AbbVie
Agios
Amgen, Inc.
Alexion Pharmaceuticals
Astellas US LLC
AstraZeneca
Bayer HealthCare
Baxter Healthcare
Biogen Idec, Inc.
Boehringer Ingelheim
Bristol-Myers Squibb
Celgene Corporation
Daiichi Sankyo
Eisai, Inc.
Eli Lilly and Company
EMD Serono
Endo Pharmaceuticals
Genentech
Gilead Sciences
GlaxoSmithKline
Incyte Corporation
Infinity Pharmaceuticals
Johnson & Johnson
Kythera Biopharmaceuticals
Merck & Co.
Millennium
Novartis
Pfizer
Roche
sanofi
Sunovion
Otsuka Pharmaceutical Co.
Takeda
Teva Pharmaceutical
Vertex, Inc.
Comments from the IQ Consortium on the FDA Draft Guidance for
Industry
“Bioanalytical Method Validation”
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulator
yInformation/Guidances/UCM368107.pdf
Docket No. FDA-2013-D-1020
Federal Register Notice: https://federalregister.gov/a/2013-22309
We are pleased to offer the following comments, prepared by the
Bioanalytical Expert Working Group of the Drug Metabolism and
Clinical Pharmacology Leadership Group of the International
Consortium for Innovation and Quality in Pharmaceutical
Development (the IQ Consortium). The IQ Consortium is a
technically focused organization of pharmaceutical and biotechnology
companies, whose mission is to advance science-based and
scientifically-driven standards and regulations for pharmaceutical and
biotechnology products worldwide.
These comments are organized into the following sections:
1. Introduction - Page 2
2. General Comments - Page 2
3. Specific Comments - 5
Please contact the IQ Secretariat with any questions: Svetlana
Lyapustina, Ph.D. (svetlana.lyapustina@dbr.com; 1-202-230-5179).
Comment Tracking Number: 1jx-899b-g0v0
Submitted to www.regulations.gov on 12 December 2013.
DC01/ 3286969.1
Introduction
The International Consortium on Innovation and Quality in Pharmaceutical Development (IQ)
appreciates the opportunity to comment on the FDA Draft Guidance For Industry on
Bioanalytical Method Validation [Docket No. FDA-2013-D-1020]). This document provides
comments and recommendations compiled from member companies participating in the IQ
Bioanalytical Working Group. The working group is comprised of bioanalytical experts working
with both small molecule and large molecule (proteins and peptides) modalities. The response
document has been organized into two sections. The first section is a General Comments section
which includes comments that pertain to the general organization and overall content of the
guidance. The second section provides specific comments on individual sections and lines in a
tabular format. Within the specific comments section, proposed changes, recommendations and
requests for clarification begin with bolded text. Furthermore, comments deemed of highest
priority nature have been noted as “Priority” in the section column.
General Comments
1. The new guidance provides the opportunity to achieve greater global harmonization in
regulated bioanalytical method validation and method use. As such, consideration of the
recommendations from the Global Bioanalysis Consortium, recent white papers and/or
the content of related regulatory guidance from other countries would aid harmonization
efforts. Appropriate incorporation of such material would make the guidance the primary
contemporary source document to guide bioanalytical method validation and use.
2. The guidance would benefit from a better delineation between the Background,
Validation and Method Use sections. Each of these sections appear to contain elements
that are not necessarily meant for the particular section where provided, but rather are
suited for other parts of the document (e.g. number of concentrations levels and replicates
used during validation vs. method use).
3. The guidance makes reference to Method Development activities throughout.
Historically, Method Development activities have not been within the scope of regulated
bioanalysis. It is recommended that “Method Development” be removed from the scope
of the guidance and that “Development” be removed from the titles of sections III.B. and
IV.B.
DC01/ 3286969.1
4. The guidance does not address a variety of topics which have become common practice
during method validation as a result of previous Crystal City discussions, white paper
recommendations, or citations in form 483s (e.g. matrix factor, impact of hemolysis,
stability testing in the presence of co-medications, etc.). Clarification of the regulatory
expectations in these and related areas is requested.
5. The guidance calls for levels that are below the LLOQ to be reported as zero. We
recommend that samples with concentrations below the LLOQ be reported as <LLOQ or
BQL, not as zeros. The use of PK data since 2001 has changed significantly, with many
more population PK and PK/PD studies now being performed. The arbitrary setting of
concentrations <LLOQ to zero may have serious implications for modeling analyses.
