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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