1. INTERNATIONAL WINE TECHNICAL FORUM, 2015 ALLERGEN METHOD VALIDATION Steve Tallman 6 th May, 2015

2. Agenda Why test for allergens? Why validate methods/processes? Method validation – ELISA test kits Definition of method performance terms Validation Data - Casein

3. Why Test for Allergens? Use of allergenic ingredients…  Ovalbumin (egg whites)  Casein (whole milk/casein)  Lysozyme (egg-based derivative) Process validation and verification…  FSMA regulations  Verification of process sanitary controls  Eliminate cross-contamination  Finished goods verification ALLERGEN METHOD VALIDATION

4. Why Validate Methods? Checks vendor performance claims Understand matrix interactions Determine lab performance capabilities Documents LOD, LOQ, % recovery values Ensures adequate analyte % recovery Provides defensibility – FSMA regulations ALLERGEN METHOD VALIDATION Principle #9 – Validation of analytical methods

5. Method Validation Protocol Define scope of test materials – Vendor selection  ELISA = enzyme-linked immunosorbent assay  r-biopharm, Neogen, Romer, Astori, Euroclone, others…  Ensure commercial viability, cost and supply  Identify additive usage that contain potential allergens  NIST certified testing materials Define acceptance criteria:  EU required detection limits:  Casein, Ovalbumin and Lysozyme LOD: 0.25 ppm LOQ: 0.50 ppm ALLERGEN METHOD VALIDATION

6. Validation Study Scheme Detection limits (LOD) - the lowest concentration level that can be determined to be statically difference from the blank  LOD = Mean value of 10 blanks + 3*SD Limit of Quantitation (LOQ) - the lowest concentration that can be quantitatively determined with suitable accuracy and precision  LOQ = Mean value of 10 blanks + 10*SD Percent recovery – methods ability to quantitate known concentration additions PRESENTATION TITLE HERE

7. Casein LOQ/LOD Data PRESENTATION TITLE HERE

8. Casein % Recovery Data PRESENTATION TITLE HERE Sample #Wine Spike Amount, materialAbsorbance Calculated ppm % Recovery 1TLV110412-Merlot 0.5 ppm Casein NIST RM0.1050.44990 2TLV110412-Merlot 5.0 ppm Casein NIST RM1.2935.06101 3TLV110412-Merlot 10.0 ppm Casein NIST RM1.4416.3163 Average % Recovery of NIST Spike85%

9. ALLERGEN METHOD VALIDATION Questions?

10. Casein % Recovery Data Sample #WineSpike Amount, materialAbsorbanceCalculated ppm% Recovery 1TLV110412-Merlot0.5 ppm Casein NIST RM0.1050.44990 2TLV110412-Merlot5 ppm Casein NIST RM1.2935.06101 3TLV110412-Merlot10 ppm Casein NIST RM1.4416.3163 Average % Recovery of NIST Spike85% PRESENTATION TITLE HERE