Pharmacokineticists require the ability to use these data in a flexible but scientifically
rigorous way.
6. The LBA section appears to contain verbiage that is specific to chromatographic assays
that should be removed. Furthermore, the LBA validation section does not recommend
using Total Error criteria for defining the LLOQ. The concept of Total Error is linked to
the 4-6-x rule advocated for in the in-study acceptance criteria and this concept should be
considered for validation as well.
7. The guidance should use relevant terms for chromatography assays and ligand binding
assays. Some terms are not universally interchangeable between these two method types.
8. The section on ISR has several changes in scope (e.g. application to PD studies, 7% of
unknown samples) that are different from white papers (AAPS), GBC recommendations
and also the EMEA guidance. It seems the scope and criteria were written specifically for
BE studies. However, ISR now is also being performed for other types of studies and
some requirements (such as ISR samples from all subjects) may not be applicable.
Additional consideration to harmonization of scope and criteria would be helpful.
9. For the first time the guidance provides information on Biomarker methods. Additional
emphasis and guidance on how study objectives (expected use of the data) can guide
appropriate fit-for-purpose validation (e.g. analytical acceptance criteria) is requested.
Clarification on requirements for multiplexed assays and ISR are also sought.
10. We would like the Agency to reconsider or clarify the request that data from new
technologies always be supported with those from established technology. This had not
DC01/ 3286969.1
been done when the industry moved from UV to MS for detection, from HPLC to
UHPLC for separation, ligand binding to MS for the detection of large molecules, etc.
We believe this requirement would essentially stifle the growth and adoption of new
technologies. Similarly, microsampling technology is evolving rapidly and therefore the
section and the requirements around DBS will likely be outdated quickly. We believe,
therefore, it is premature to include specific guidance around DBS in this document.
These new technologies should be allowed to evolve further in conjunction with robust
industry and agency interactions. (Comment provided by the IQ Consortium
Microsampling Working Group)
11. Several sections in the guidance discuss documentation requirements, but it is not always
clear whether the guidance is referring to documentation that should be part of the raw
data (study file), the bioanalytical report or a submission. Furthermore, the guidance
recommends additional extensive hyperlinking in reports and submissions the extent of
which may not always be practical.
DC01/ 3286969.1
Specific Comments
Line
Section Title
Specific Comments by Section and Line
I. INTRODUCTION
16
18-26
28-33
Line 33 of the guidance currently includes tissue and skin as matrices in scope. Calibration
and QC tissue samples may not mimic the incurred samples and/or data are often generated
for internal decision making.
Proposed Change: remove tissue, and skin from line 33 so it reads “…proteins in
biological matrices, such as blood, serum, plasma, and urine.”. Also add appropriate text
describing expectations for fit for purpose validation of tissues.
42-46
DC01/ 3286969.1
Line 23 states: “This guidance also applies to bioanalytical methods used for nonclinical
pharmacology/toxicology studies.”
Proposed Change: “This guidance also applies to bioanalytical methods used in support of
nonclinical safety pharmacology/toxicology studies.”
Line 45 of the guidance indicates that the word “should” means that something is suggested
or recommended but not required. However, throughout the document the term “should” is
used in places where an activity is clearly a regulatory expectation for acceptance by the
agency (e.g. Line 77 where a BA method “should” be fully validated to support a BE
study).
Recommendation: Provide alternate verbiage where the agency is indicating a
requirement.
II. BACKGROUND
48
59-71
Proposed change: Remove “and/or biopharmaceutics” from line 61 as it is out of context.
The sentence would then read: “Selective, sensitive, and validated analytical methods for
the quantitative evaluation of drugs and their metabolites (analytes) and biomarkers are
critical for the successful conduct of nonclinical and/or clinical studies for small-molecule
drugs and biopharmaceutics.”
The guidance does not mention specificity as one of the fundamental parameters for
validation of LBA assays.
Proposed change: Add “Specificity in case of LBA” to the list of fundamental parameters.
73-78
The guidance suggests that for exploratory methods “less validation may be sufficient” (line
78). Historically, exploratory methods (screening methods and those applied to nonregulated studies) have not been within the scope of regulated bioanalysis and therefore
have not been subject to formal validation.
Recommendation: Remove this sentence. Alternatively, further clarify how fit-for-purpose
validation should be applied.
