Validation strategies and which parameters to focus on€¦ · −ICH guideline Q2(R1) does not cover relative test methods (e.g. area%) −Validation parameters need to be aligned

CE Pharm 2018

Dr. Angelina Rafai, PD Dr. Maria Schwarz

12 September 2018

Validation strategies and which parameters to focus onSpecial characteristics of area percentage test methods

Introduction

2 Angelina Rafai | CE Pharm 2018

Method feasibility/

setup

Method development

Method

Qualification / Validation

Method performance verification

Method design

− Method purpose

− Separation mode

− Impurity identification

− Availability of impurities

− Evaluation of method characteristics (e.g.

precision, robustness)

− Future specifications?

− Can method meet these specifications?

� Preparation of method validation!

Routine analysis

− Stability

− Release

Change

3

Validation of Analytical Procedures: Text and Methodology

ICH Q2(R1) - Analytical Validation

Type of testmethod

Identification Testing for impurities Assay

Characteristic Quantitative Limit

Specificity + + + +

Precision

Repeatability

Intermediate

precision

LOD

LOQ

Linearity

Accuracy

Range +

Robustness + +

Angelina Rafai | CE Pharm 2018

Quantitative methods

(response ~ conc.)

4

Test methods in biopharmaceutical analysis


Size variants

Variants differing

in hydrophobicity

Charge variants

fragments

aggregates

chemical modifications

deamidation

AA modificationssialylation

glycosylation

glycation

oxidation

peptide mapping

SEC

CGE

RP-HPLC/

HIC

HILIC

CZE/cIEF

IEX

Relative (e.g. area%)

Method characteristics


Relative methods (e.g. area%)

− Response (e.g. area%) not related to

the test sample concentration

− Response related to relative content of

each component

− Extinction coefficient known for entire

protein solution, not for each single

component

− Protein variants might differ in

extinction coefficient (depending on

modification)

Quantitative methods

− Response (e.g. area) related to

concentration

− Extinction coefficient known

− All variants are identified and

characterized


Test methods in biopharmaceutical analysis

Relative (e.g. area%)

Quantitative

7

Common procedure for relative methods (e.g. area%)

Analytical Validation


Type of testmethod

Identification Testing for impurities AssayPurity/Impurity

based on e.g. area%


Specificity

Precision

Repeatability

Intermediate

precision

LOD

LOQ

Linearity

Accuracy

Range

Robustness

ICH guideline does not cover

relative (area%) methods

(biopharmaceuticals)

8




Type of testmethod


based on e.g. area%


Specificity

Precision

Repeatability

Intermediate

precision

LOD

LOQ

Linearity

Accuracy

Range

Robustness

9




Type of testmethod


based on e.g. area%


Specificity Interfering signals

Precision

Repeatability RSD (area%), n = 6

Intermediate

precisionRSD (area%), n = 9

LOD S/N ≥ 3

LOQ S/N ≥ 10, RSD (area)

Linearity LOQ – 120 % (area)

Accuracy Recovery/Diff (area%)

RangeDeduced from Spec,

Lin, Prec

Robustness Recovery/Diff (area%)

10




Type of testmethod


based on e.g. area%


Specificity Interfering signals

Precision

Repeatability RSD (area%), n = 6

Intermediate

precisionRSD (area%), n = 9

LOD S/N ≥ 3

LOQ S/N ≥ 10, RSD (area)

Linearity LOQ – 120 % (area)

Accuracy Recovery/Diff (area%)

RangeDeduced from Spec,

Lin, Prec

Robustness Recovery/Diff (area%)

11

Suggested procedure for relative methods (e.g. area%)



Type of testmethod


based on e.g. area%


Specificity

Precision

Repeatability

Intermediate

precision

LOD

LOQ

Linearity

Accuracy

Range

Robustness

− Sample dilution to LOQ level (e.g. 0.1 %)

− Evaluation of RSD for area (Main variant)

− Evaluation of S/N

− LOQ level is determined by serial dilutions (calculating the S/N)

− Determined LOQ is applied to test method (one LOQ for all variants)


LOQ – common procedure and suggestion

suitable

signals for

LOQ

evaluation

− Undiluted test solution (100 % target load)

− Possibly using stressed material

− Differentiation between fragments/aggregates and acid/basic variants

− Evaluation of RSD for area% (suitable LOQ signals)

− Separate LOQs for different variants


Linearity – common procedure and suggestion

− Sample dilution (LOQ – 150 % of target load)

− Evaluation of response (e.g. area)

− Linear plot %target load / response (e.g. area)

− Linear range of detector (also for relative methods)


Linearity – suggestion

− Linear range of detector (also for relative methods)

− 50 – 150 % of target load

− If suitable material is available (purified variants)

− Differentiation between fragments/aggregates and acid/basic variants

− e.g. 60 – 120 % of specification

− Evaluation of residuals

− Deduce accuracy

Unspiked sample

Spiked sample

Accuracy - suggestion

Size-based separation (CGE, SEC)

− Origin and identity of impurities should

be clear

− Non-red: fragments

� spiking of reduced material

− Red: deglycosylated variants

� spiking of deglycosylated material

Charge-based separation (cIEF, CZE, IEX)

− Identity of impurities should be clear

− Charge variants may include fragments

(ε ≠ const.) and amino acid modifications(ε ≈ const.)

� spiking material with equal features

(produced material has to be

characterized first)


Examples for mAb


Summary

− Relative methods (e.g. area%) are not quantitative

− ICH guideline Q2(R1) does not cover relative test methods (e.g. area%)

− Validation parameters need to be aligned with analytical result (no evaluation of

absolute values, e.g. area)

− LOQ, linearity, and accuracy have to be addressed appropriately (extinction

coefficient)

Acknowledgements

− Solvias AG, Biopharma Department

− PD Dr. Maria Schwarz

− Dr. Alexander Beck

17 Angelina Rafai | CE Pharm 2018

Römerpark 2, 4303 Kaiseraugst, Switzerland

www.solvias.com

Angelina Rafai, [email protected], T +41 61 845 6501

Solvias AG

Documents

Validation strategies and which parameters to focus on€¦ · −ICH guideline Q2(R1) does not cover relative test methods (e.g. area%) −Validation parameters need to be aligned