VALIDATION GUIDE FOR CHROMOGENIC ASSAY FOR ACTIZYME Anti-FIIa
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1 VALIDATION GUIDE FOR CHROMOGENIC ASSAY FOR ACTIZYME Anti-FIIa Chromogenic assay for testing heparins (UFH, LMWH and Enoxaparin) in purified systems by measurement of FIIa Inhibition, in compliance with pharmacopoeias (EP/BP) and FDA guidelines. Document History : CodificatIon History Date Version#1.0 VALIDATION DATA OF Heparin (anti-fiia) Cat No # KBBA Approved Quality Approved Approved Operations Control Product Development Head Mihir Parmar Trupti B. Kalpesh Jain
2 Table Contents : 1 Introduction 2 Experimental procedures 2.1 Reagents 2.2 Material to be provided by the End-User 2.3 Storage and Stability Information 2.4 Health Hazard Warning 2.5 Assay Protocol 2.6 Statistical analysis 2.7 Validation of the anti-factor IIa method Linearity Precision Accuracy 3 Result and Discussion 4 Conclusion 1. Introduction: Heparin (anti F-IIa) is a chromogenic assay intended for the quantitative determination of therapeutic heparin in purified solutions by measurement of factor IIa (thrombin) activity. The kit can be used for 200 test reactions as per microtiter plate protocol and 100 test reactions as per test tube protocol. The inhibitory effect of anti-thrombin III (AT-III) on factor IIa (thrombin), factor Xa and other coagulation serine proteases in plasma is increased several thousand-fold by heparin. This inhibition accounts for the anticoagulant effect of heparin. The quantitative determination of heparin levels by the measurement of their anti-factor IIa activity is a necessary tool for monitoring treatment efficacy. Low molecular weight heparin (LMWH) preparations appear to catalyze the reaction between thrombin (factor IIa) and AT-III less readily than the reaction between factor Xa and AT-III. Unfractionated heparin (UFH) catalyzes both reactions equally. In the assay, the rate of factor IIa inhibition is directly proportional to the heparin concentration since both factor IIa and AT-III are in excess. The residual factor IIa activity is inversely proportional to the heparin. 2. Experimental Procedure: 2.1. Reagents 1. Human Thrombin Reagent 2. Human Anti-thrombin III Reagent 3. Chromogenic Substrate 4. Instruction Manual 2.2 Materials to be provided by the End-User: 1. Microplate Reader / Spectrophotometer able to measure absorbance at 405nm 2. Adjustable pipettes to measure volumes ranging from 25μl to 2500μl, duly calibrated 3. Deionized (DI) water 4. Parallel line software for data analysis 5. Plastic tubes or cuvettes or microtiter plates with overflow capacity 350µl/well C water bath or microplate incubator preferred (for microplate method) 7. Timer/Stop watch 8. Glacial Acetic Acid 9. Absorbent paper 10. Tris (hydroxymethyl) aminomethane 11. Sodium Chloride 12. EDTA 13. Bovine Serum Albumin (BSA) 2.3 Storage and Stability Information: Un-reconstituted reagents are stable until the expiration date indicated on the label when stored at 2 to 8 C.
3 1. Human Thrombin Reagent: Reconstituted reagent is stable for 2 weeks at 2 to 8 C and for 4 months at -20 C. 2. Human Anti-thrombin III Reagent: Reconstituted reagent is stable for 2 weeks at 2 to 8 C and for 4 months at -20 C. 3. Chromogenic Substrate: Reconstituted reagent is stable for 2 weeks at 2 to 8 C and for 4 months at -20 C. 4 Dilution Buffer and Acetic acid are to be freshly prepared, prior to use. 2.4 Health Hazard Warnings: 1. The source material for the human anti-thrombin III has been found to be non-reactive for Hepatitis B Surface Antigen (HBsAg), Hepatitis C Virus (HCV) and Human Immunodeficiency Virus Type 1 and Type 2 (HIV-1, HIV-2) using FDA approved methods. 2. The Heparin (anti-fiia) antithromin III reagent contains sodium azide that may react with lead or copper plumbing to form highly explosive azides. Specimen Collection and Handling: Purified Samples: Dilute the heparin preparation with Dilution Buffer in order to bring it at a concentration within the assay working range. Reagent Preparation (all reagents should be diluted immediately prior to use): 1. Human Thrombin Reagent: Human Thrombin Reagent is a lyophilized preparation. Allow reagent to equilibrate to room temperature. Reconstitute with 5 ml of deionized water. Gently mix and leave to stand for 15 mins. 2. Human Anti-thrombin III Reagent: Anti-thrombin III is a lyophilized preparation. Allow reagent to equilibrate to room temperature. Reconstitute with 5 ml of deionized water. Gently mix and leave to stand for 15 mins. 3. Chromogenic Substrate: Chromogenic Substrate is a lyophilized preparation. Allow reagent to equilibrate to room temperature. Reconstitute with 5 ml of deionized water. Gently mix and leave to stand for 15 mins. 4. Dilution Buffer: (Not provided in the kit) to be prepared separately as follows: Dissolve gm of Sodium Chloride, 6.10 gm of tris (hydoxymethyl) amino methane, 2.80 gm of EDTA and 2.0 gm of bovine albumin in 800 ml of water. Adjust the ph 7.4 with hydrochloric acid and dilute to 1000 ml with water. 