80-86
The guidance states: “These modifications should be validated…”,
Recommendation: Rather than using this statement, reference the partial validation section
98-116, which provides further detail on appropriate levels of validation for method
changes.
88
DC01/ 3286969.1
The details on full, partial and cross-validation should be moved from the Background
section to the Validation section.
Proposed change: Replace “During development and implementation….” with “Prior to
implementation of a novel bioanalytical method…”. (line 94)
90-96
DC01/ 3286969.1
In line 96, the guidance states: “For revisions to an existing method that add metabolite
quantification”
Please clarify: Whether a full validation or partial validation is needed when a metabolite is
removed from an assay.
In line 105, the guidance states: “Bioanalytical method transfers between laboratories or
analysts”. A change in analysts within a laboratory should not be considered a method
change.
Proposed change: Change bullet point to “Bioanalytical method transfers between
laboratories”.
In line 105, the guidance states: “Bioanalytical method transfers between laboratories or
analysts”.
This is in conflict with lines 117-131 (cross-validation) which states that a cross-validation
is required if sample analysis is performed at more than one laboratory. It is also not clear
what exactly has to be done for a method transfer between laboratories. Is stability testing at
both sites required or can stability testing at one site be referenced.
Please clarify: What exactly has to be done when methods are transferred between
laboratories and samples within one study are analyzed at more than one site.
98-115
In line 107, the guidance states: “Change in anticoagulant in harvesting biological fluid
(e.g., heparin to EDTA)”.
Recommendation: Addition of statement clarifying that change in anti-coagulant counter
ion does not require partial validation.
In line 108 and 110, the guidance states: “Change in matrix within species” and “Change in
species within matrix”. These usually require full validation and the EMA guidance
recommends full validation for change of species.
Recommendation: Where possible please be consistent with the EMA guidance. If a partial
validation is only required in these situations, it needs to be clarified which parameters need
to be tested and for which assay format (e.g. LBA versus LC-MS/MS).
In line 112, the guidance states: “Changes in instruments and/or software platforms”.
We assume that the guidance means the change in instrument platforms (same as software
platforms) rather than instruments. This sentence could be misleading.
Proposed change: “Changes in instrument platforms and/or software platforms”
DC01/ 3286969.1
Priority
98-115
DC01/ 3286969.1
In line 115, the guidance states: “Selectivity demonstration of an analyte in the presence of
concomitant medications”. Selectivity of an analyte in the presence of concomitant
medications may not be needed if a SLIS is used in LC-MS/MS and if the concomitant
medication is non-isobaric. Rather, partial validation is needed on a case-by-case basis.
Proposed change: “Selectivity demonstration of an analyte in the presence of concomitant
medications if needed”.
In addition, there is no description of the requirement for conducting the long-term storage
stability assessment of the analyte in the existence of the concomitant medications, Please
clarify.
Priority
For cross-validations, the document should provide more guidance on acceptance criteria
“equivalency” for cross-validations and when to perform cross-validations as well as
guidance on parameters like number of samples and target concentration of samples. For
example, are criteria used for ISR experiments sufficient?
In line 126, the guidance states: “cross-validation with spiked matrix standards and subject
samples…” Generally, QCs are used and subject samples are pooled for cross validation
activities.
Proposed change: “…, cross validation with spiked QCs and incurred subject samples
(pooled)….”
117-131
In lines 119-123, the verbiage used to introduce cross-validation is confusing. For example,
line 120 is not consistent with line 128, as line 128 specifically states that cross validation is
needed when different analytical techniques are used across different studies. In addition, it
may not be necessary to conduct cross-validation for methods applied to different studies.
Recommendation: Remove this paragraph since appropriate scenarios are given in the
subsequent paragraph (lines 125-131). Alternatively reword paragraph.
In line 123, the guidance states: “…comparisons should be done both ways.”
If the paragraph is not deleted but reworded, this needs further clarification.
Please clarify: What exactly “both ways” means and should it not rather be one way? For
example, it is unclear if QC samples need to be prepared in both labs (reference lab and
comparator lab), analyzed, and then shipped to the other lab.
In lines 128-129, the guidance states: “Cross-validation should also be considered when
data generated using different analytical techniques (e.g., LC-MS/MS vs. ELISA) in
different studies are included in a regulatory submission.”
Please clarify: For data generated using different analytical technologies (e.g., LC-MS/MS
vs. ELISA) used for different studies, it may not always be necessary to conduct cross
validation between the different methods employing the different technological platforms.
