Assay Qualification/Validation –

a Reviewer’s Expectations

Sarah Kennett
Division of Monoclonal Antibodies
Office of Biotechnology Products
OPS/CDER/FDA

1
October 20, 2010
 Disclaimer
The views and opinions expressed here are
my own and should not be used in place of
regulations, published FDA guidances or
discussions with the Agency.
These views reflect CDER/OPS/OBP
discussions.
This talk is focused on analytical assays used
to assess drug substance, drug product, and
in-process materials.
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 Overview
Why this subject?
What does FDA say – officially?
What do we OBP product reviewers
mean by “qualification” and
“validation”?
Some expectations

3
• Person X: Someone told me assays need to be validated to start
Phase 2 studies.
• Me: No, unless there is a scientific reason, submission of assay
validation isn’t required until the BLA is submitted. However, you
need to be using qualified assays from the beginning. If the
assays you’re using during later phases of clinical development
can’t be validated, you might be in trouble regarding setting of
acceptance criteria and dosing/efficacy assessment.
• X: So, we need to show you validated assays by Phase 3.
• Me: Uhh, no, not exactly. Formal validation studies CAN be
performed as early as you want, but with a few exceptions, the
REQUIREMENT regarding timing of the original validation
{exceptions include assays critical for safety!} is that it be
completed prior to submission of the BLA to be included in the
submission. For phase 3, you probably need to be using assays
that can be validated, though, so they need to at least be well
qualified.
• X:… 4
• Me:…
What does FDA say regarding BLA/NDA stage
assays?
The accuracy, sensitivity, specificity, and reproducibility of test
methods employed by the firm shall be established and
documented. Such validation and documentation may be
accomplished in accordance with …
21 CFR 211.165(e) (Testing and release for distribution)
At the time the application is submitted to the regulatory
authorities, applicants should have validated the analytical
procedures uses in the specifications.
ICH Q6B
Analytical methods should be validated…
ICH Q7
The stability program shall include…reliable, meaningful, and
specific test methods.
5
21 CFR 211.166(a)(3)
What does FDA say regarding IND stage assays?
Although in each phase of the investigation sufficient
information is required to assure the proper identification,
quality, purity, and strength of the investigational drug, the
amount of information needed to make that assurance will
vary with each phase of the investigation, the proposed
duration of the investigation, the dosage form, and the amount
of information otherwise available.
21 CFR 312.23(a)(7)(i)
The {IND} submission is required to contain:
…the acceptable limits and analytical methods used to assure
the identity, strength, quality, and purity of the drug substance
[drug product]…
21 CFR 312.23(a)(7)(iv)(a) [21 CFR 312.23(a)(7)(iv)(b)]
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 One of the clearest statements FDA makes
regarding a requirement for ASSAY validation at
the IND stage is in the PROCESS validation
guidance?
While validated analytical methods are not required
during product- and process-development activities,
methods should be scientifically sound (e.g., specific,
sensitive, and accurate), suitable, and reliable for the
specified purpose.

Guidance for Industry: Process Validation: General Principles and Practices.

FDA/CDER, CBER, CVM. 2008. 7
 Validation vs. Qualification
Are we speaking the same language?
Process validation is defined as the collection and evaluation of data,
from the process design stage throughout production, which
establishes scientific evidence that a process is capable of
consistently delivering quality products. Process validation involves
a series of activities taking place over the lifecycle of the product
and process. This guidance describes the process validation
activities in three stages.
• Stage 1 – Process Design: The commercial process is defined during this
stage based on knowledge gained through development and scale-up
activities.
• Stage 2 – Process Qualification: During this stage, the process design is
confirmed as being capable of reproducible commercial manufacturing.
• Stage 3 – Continued Process Verification: Ongoing assurance is gained
during routine production that the process remains in a state of control.

Guidance for Industry: Process Validation: General Principles and Practices.

FDA/CDER, CBER, CVM. 2008.
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 Validation vs. Qualification
Are we speaking the same language?
Lifecycle
Stage 1 – Process Design: The commercial process is
defined during this stage based on knowledge gained
through development and scale-up activities.
Stage 2 – Process Qualification: During this stage, the
process design is confirmed as being capable of
reproducible commercial manufacturing.
Stage 3 – Continued Process Verification: Ongoing
assurance is gained during routine production that the
process remains in a state of control.

