = 7-Methoxycoumarin-4- acetamide as fluorophore, Dnp=dinitrophenyl residue as quencher) separated by a peptide library. The peptide library is five amino acids long, while each position is statistically occupied by one of all proteinogenic amino acids except cysteine. Therefore, the P-CHECK assay contains 19 5 = different possible substrates which detect a broad spectrum of proteases. In the uncleaved substrate, the nearby Dnp residue quenches the Mca fluorescence by Förster resonance energy transfer (FRET, Reference 1). pon cleavage, fluorophore and quencher are separated and therefore, FRET efficiency decreases and fluorescence increases (see Figure 1, References 2, 3, 5 and 6). 2. Materials Figure 1: Schematic illustration of the assay principle. X=one of 19 amino acids. P-CHECK kit contains: P-CHECK Substrate (FRET substrate) Mca (Mca standard) 10 ml of dissolving solution for P-CHECK Substrat and Mca standard 50 ml Working Buffer I for protease assays at ph ml Working Buffer II for protease assays at ph 2.8 Microtiter plate (Nunc F96 MicroWell Plates, non-treated, black) 2 11

3 3. Materials required but not provided Store kit at 2-8 C. Kit is designed for immediate use after dissolving of P-CHECK Substrate. 3. Materials required but not provided Microtiter plate reader/elisa reader, equipped with excitation filter of 320 nm and emission filter of 405 nm. Standard laboratory equipment (e.g. pipettes, pipette tips, reaction tubes) 4. Experimental Protocol 4.1. Instrument settings Incubate and measure your samples at 37 C (98.6 F) if possible. For detecting less thermostable proteases, you can also carry out the assay at room temperature. Adjust the excitation wavelength to 320 nm and the emission wavelength to 405 nm. We recommend measuring in intervals of five minutes for four hours. However, since this can cause photobleaching depending of the instrument, you can also use longer intervals or starting point and endpoint measurement only for higher sensitivity. Avoid drying of the samples, especially if you measure for more than four hours Fluorophore standard curve Dissolve the complete Mca standard in 2 ml of dissolving solution by vortexing thoroughly and incubation in an ultrasonic waterbath for about ten minutes. This gives a 0.25 mg/ml (corresponding to 1.07 mm) solution. We recommend measuring one column of standard s on the microtiter plate used for the protease assay. Therefore, generate six serial s of 1:2 in dissolving solution. Then, dispense 190 µl of assay buffer (either Working Buffer I or Working Buffer II or both in parallel) into each well, then add 10 µl of the respective Mca s or 10 µl dissolving solution for the blank. This gives the following final Mca amounts per well: 1st 2nd 3rd 4th 5th 6th 10 μl dissolving solution 5337 pmol 2669 pmol 1334 pmol 667 pmol 334 pmol 167 pmol 0 pmol 4.3. Preparation of P-CHECK Substrate solution Add 1 ml of dissolving solution to the lyophilized P-CHECK Substrate and completely dissolve the pellet by vortexing thoroughly and incubation in an ultrasonic waterbath for about 10 minutes. 10 3

4 5. Measurement 4.4. activity standard curve (optionally) If desired, prepare a series of a protease with known activity (e.g. trypsin, pepsin) and add 10 µl of these s to 180 µl of buffer in each well and start the reaction with 10 µl of P-Check Substrate solution. As negative control, add 10 µl of buffer instead of protease. Measure the emerging fluorescence over time Sample analysis For solid samples, first dissolve your samples at up to 20 mg/ml in the desired Working Buffer. Then add 140 µl sample to 50 µl of the Working Buffer in each well. For liquid samples, add µl of sample and add buffer to a final volume of 190 µl to each well. If the samples are already dissolved in an adequate buffer/ph for protease detection, no additional buffer is needed. For liquid samples in a buffer differing from desired measurement conditions, add only 50 µl sample to 140 µl of the corresponding Working Buffer in each well. The assay will get more sensitive if you add more sample. However, the ph values will differ from the buffer ph if the sample is more concentrated. If your sample is buffered and you want to perform the protease assay at a specific ph, you should either take less sample so the buffer in your sample gets diluted, or you should adjust the ph of your samples in advance. If you want to detect proteases impairing the stability of your protein in the specific buffer of your sample solution, you do not have to dilute your sample Negative controls Add appropriate negative controls to your plate. In the simplest case, this is just 190 µl of either Working Buffer I or Working Buffer II or both in parallel plus 10 µl of P-CHECK Substrate solution. If you want to measure raw materials and you have protease free control, you can also use this as negative control. We recommend to prepare the negative control in triplicates and to calculate the standard deviation to get a confidence interval. References References 1. Theodor Förster (1948). Zwischenmolekulare Energiewanderung und Fluoreszenz, Ann. Physik 6(2): Kapprell, H.P. et al. (2011). Development of a Fluorescence Resonance Energy Transfer Peptide Library Technology for Detection of Contaminants in Protein-Based Raw Materials sed in Diagnostic Assays. ASSAY and Drug Development Technologies. 9(5): Filippova, I. Y., E. N. Lysogorskaya, et al. (1986). Intramolecularly Quenched Fluorescent Substrates For Aspartic Proteinases. Bioorganicheskaya Khimiya 12(9): Furka, A., F. Sebestyen, et al. (1991). General-Method For Rapid Synthesis Of Multicomponent Peptide Mixtures. International Journal of Peptide and Protein Research 37(6): Gershkovich, A. and V. V. Kholodovych (1996). Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). Journal of Biochemical and Biophysical Methods 33(3): Knight, C. G. (1995). Fluorometric Assays Of Proteolytic-Enzymes. Proteolytic Enzymes: Aspartic and Metallo Peptidases 248: Twining, S. S. (1984). Fluorescein Isothiocyanate-Labeled Casein Assay For Proteolytic-Enzymes. Analytical Biochemistry 143(1): Measurement 5.1. Measurement of Mca standard Measure the Mca standard wells with the same setting as the samples. Do NOT use automatic gain settings for your instrument if you also measure the standard curve since this will set the gain much too low for the sample measurement. If you want to use automatic gain setting, you should only use much lower amounts of Mca in your standard wells. 4 9