For example, two validated LC-MS/MS methods used independently in different studies
should not require cross-validation.
DC01/ 3286969.1
117-131
In lines 129-131, the guidance states: “All modifications to an existing method should be
assessed to determine the recommended degree of validation.” Modification to an existing
method should be assessed with a partial validation, instead of cross-validation.
Recommendation: Propose to place this sentence in the section of partial validation.
133-136
In lines 133-135, the guidance states: “The analytical laboratory conducting nonclinical
pharmacology/toxicology studies for regulatory submissions should adhere to FDA's Good
Laboratory Practices (GLPs) requirements (21 CFR Part 58).” Pharmacology studies are
generally not done under GLP.
Proposed change: Remove “pharmacology” from line 133 or insert “safety” prior to
“pharmacology”, if that was the intent. Also, In lines 133-135 replace “(21 CFR Part 58)”
with “(relevant sections of 21 CFR Part 58).”
In lines 135-136, the guidance states: The bioanalytical method for human BA, BE, PK, and
drug interaction studies must meet the criteria specified in 21 CFR 320.29. However, 21
CFR 320.29 does only apply to analytical methods for an in vivo bioavailability or
bioequivalence study.
Please clarify: Why 21 CFR 320.29 should also apply to PK and drug interaction studies.
Priority
138-144
DC01/ 3286969.1
In lines 139-140, the guidance states: “SOPs should cover all aspects of analysis from the
time the sample is collected and reaches the laboratory until the results of the analysis…”.
Bioanalytical SOP’s typically do not describe activities related to the collection, storage or
shipping of samples from the in-life site. These activities are described in the study
protocols or related manuals.
Proposed change: “Bioanalytical SOPs should cover all aspects of analysis and storage
from the time samples reach the laboratory until the results of the analysis are reported”.
150
III.
CHROMATOGRAPHI
C METHODS
A. Reference
Standards
152
154-160
DC01/ 3286969.1
Reference standard requirements are described based on the analytical technique (in Section
III or IV).
Recommendation: provide a separate section for Reference Standards (similar to the
current 2001 guidance) and discussing their requirements based on whether they are small
or large molecules, not the technique used to measure them.
Recommendation: Remove free base or acid and ester from example on line 159-160.
These are examples of alternative forms of the analyte (typically a free base or acid) and
esters of the parent are not typically used.
162-169
171
173-177
DC01/ 3286969.1
Priority
Line 167. The requirements for analytical reference standards should not be applied to
internal standards. Internal standards serve a different purpose, are not quantified, and
frequently are prepared in much smaller quantities, insufficient for continued stability
testing. When using LC-MS/MS, the majority are stable isotope-labeled versions of the
analyte. It should be demonstrated that the internal standard is suitable (i.e., does not
interfere with measurement of the analyte).
Recommendation: Revise line 167 to remove the internal standard. This would be
consistent with the Crystal City 3 recommendations.
Priority
Line 168. Stability of a stock solution is independent of the reference standard material.
They are different physical forms—solution vs. solid state. Expiration of a stock solution
should depend on solution stability data. Expiration or re-test of a reference material should
not prevent using previously prepared stock solutions within the time that stability has been
established. New stock solutions should not be prepared using an expired reference
material.
Proposed Change: Revise line 169 to “…should not be used unless purity is re-established
or stock solution stability has been demonstrated.”
B. Bioanalytical
Method Development
and Validation
The guidance should not apply to method development.
Recommendation: Change section title to “Bioanalytical Method Validation” and
removing references to development activities.
Proposed Change: Suggest rewording ‘a priori’ to ‘prior to initiation of validation’ to
clarify the meaning. (line 173)
Line 174-177. This text is an introduction and it is recommended to revise to that effect.
Investigation of each method step and consideration of any variables from collection to
analysis is unclear. BA groups can evaluate critical steps in the method and provide
information (to clinical or non-clinical groups) concerning sample collection, processing,
and shipping requirements.
Proposed Change: Line 180, replace “for production batches” with “during sample
analysis”.
179-185
Line 179-183. This introductory section discusses matrix effects, but there is no section
below for this topic. Matrix effects should be discussed in further detail in an appropriate
validation section below (its own section or under Selectivity).
Recommendation: Remove the first two sentences of this paragraph. Also the impact of
matrix effect on extraction efficiency is not normally evaluated.
Please clarify: On Line 185, “description of validation runs that failed”. Is a listing (table)
of failed runs in the report and the reason for failure sufficient?