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 Validation vs. Qualification
Are we speaking the same language?
Lifecycle (borrowing from Process Validation)
Stage 1 – Assay Design: The assay is defined during this
stage based on knowledge gained through development
activities.
Stage 2 – Assay Qualification: During this stage, the assay
design is confirmed as being capable of producing
reproducible results suitable for the specified purpose.
Stage 3 – Continued Assay Verification: Ongoing
assurance is gained during routine use that the assay
remains in a state of control.

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 Validation vs. Qualification
Are we speaking the same language?

For a bioanalytical [BA, BE, PK] method to be considered

valid, specific acceptance criteria should be set in
advance and achieved for accuracy and precision for the
validation of QC samples over the range of the
standards.
The validity of an analytical method should be established
and verified by laboratory studies, and documentation of
successful completion of such studies should be
provided in the assay validation report.

Guidance for Industry: Bioanalytical Method Validation.

FDA/CDER, CVM. 2001.
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 Validation vs. Qualification
Are we speaking the same language?
Qualification: The assay design is confirmed as being capable of
producing reproducible results suitable for the specified purpose.
Test how the assay performs and decide if that performance is
suitable for that point in development. If it isn’t good enough,
change the assay.
Work in progress, but scientifically sound and suitable for its
purpose.
Validation: The assay is tested against specific acceptance criteria
set in advance to verify that the performance characteristics
(those listed in ICH Q2(R1) and potentially others, depending on
the assay and the product) of the final method are suitable and
reliable for the intended applications. Documentation of
successful completion of such studies should be provided in an
assay validation report.
Final product, documented to meet performance acceptance 12
criteria.
 Validation vs. Qualification
Are we speaking the same language?
Lifecycle (borrowing from Process Validation)
Stage 1 – Assay Design: The assay is defined during this stage based
on knowledge gained through development activities.
Stage 2 – Assay Qualification: During this stage, the assay design is
confirmed as being capable of producing reproducible results
suitable for the specified purpose. Scientifically sound work in
progress.

Stage 2 ½ - Formal Assay Validation Study: The assay is tested

against specific acceptance criteria set to verify that the
performance characteristics of the final method are suitable and
reliable for the intended applications.
Stage 3 – Continued Assay Verification: Ongoing assurance is gained
during routine production that the assay remains in a state of
control.
(system suitability, assay suitability, trending of results, OOS, etc.) 13
 Early Development
Submissions need to contain enough information
for reviewers to determine whether or not the
methods are scientifically sound (e.g., specific,
sensitive, and accurate), suitable, and reliable
for the specified purpose.
The specified purpose is to provide sufficient
information to assure the proper identification,
quality, purity, and strength. The assay needs to
be sufficiently developed to ensure that
acceptance criteria are meaningful.

What does this really mean?

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 Early Development
Reviewers need enough information to make these
assessments.
How much information is needed will be product
and assay dependent.

concentration potency

less CE methods more

UV spectroscopy cell based assay

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 Early Development
Reviewers need enough information to make these
assessments.
How much information is needed will be product
and assay dependent.

cIEF cIEF cIEF

less more

Charge variants Antibody drug conjugate

of an IgG1 Charge variants drug:antibody ratio
of a Fc fusion protein

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 Early Development
Reviewers need enough information to make these
assessments.
How much information is needed will be product and
assay dependent.

cIEF for identity example: If the product is a second

generation product, a sponsor may need to include
some specificity data in the IND original submission.

The data themselves can sometimes provide information

regarding the suitability/reliability of the assay.
(don’t forget about system suitability, equipment qualification, 17
critical reagent variability)
 Later Development
Submissions need to contain enough information
for reviewers to determine whether or not the
methods are scientifically sound (e.g., specific,
sensitive, and accurate), suitable, and reliable
for the specified purpose.
The specified purpose is to provide sufficient
information to assure the proper identification,
quality, purity, and strength. The assay needs to
be sufficiently developed to ensure that
acceptance criteria are meaningful.