5 Notes 7. Recommended data evaluation Pepsin (porcine gastric mucosa) Cathepsin D (bovine spleen) Aspartic Aspartic 7.81 x x M Glycin/HCl, ph M Glycin/HCl, ph Measurement of samples Initiate the reaction by the addition of 10 µl of P-CHECK Substrate solution, without delay start the software of your Microtiter plate reader/elisa reader and follow the fluorescence over time. Trypsin (porcine pankreas) ng 6. Assay flow-chart Chymotrypsin (bovine pankreas) ng Glu-C V8 protease (Staphylococcus aureus, recombinant) 3.91 ng Lys-C (Lysobacter enzymogenes) 6.25 x 10-2 ng Notes If you use the same sample volume and the same buffer for all samples, you can prepare a master mix of buffer and P-CHECK Substrate solution for less pipetting steps and better mixing. In this case, just add 10 µl of sample per well and start the reaction by adding 190 µl of master mix containing 180 µl buffer and 10 µl substrate solution. You can also use other buffers if desired. Working Buffer I and Working buffer II are supplied for your convenience. Due to its design, this assay only detects endopeptidases. Most carboxyand aminopeptidases will not cleave the P-CHECK Substrate. Assay sensitivity decreases if your sample is autofluorescent at this particular wavelength combination. You have to pay special attention to samples that show high background fluorescence. E.g. spike a known protease amount to your sample and check if it is still detected. Assay sensitivity also decreases if your sample contains a dye quenching the fluorescence. As a control, spike a known protease amount to your sample and check if it is detected. The fluorophore calibration curve only allows for very rough approximations of the released Mca since it doesn t contain free quencher. It is mainly used to compare results between plates and readers. 7. Recommended data evaluation 7.1. Only use this evaluation mode if the fluorescence background (starting values) is about the same as the negative controls (so you don t have autofluorescent samples). Calculate the mean value and standard deviation of the final time point of your negative controls. If the final time points of your samples are smaller than mean value plus threefold standard deviation of your negative control, no protease activity could be detected. If the values are higher than this threshold, there is only a statistical chance of 5% that this happened by chance, so the sample carries a protease with a probability of 95%. You can adjust the threshold to your needs (less false positives/less false negatives) Subtract the mean value of your negative controls from each sample time point and then plot fluorescence vs. time. Calculate the slope for each sample in the linear interval by linear regression analysis. If no quantitative conclusion is to be drawn, you can also subtract the starting value from the endpoint value. Note: For higher protease activity, only the initial values are linear, then the observed activity decreases. se the first values in this case to calculate the slope. The slope (RF per minute) directly correlates with protease activity. You can also 8 5

6 8. Anticipated results 9. detection limits approximate the amount of released Mca with the help of the Fluorophore standard curve (pmol Mca per minute). 8. Anticipated results 8.1. Fluorophore standard curve Working Buffer I ph 7.4 Trypsin (25 ng) Pepsin (250 ng) Working Buffer II ph 2.8 Note that the curve might not be linear at higher concentrations. 8.2 Repeated measurement of the same sample 25 ng of Trypsin and 250 ng of Pepsin were measured in three wells (black lines) in two different buffers and plotted together with three wells of negative control (grey lines). Median plus threefold standard deviation of the negative control (detection limit) is shown as dashed line. Samples were measured with a Tecan fluorescence reader infinite F200 (software: Magellan Tracker V6.6) and a manual gain setting of detection limits We elaborated detection limits for some selected proteases. We added 10 µl of a serial of each protease to 180 µl of corresponding Working buffer and started the reaction by the addition of 10 µl P-Check Substrate solution. We regarded a protease as detected if the final value was over the mean value of the negative controls plus the threefold standard deviation of the negative controls. Enzyme (Source) Elastase (human leukocytes) Proteinase K (Tritirachium album) Subtilisin (Bacillus licheniformis) Thermolysin (Bacillus thermoproteolyticus rokko) Class Metalloprote ase Detection Limit Buffer Conditions 2 x x x x 10-4 Bromelain (pineapple stem) Papain (papaya latex) Cysteine Cysteine 5 x x