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
Angelina Rafai | CE Pharm 2018
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
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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
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Test methods in biopharmaceutical analysis
Relative (e.g. area%)
Quantitative
7
Common procedure for relative methods (e.g. area%)
Analytical Validation
Angelina Rafai | CE Pharm 2018
Type of testmethod
Identification Testing for impurities AssayPurity/Impurity
based on e.g. area%
Characteristic Quantitative Limit
Specificity
Precision
Repeatability
Intermediate
precision
LOD
LOQ
Linearity
Accuracy
Range
Robustness
ICH guideline does not cover
relative (area%) methods
(biopharmaceuticals)
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Common procedure for relative methods (e.g. area%)
Analytical Validation
Angelina Rafai | CE Pharm 2018
Type of testmethod
Identification Testing for impurities AssayPurity/Impurity
based on e.g. area%
Characteristic Quantitative Limit
Specificity
Precision
Repeatability
Intermediate
precision
LOD
LOQ
Linearity
Accuracy
Range
Robustness
9
Common procedure for relative methods (e.g. area%)
Analytical Validation
Angelina Rafai | CE Pharm 2018
Type of testmethod
Identification Testing for impurities AssayPurity/Impurity
based on e.g. area%
Characteristic Quantitative Limit
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
Common procedure for relative methods (e.g. area%)
Analytical Validation
Angelina Rafai | CE Pharm 2018
Type of testmethod
Identification Testing for impurities AssayPurity/Impurity
based on e.g. area%
Characteristic Quantitative Limit
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%)
Analytical Validation
Angelina Rafai | CE Pharm 2018
Type of testmethod
Identification Testing for impurities AssayPurity/Impurity
based on e.g. area%
Characteristic Quantitative Limit
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)
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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
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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)
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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)
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Examples for mAb
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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
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