analytical method development & validation for therapeutic proteins 1 jichao (jay) kang, ph.d.,...
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Analytical Method Analytical Method Development & Validation for Development & Validation for
Therapeutic ProteinsTherapeutic Proteins
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Jichao (Jay) Kang, Ph.D., RAC
Director, Analytical and Formulation Development
Gallus Biopharmaceuticals NJ, LLC
Course ObjectivesCourse 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 CharacterizationGet familiar with method validation and
method transfer: principle, common practice, and potential problems
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Basic Structure of MAbBasic Structure of MAb
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Major N-linked GlycansMajor N-linked Glycans
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Classified depending on the extent of processing:High mannose: lack Gal or GlcNAc at the end of antennaeComplex: GlcNAc after the Man at the antennaeHybrid: have both Man and Gal/GlcNac at end of the antennae
Hybrid and Complex types can have two or more branches, termed Bi-antennary, tri-antennary, or Tetra-antennary
Reduced CE-SDS of MAbReduced CE-SDS of MAb
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SEC-HPLC-MALSSEC-HPLC-MALS
Three detector monitoring
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Hydroxylated Proline and Lysine by Hydroxylated Proline and Lysine by AAAAAA
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Protein Structure Elucidation by Protein Structure Elucidation by MS/MSMS/MS
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Roepstorff Nomenclature Scheme
Secondary Structure by CDSecondary Structure by CD
9T Estey, J kang et al., Pharm Sci , 2006, 95: 1626–1639
Timing for Method Timing for Method Qualification/ValidationQualification/Validation
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Preclinical Clinical CommercialPhase I Phase II Phase III
Method Development and Qualification
Qualification Validation Verification
Measurement defined
Scientifically Defendable
Ready for validation
Fully ValidatedMethod 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
Case Study: Binding Assay Case Study: Binding Assay QualificationQualification
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x axis
1 10 100 1000 10000 100000
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Parallel Line Analysis
4-P Fit: y = (A - D)/( 1 + (x/C)̂ B ) + D: A B C D Rel. Pot.
Standard (Standard: Concentration vs Mean... 0.0395 1.31 202 2.61 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 2̂ = 1 F-stat = 2.14 F-prob = 0.712
Binding ELISA curves at different level
Quiz # 15Quiz # 15
For an identification method, what are the validation parameters?
How to determine if a method is stability-indicating?
How to determine LOQ?How to calculate method supported
specification (SS)?List three considerations in setting SSTList three method transfer pitfalls
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