Validation of an Endpoint Enzyme Activity Assay to Evaluate Potency for Lot Release and Stability Testing. Loc Vo, PhD

Transcription

1 Validation of an Endpoint Enzyme Activity Assay to Evaluate Potency for Lot Release and Stability Testing Loc Vo, PhD

2 Background: Enzyme replacement therapy for a lysosomal storage disease

3 Timeline for Approval IND - Assay Qualified Phase 1 Phase 2 Phase 3 Validation Started Validation Completed - BLA FDA Approval

4 Potency Potency definition Specific capacity of a product to achieve its intended effect Based on the measurement of some attribute of the product and is determined by a suitable quantitative method Characterizes effectiveness of the medicine Potency for Enzyme Replacement Therapy Enzyme activity assay Cellular uptake assay

5 Key Characteristics of Lot Release Assays and Stability-indicating Assay Lot Release Accuracy Good Precision Stability Able to detect change in drug Accuracy Good Precision Reliability / Robustness Ease/Time Scientifically Accepted Sensitivity Highthroughput Result Output Biological Relevance Enzyme Cell- Based Range Relative + +++

6 What is Assay Validation? Demonstrate that an assay is suitable for its intended purpose Assign mathematical values to the characteristics/properties of an assay

7 Composition of an Assay Validation Type of Assay ID Purity (Quant.) Purity (Limit) Content/ Potency Accuracy Repeatability Intermediate Precision Specificity Detection Limit Quantitation Limit Linearity Range Robustness System Suitability Testing Stability-Indicating Properties

8 Enzyme Activity Assay Principle The Activity Assay determines whether the product contains functional enzyme, at least within the parameters of the assay. Activity of each lot of product must be determined to be within specification prior to release. 4-methylumbelliferyl sulfate (4MUS) 2 4-MUS (with ENZYME) 4-MU + S

9 Enzyme Activity Assay Enzyme Activity is a measurement in Velocity Defined as µmoles of product converted/min/ml or Units/mL Specific Activity is Units/mg of protein blank-subtracted fluorescence nm ASB 1 nm ASB [4-MUS], mm

10 Basic Steps of the Enzyme Activity Assay Dilute Enzyme & MU Standard Read Plate Load Plate STOP The Reaction with GSB Add 4-MUS & Incubate Pre-Incubate

11 Acceptance Criteria for Enzyme Assays Specificity Precision Accuracy Robustness Demonstrate Specificity Repeatability: 10% RSD Intermediate precision: 15% RSD Reproducibility reference trend data % of expected Incubation Time (90-110% of expected activity and 10% RSD ) Hydrolysis temperature (90-110% of expected activity and 10% RSD ) Substrate Preparation (90-110% of expected activity and 10% RSD ) Stability Standard Preparation (90-110% of expected activity and 10% RSD ) Detect 20% changes in activity in samples subjected to accelerated degradation conditions

12 Initial Validation Results Intermediate Precision: ~18% RSD Assay qualified at 9% RSD Precision Assay Optimization Change incubation source Jitterbug

13 Consequences of Assay Optimization Change SOP Re-start Validation Activity values now 50% higher Impact Change in product specification Comparability to all previously generated data (lot release and stability)

14 Precision of the Optimized Activity Assay Intermediate Precision Repeatability % RSD 25% 20% 15% 10% 5% 0% Old Assay New Assay Replicate Activity (U/mL) Average 74 SD 3 %RSD 4

15 Reproducibility of the Reference Material Density Density Lab Lab 1 (n=37) Average 71 SD 7 %RSD 10% Lab 2 (n=30) Average 63 SD 5 %RSD 9% Inter-Lab Average 68 SD 7 %RSD 11% Lab 2

16 Assay Technique Standardization Analyze each lab s sample handling techniques Greatest variation found during enzyme dilution steps One pre-rinse of pipette tips SOP modified to include pipetting instructions

17 Reproducibility of the Reference After Standardization of Lab Techniques Lab 1 (n=11) Density Average 67 SD 7 %RSD 10% Inter-Lab Lab 1 Average 67 Density Lab 2 (n=20) Average 67 SD 3 %RSD 4% SD 4 %RSD 7% Lab 2

18 Defining Stability-Indicating Properties by Forced Degradation Mode of Degradation H 2 O 2 TBHP Agitation Oxidation 4 C 25 C Temperature 50 C Freeze/Thaw ph 3 ph 10 ph

19 Stability Indicating Properties 120% 100% 80% 60% 40% % Activity of Control 20% 0% Agitation 25 C Agitation 4 C TBHP Oxidation Oxidation H2O2 Freeze/Thaw 50 C 14 days 50 C 30 days ph 3 ph 10

20 Validation Summary Test Parameters Specificity Precision Accuracy Linearity and Range Robustness Stability Indication Result Pass 4 % RSD (Repeatability) % RSD (Intermediate) 11% Before; 7% After (Reproducibility) % at U/mL 89% at 97 U/mL Standard Curve: R ; Standard Curve Back calculations: % Linearity for dilutions of ; R % for ± 1 min incubation; 6% RSD % for ± 1 ºC temperature; 7% RSD 110% for substrate variation; 6% RSD 93% for standard variation; 9% RSD Sensitivity to oxidation, heat, bases

21 Critical Factors of the Activity Assay Temperature dependence Sample handling (pipetting) Substrate variation

22 Regulatory Interactions Post-marketing commitment to develop an assay with a more BIOLOGICALLY relevant SUBSTRATE Chondroitin Sulfate Tightening of Activity Specifications

23 Acknowledgements QCA Lab QCIP Lab Bill Prince, PhD Victoria Sluzky, PhD Doug Tingley Andrea Van Tuyl Joanne Liang