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Analytical Method Development & Validation for Therapeutic Proteins Jichao (Jay) Kang, Ph.D., RAC Director, Analytical and Formulation Development Gallus Biopharmaceuticals NJ, LLC 1 Course Objectives Basic scientific and industrial knowledge of working on proteins (including antibodies) Get familiar with commonly used analytical methods: purpose, advantages, limits, pitfalls Get familiar with Protein Characterization Get familiar with method validation and method transfer: principle, common practice, and potential problems 2 Basic Structure of MAb 3 Major N-linked Glycans Classified depending on the extent of processing: High mannose: lack Gal or GlcNAc at the end of antennae Complex: GlcNAc after the Man at the antennae Hybrid: have both Man and Gal/GlcNac at end of the antennae Hybrid and Complex types can have two or more branches, termed Bi-antennary, triantennary, or Tetra-antennary 4 Reduced CE-SDS of MAb 5 SEC-HPLC-MALS Three detector monitoring 6 Hydroxylated Proline and Lysine by AAA 7 Protein Structure Elucidation by MS/MS Roepstorff Nomenclature Scheme 8 Secondary Structure by CD T Estey, J kang et al., Pharm Sci , 2006, 95: 1626–1639 9 Timing for Method Qualification/Validation Preclinical Clinical Phase I Phase II Qualification Commercial Phase III Validation Verification Method Development and Qualification Measurement defined Scientifically Defendable Ready for validation Fully Validated Method Transfer Methods related to product safety require qualification even at phase I Other methods need to be validated prior to BLA, but by phase III, you should have the methods that can be validated 10 Case Study: Binding Assay Qualification Parallel Line Analysis 3 2 1 0 1 10 100 1000 10000 100000 x axis B C D Standard (Standard: Concentration vs Mean... 0.0395 4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A 1.31 202 2.61 Rel. Pot. 1 Sample3 (150%: Concentration vs MeanVal... 0.0395 1.31 127 2.61 1.59 Sample1 (50%: Concentration vs MeanValue) 0.0395 1.31 372 2.61 0.541 Sample2 (100%: Concentration vs MeanVal... 0.0395 __________ 1.31 200 2.61 1.01 Curve Fit Option - Fixed Weight Value PLA (Std. Curve: Standard) Degrees of Freedom: parallel = 37 free = 28 non-parallel = 9 R^2 = 1 F-stat = 2.14 F-prob = 0.712 Binding ELISA curves at different level 11 Quiz # 15 For an identification method, what are the validation parameters? How to determine if a method is stabilityindicating? How to determine LOQ? How to calculate method supported specification (SS)? List three considerations in setting SST List three method transfer pitfalls 12