What does this really mean?

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 Later Development
Submissions need to contain enough information
for reviewers to determine whether or not the
methods are scientifically sound (e.g., specific,
sensitive, and accurate), suitable, and reliable
for the specified purpose.
The specified purpose is to provide sufficient
information to assure the proper identification,
quality, purity, and strength. The assay needs to
be sufficiently developed to ensure that
acceptance criteria are meaningful.

What does this really mean?

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 Later Development
Need to be able to correlate commercial product
with the clinical study material.
Efficacy data and safety data from clinical studies
are associated with quality attributes.
There is a sort of link between the safety and
efficacy data and the acceptance criteria set at
licensure.
Specification acceptance criteria are initially set
using historic data from clinical lots with an
emphasis on the product used in pivotal studies.
The historic data need to be “correct.”
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 Later Development
LOQ (or LOD), range, precision, etc. may be
extremely important, especially if a safety
risk or dosing issue is involved.
e.g. an impurity that is detected using a
CE method

If the assay that was in place during testing

of late stage clinical material cannot be
validated, you may have difficulty setting
acceptance criteria.
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 BLA/NDA submission
The application for licensure/approval should include a validation
report for each non-compendial assay (suitability
verification/conditions of actual use) used for lot release and
stability. Characterization assays used during the generation of
new reference material should also be validated, as should
additional characterization assays depending on their intended
use.

The studies performed should include the appropriate

performance parameters from those listed in ICH Q2(R1) and
potentially others, depending on the assay and the product) .

Information included in/with the report should include the study

protocols with the pre-defined acceptance criteria and enough
information regarding the assay protocol and prior
characterization/qualification to allow for an assessment of the
validation protocol. 22
 Post-licensure Changes
Modification of any particular test method or
manufacturing process or the conditions under
which it is conducted…shall be permitted only
under the following conditions
The applicant presents evidence…demonstrating that
the modification will provide assurances of the safety,
purity, potency, and effectiveness of the biological
product equal to or greater than the assurances
provided by the method or process specified…
21 CFR 610.9(a)

To us (FDA), this means re-validation (sometimes

partial revalidation) is likely to be necessary.
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 Post-licensure Changes
Revalidation may be necessary for
Changes in the synthesis of the drug substance
Changes in the composition of the finished product
Changes in the analytical procedure

The degree of revalidation required depends on

the nature of the changes.

Certain other changes may require revalidation as

well.

ICH Q2 (R1) 24
 Method Transfer
Transfer of validated analytical methods from the originating laboratory to a
new laboratory
To ensure comparability in the validation characteristics between laboratories
To prevent and/or detect changes in data trend
To improve transfer success, historical data from the originating site can be
used to identify the greatest causes of variance in an assay.
Assess a subset of validation parameters with pre-defined acceptance criteria
Using Equivalence Testing (Precision and Accuracy)
Using Key attributes (Precision, LOD/LOQ, Accuracy, Identity, Linearity)
Typical Transfer should include:
More than one lot of material (Reference Standards, Samples at extremes of the
established acceptable limits, Stress samples)
Testing of the same samples at both sites
At least one analyst at the originating laboratory and multiple analysts in the
receiving laboratory

(From Kathy Lee talk “Analytical Method Transfer”) 25

 References
21 CFR 211, 21 CFR 312, 21 CFR 610
Guidance for Industry: Bioanalytical Method Validation. FDA/CDER,
CVM. 2001.
Guidance for Industry: Process Validation: General Principles and
Practices. FDA/CDER, CBER, CVM. 2008.

ICH Q2(R1). Validation of Analytical Procedures: Text and

Methodology. 2005.
ICH Q6B. Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products. 1999.
ICH Q7. Good Manufacturing Practice Guide for Active Pharmaceutical
Ingredients. 2000.

Other interesting reading:

Nadine Ritter, et al. What is Test Method Qualification: Proceedings of
the WCBP CMC Strategy Forum, 24 July 2003. BioProcess
International 32-46. 2004. 26