This document discusses the importance of monitoring reference standards, controls, and reagents over the analytical method life cycle to ensure consistent and reliable method performance. It provides examples of how changes can affect method validity and examples of proper qualification and monitoring practices for these critical materials. Qualification establishes acceptance criteria that reference standards, controls, and reagents must meet for the method, and ongoing monitoring ensures continued acceptable performance over the method life cycle.

1. Qualification of Reference Standards,
Controls and Reagents as a part of
Analytical Method Life Cycle
Management
Grainne Tobin
LACBRP/DBSQC/OCBQ/CBER
US Food and Drug Administration

2. 2
Overview
• Importance of monitoring the performance of the method over
the life-cycle
• Factors affecting method performance
• Considerations for qualification of:
– Reference Standards
– Controls
– Reagents
www.fda.gov

3. 3
Drug Product Development Process
www.fda.gov
Discovery and Development
Preclinical – in vitro and in vivo studies
Clinical – Phase I, II, III
FDA Review
FDA Post Market Safety Monitoring
IND
NDA / BLA
Approval

4. 4
Analytical Procedures
• Analytical procedures are developed to ensure the identity,
strength, quality, purity and potency of the drug substance and
drug product
• The Biologics License Application (BLA) must include a full
description of the manufacturing process, including the
analytical procedures
• Data must be available to establish that the analytical
procedures used in testing meet proper standards of accuracy,
sensitivity, specificity and reproducibility and are suitable for
their intended purpose
Reference - CDER, CBER Guidance for Industry: Analytical Procedures and Methods Validation for
Drugs and Biologics, July 2015

5. 5
Specifications
• The setting of specifications for drug substance and drug
product is part of an overall control strategy which includes
control of raw materials and excipients, in-process testing,
process evaluation and validation, adherence to good
manufacturing practices, stability testing, and testing for
consistency of lots.
• Assures appropriate identity, strength and quality of the product
is maintained
Reference – ICH Harmonised Tripartite Guideline: Q6B Specifications: Test Procedures and
Acceptance Criteria for Biotechnological/Biological Products, March 1999

6. 6
Specifications
• Specifications are necessary to define critical quality attributes
– For example, potency, the nature and quantity of product-related
substances (e.g., molecular variants, IgG dimer), product-related
impurities, and process-related impurities.
• Specifications reflect
– manufacturing consistency
– the stability of drug substance and drug product
– the quality of the material; specifications provide a way to
ensure lots produced at commercial scale are representative
of the lots used in preclinical and clinical studies
– analytical method attributes, e.g., assay variability

7. 7
Factors that may affect method performance
• Different analysts
– No two analysts are the same!
• Change in instrumentation
– Discontinuation of instrument or introduction of newer model
• Change in laboratory location
– Testing assigned to a new Contract Research Organization
– Construction of new Quality Control laboratory
• Change in product matrix
– New manufacturing equipment, source of raw materials
• Change in standard or critical reagent
– Adequate qualification

8. 8
Reference Standards
“A reference standard, or reference material, is a substance
prepared for use as the standard in an assay, identification, or
purity test. It should have a quality appropriate to its use. It is often
characterized and evaluated for its intended purpose by additional
procedures other than those used in routine testing.”
• Two types of Reference Standard:
– Primary Reference Standard
– Working Standard
Reference: ICH Harmonized Tripartite Guideline: Specifications: Test Procedures and Acceptance Criteria
for New Drug Substances and New Drug Products: Chemical Substances, Q6A, October 1999

9. 9
Primary Reference Standard
(a) Authoritative Standard:
1. International Standard
• Available through World Health Organization (WHO)
2. US Standard
• Available through CBER, National Institute of Standards and
Technology (NIST), and United States Pharmacopeia (USP)
• Potency assigned in collaborative study
– Potency measured in a number of different of laboratories using
different procedures
– Potency compared to previous Standard
• May have different matrix than test sample

10. 10
Primary Reference Standard
(b) Standard from Commercial Source
(c) In-House Standard
• Developed by manufacturer, even when International Standard
is available
• Usually drug product or drug substance lot
– Advantage: Same or closely related matrix as test sample
• The In-House Standard should be qualified against the
International Standard
– Measure multiple (at least 10) replicates relative to Authoritative
Standard to establish potency
– Need to have sufficient quantity to last through life-cycle of product

11. 11
Working Reference Standard
• Working Reference Standard
– Derived from a batch of drug product
– Used to enable the Primary Reference Standard to last through the
product life-cycle
• Qualified against Primary Reference Standard, NOT the previous
Working Reference Standard
– Removes propagation of error