5. Acetic Acid Solution: (Not provided in the kit) to be prepared separately as follows: Dilute Glacial Acetic Acid with Distilled Water or Deionized Water in the ratio of 42: Assay Protocol Two step protocol for measurement of factor IIa activity in purified systems, in compliance with Pharmacopoeias (EP/ BP) and FDA guidelines. Note: 1. The Standard Dilution Samples (S1, S2, S3, and S4) and Test Dilution Samples (T1, T2, T3, and T4) have to be diluted in same ratios to the nearest third decimal concentrations to assure high accuracy and estimation by extrapolation. 2. The dilution steps prior to the Standard and Test Dilution also should be to the nearest concentration possible to assure high accuracy. 3. Please see an example of the dilution steps followed to get a high degree of accuracy when assaying the Test Samples. 4. Dilute Standard and Test Samples with the Dilution buffer ph 7.4, mentioned in Reagent Preparation. For Example: Preparation of Standard Stock Standard Concentration IU/mg is to be diluted to make Stock Solution strength of IU/ml
4 Standard Dilution Test Sample Dilution Stock Solution strength: IU/ml ml 2 ml IU/ml 0.1 ml 2 ml 5 IU/ml 1.5 ml 2 ml IU/ml 0.2 ml 2 ml 0.5 IU/ml 0.8 ml 2 ml IU/ml ml 2 ml IU/ml 1.5 ml 2 ml IU/ml 0.8 ml 2 ml IU/ml S ml 2 ml IU/ml T ml 2 ml IU/ml S ml 2 ml IU/ml T ml 2 ml IU/ml S ml 2 ml IU/ml T ml 2 ml IU/ml S ml 2 ml IU/ml T ml 2 ml IU/ml Add the reagents into the microwell or plastic test tube, as per following steps: microwell test tube Standard or Test Sample 25µl 50µl Anti-thrombin III 100µl 125µl Mix but do not allow bubbles to form. Incubate at 37⁰C, for 1 minute Human Thrombin Reagent 50µl 100µl Mix and incubate at 37⁰C, for exactly 1 minute Chromogenic Substrate 50µl 175µl Mix and incubate at 37⁰C, for 4 minutes Acetic Acid 125µl 250µl Mix and measure the absorbance at 405nm 2.6. Statistical analysis Statistical analysis of the assay data were carried out according to PLAv28 (Excel based worksheet/software based on DJ Finney, Statistical Method in Biological Assay, 3rd edition by parallel line methods). Analysis of variance was performed for each independent, assay and the assumption of linearity and parallelism of the log dose log response lines was tested. 2.7 Validation of the anti-factor IIa method In this procedure we followed the microwell method and the parameters were evaluated according to the EP/USP guidelines Linearity The Standard of low molecular weight heparins specifying the activity as IIa/ vial, was reconstituted with 5 ml of distilled water. Appropriate amounts of this stock solution were diluted in Tris buffer (0.05 M, ph 7.4) yielding concentrations of , , , , , , and IU/ml. Two independent calibration curves were constructed and the linearity evaluated by linear regression analysis, which was calculated by the least square regression method Precision The precision of the method was determined by repeatability and intermediate precision and was expressed as the relative standard deviation. The repeatability was examined by assaying one samples of Heparin on the same day (intra-day) and under the same experimental conditions, against Standard of low molecular weight heparins. The intermediate precision of the method was evaluated through the performance of the analysis on two different days (inter-days) and by having another analyst performing the analysis in the same laboratory (between-analysts).
5 ACTIZYME Anti-FIIa Intra Assay Run#1 Run#2 Run#3 Run#4 %C.V Sample Concentration (IU/ml) Standard Concentration (IU/ml) Inter Assay Run#1 Run#2 %C.V Sample Concentration IU/ml Standard Concentration IU/ml Accuracy To determine the accuracy of the proposed method, the test was performed over 4 concentration levels covering the specified range, and carrying out assay; each one in duplicate with the results being combined statistically. ACTIZYME Anti-FIIa Intra Assay Inter Assay Standard Standard Concentration Run#1 Run#2 Run#3 Run#4 % CV Concentration Run#1 Run#2 % CV Sample Run#1 Run#2 Run#3 4 % CV Sample Run#1 Run#2 % CV Concentration Concentration Results and Discussion: For each series, we calculate the regression of the absorbance against log concentration of the sample solutions and the standard solutions. The four independent log relative potency estimates are then combined to obtain the final geometric mean. Its confidence limits are calculated. Standard and Test Samples being serial diluted should pass the test for linearity and parallelism as the interpretation is done by extrapolating the data. The kit indicates a precision of <=1% and an accuracy of <=1%. The confidence limits were set at +/- 10%. 4. Conclusion: The Anti-Factor IIa assay was validated and the kit is accurate and complies with the parameters for bioassays as per EP/USP guidelines.
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