12. 12
Case Study #1: In-support Testing using
FVIII Chromogenic Assay
• 5 lots of recombinant FVIII drug product and In-house standard
provided by manufacturer to CBER for FVIII chromogenic assay
in-support testing for BLA licensure
• Manufacturer provided information on Reference Standard
qualification: In-House standard - batch of drug product which
had been qualified against the 8th WHO IS for FVIII concentrate
• CBER testing performed using In-House standard and FVIII
chromogenic assay kit

13. 13
CBER In-support Testing Results using
FVIII Chromogenic Assay
Lot
Number
Manufacturer’s
Calculated Result
(vs In-House
standard) (IU/mL)
CBER FVIII Potency Value
(using In-House standard)
(IU/mL)
Kit 1 Kit 2
1 270 340 279
2 270 257 346
3 294 335 327
4 302 265 357
5 286 314 305
• CBER results were 88 – 128% manufacturer’s results
• Difference in test results at CBER show inconsistent
performance of the assay kits
• Manufacturer’s results were theoretically calculated results
• FVIII potency measured at CBER vs manufacturer’s results

14. 14
Investigation
• Many of the assay validity criteria were not met: R2, slope ratio
between standard and sample, % RSD of triplicate sample
measurements
• FVIII Chromogenic assay gave variable results for CBER and
manufacturer
• Manufacturer’s batch release testing performed using in-house
standards
• Manufacturer noted a change in the potency of successive
reference standards with time
– in-house standard B qualified against prior in-house standard A, etc.
– This led to a progressive decrease in potency of the standard
– Needed to apply “Correction Factor” which changed with time to be
able to compare the potency values of lots measured using standard
A and B to potency values of lots measured using the In-House
standard since standard A and B stocks were depleted

15. 15
Outcome
• Due to the variable potency measurements using the FVIII
Chromogenic assay by the manufacturer and CBER using
different lots of kits, and the problems with the standard in this
test, the FVIII Chromogenic assay was not approved for use as
part of the lot-release protocol and could only be used for
information only
• The FVIII One Stage Clotting Assay, which gave more
reproducible results, was approved for potency measurement
for lot-release

16. 16
Controls
• A Control is tested with the test article to verify the validity of
the test performance
• Source of Controls
– A lot of drug product
– Commercially available
• Can include negative, positive, or both
• Acceptance criteria for the negative control are defined from a
number of test repeats and is generally < Limit of Quantitation
(LOQ) of the assay

17. 17
Positive Controls
• Positive controls may be used at low, medium and high dilutions
to cover the range of the method
• Positive controls may be used in qualitative and quantitative
assays
• Positive Control is qualified against the Reference Standard
• Qualification of the Positive Control sets the calculated range as
Mean ± k.SD (where, k = tolerance factor at 95% CI)

18. 18
Case Study #2: Qualification of Sigma Thrombin as
Positive Control in Thrombin Clotting Assay
• Qualification of the positive control against the International Standard
(IS) prior to use is required as there is a difference in how the potency
of the IS and commercially available thrombin are measured
• Thrombin Potency by Clotting Assay
• Measured in a STA Compact coagulation analyzer
• Reference Standard – WHO 2nd IS for Thrombin 01/580
– 5 point standard curve
• Positive Control – Thrombin (Sigma-Aldrich or equivalent)
– 2 independent measurements
• Test sample – 3 independent measurements of one concentration
• Reagents – Pooled normal plasma and dilution buffer
• Assay validity criteria
– R2 for the standard curve ≥ 0.98
– The mean potency of the thrombin positive control must be within ± 3SD
of the qualified thrombin positive control (IU/mL)
– % RSD of triplicate measurements for each sample must be ≤ 10%

19. 19
Qualification of Control for Thrombin
Potency Assay
• Prepare serial dilutions of the Control within the range of the
standard curve
• Measure 6 replicates of the test sample
• Test results for Control must be within pre-established
acceptance criteria of assay
– Range
• Measured clot time must be within range of standard curve
– Linearity of Control curve
• R2 ≥ 0.98
– Dilution parallelism between Standard and Control
• 0.80 ≤ Slope Ratio ≤ 1.20
– Intermediate precision
• % RSD of results ≤ 15%

20. 20
Control Qualification
Figure 1: Standard Curve Table 1: Acceptance Criteria
• Assay validity criteria for the 6 dilution series were met

21. 21
Control Qualification
• Qualified Potency for Positive Control
• The qualified potency of the lot of Sigma Thrombin is 132.1
IU/mL
• The acceptance criteria of the lot of Sigma Thrombin is 109.4 –
154.8 IU/mL
Dilution
Number
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6
1 130.7 122.8 117.1 132.8 121.1 129.8
2 133.2 129.0 136.8 131.6 130.5 130.1
3 134.3 143.8 132.1 138.7 130.0 135.3
4 139.7 145.1 125.1 144.4 129.9 129.4
5 141.2 136.8 131.0 139.5 112.8 128.5
Qual. Potency (IU/mL) 132.1
SD 7.57
%RSD 5.73
HIGH LIMIT (+3SD) 154.8
LOW LIMIT (-3SD) 109.4

22. 22
Qualified Potency of Two Different Lots of
Positive Control
Lot 1: Measured using 1 lot Pooled
Normal Plasma
Lot 2: Measured using 3 lots Pooled
Normal Plasma
• Two lots of positive control have been qualified.
• All measured potencies are within the acceptance criteria
• Results demonstrate effect of different lots of reagent (Pooled
Normal Plasma) on test result

23. 23
Critical Reagents
• A critical reagent is a reagent that impacts the performance
characteristics and results of the assay
• Need to qualify each new lot of critical reagent prior to use in its
official testing unless it is from an authoritative source
– Lot-to-lot variation of reagents
– Biological reagents are variable, chemical reagents are less-so
– Identifies reagents that may bias or compromise the potency
measurement

24. 24
Qualification of Reagents
• Qualify reagents using System Suitability / Assay Validity test of
the instrument
– Routinely performed before initiation of test
– Demonstrates that the measured potency is within the
System Suitability / Assay Validity Criteria for the assay
• Reagent must be qualified using the assay procedure, including
the reference standard

25. 25
Case Study # 3: von Willebrand Factor:Ristocetin Cofactor
(vWF:RCo) Activity Assay
• Assay measures the ability of vWF in the test sample to cause
platelet agglutination
• Measured using an aggregometer and detected as rate of
increase in light transmission
– Critical reagents:
• AggRecetin
• Lyophilized platelets
• Human von Willebrand Plasma –Type 2 (vWD Trait Severe)
– WHO International Standard used as the reference standard in the
assay is not subject to qualification since obtained from an
authoritative source
• System Suitability test: First measurement of standard curve
(1:2, 1:4 and 1:8 dilutions) used to establish the standard curve
against which all other samples are compared
– Acceptance criteria of curve established by instrument manufacturer

26. 26
System Suitability Results
• All Assay Validity criteria are met using these lots of reagents

27. 27
Case Study #4: Thrombin Potency by
Clotting Assay
• Thrombin Potency by Clotting Assay adopted in 2016
• Verification of method using three lots each of four different
drug products
• 27 tests have been performed at DBSQC since test method
approved in 2016
• 5 of these tests were aborted due to invalid standard curve (R2 ≤
0.98: 4 tests), or invalid control sample (clot time of lowest
concentration of positive control < lowest point on standard
curve: 1 test)

28. 28
Clot Times of WHO IS 01/580
in Thrombin Potency by Clotting Assay
www.fda.gov
20 IU/mL
15 IU/mL
10 IU/mL
5 IU/mL
3.75 IU/mL

29. 29
Monitoring Assay Performance
• The four higher concentrations show little variation in clot time
• In contrast, the lowest concentration of standard curve gives
variable clot times
• For the four invalid standard curves, the clot time of the lowest
concentration of the curve was the reason the acceptance
criteria for linearity (R2 ≥ 0.98) was not met
• Suggests lowest concentration of the standard curve may be
below the sensitivity of our instrument
• Result - Revise method to exclude lowest concentration point

30. 30
Importance of Monitoring Standards, Controls
and Reagents over the Life Cycle
• If the standards, controls and critical reagents are meeting
criteria
– Confidence in assay performance
• Monitor stability of standards, controls and critical reagents
– Test to confirm consistent performance at regular intervals
– Store under conditions that maintain stability
– Apply appropriate expiration dates
– Qualify new sources and lots of material

31. 31
Importance of Monitoring the Method
over the Life-Cycle
• If the method is performing within the acceptance criteria
– Trust the validity of the results
– Confidence in laboratory performance
• If the method is not performing as expected
– Allows improvement of factors that are causing variability
• Ensures validity of the method over the life-cycle

32. 32
Acknowledgements
DBSQC
Maryna Eichelberger
Lokesh Bhattacharyya
Kori Francis
Leslyn Aaron
Parmesh Dutt
Karen Smith
Ishrat Sultana
Sean Younker