187-190
Recommend removing the reference to ‘development’. These items should be demonstrated
during validation.
1. Selectivity
192
DC01/ 3286969.1
EMA and Anvisa guidances require the assessment of selectivity in hemolyzed and lipemic
matrix sources when serum/plasma is the target matrix.
Recommendation: In order to align the draft guidance with those of the other agencies we
recommend that lines 196-197 be revised to state “Analyses of blank samples of the
appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at
least six independent sources; in the case of plasma or serum, these six sources may include
those that are hemolyzed and/or lipemic”.
194-199
We suggest that the agency consider aligning the need for method selectivity assessment to
the inherent specificity of the detection approach that the method employs. Specifically,
retention of the following text from the 2001 guidance should be considered: “for
hyphenated mass spectrometry-based methods, however, testing six independent matrices
for interference may not be important.”
Finally, we request that agency expectations with respect to the evidence needed to prove
that the substance quantified is the intended analyte be stated. Is a match of the retention
time of analyte peaks in standards and post-dose samples sufficient evidence for SRM
LC/MS methods, or is additional information required?
DC01/ 3286969.1
Additional clarity on the methodology for demonstrating lack of interference from
concomitant medications and other xenobiotics is requested; is it expected that data
generated by comparing the response from samples containing analyte at the LLOQ with
those containing the analyte at the LLOQ together with the concomitant medication be
presented? If so, an acceptance criteria for this assessment should be provided.
Priority
201-204
Furthermore, it is proposed that, in general, specific testing for concomitant medications be
limited to those compounds planned in a study protocol to be administered together.
Examples include drugs to be administered together as part of a drug-drug interaction study
and drugs being developed as components of fixed dose combination therapies.
In the case of studies in patients, where the number and identity of concomitant medications
may be highly variable, demonstration that there are no interfering peaks observed in a predose sample obtained prior to the administration of test compound, but at a point in time
where concomitant medications would be expected to be at steady state levels, should be
recognized as sufficient evidence to confirm a lack of interference.
2. Accuracy,
Precision,
Recovery
206
DC01/ 3286969.1
Line 179 refers to matrix effects, but this topic is absent from the subject heading and the
topic is better aligned with Recovery.
Recommendation: We recommend that Matrix Effect be addressed in this section and the
heading be “2. Accuracy, Precision, Recovery, and Matrix Effects”
Recommendation: To add clarity to the A&P discussion, the following statement should be
added to the start of the this section. “A validation should contain a minimum of 3
precision and accuracy runs conducted over at least 2 days followed by as many additional
runs as are necessary to establish the required validation criteria.”
208-215
217-227
DC01/ 3286969.1
The current description of the accuracy requirements in lines 208-215 lacks sufficient
clarity. In addition, the lines are at variance with the EMEA recommendations.
Proposed change: We propose the following text to clarify this section. “Accuracy is
determined by replicate analysis of validation samples containing known amounts of the
analyte. Intra-run accuracy should be measured using a minimum of five determinations at a
minimum of four concentration levels (e.g., LLOQ, low, middle and high). The mean
measured value should be within ±15% (bias) of the nominal value for all validation
concentration levels except for the LLOQ, where the bias (%) should be within ±20%. For
the validation of the inter-run accuracy, LLOQ, low, middle and high validation samples (n
 5 each) from at least three runs analyzed on at least two different days should be
evaluated. The mean concentration should be within ±15% of the nominal values for all
validation samples, except for the LLOQ which should be within ±20% of the nominal
value.”
The current description of the precision requirements in lines 217-227 lacks sufficient
clarity. Furthermore we feel that an assessment of precision that involves different analysts,
equipment, reagents, and laboratories is beyond the scope of an assay validation. We
propose the following text to clarify the recommendations pertaining to precision
assessments during assay validation.
Proposed change: The precision of an analytical method describes the closeness of
individual measures of an analyte when the procedure is applied repeatedly to multiple
aliquots of a single volume of biological matrix. Intra-run precision should be measured
using a minimum of five determinations at a minimum of four concentration levels (e.g.
LLOQ, low, middle and high). The precision determined at each concentration level should
not exceed a CV of 15% except for the LLOQ, where the CV should not exceed 20%. For
inter-run precision, LLOQ, low, middle and high validation samples (n  5 at each
concentration in each run) from at least three runs analyzed on at least two different days
should be evaluated. The inter-run CV value at each concentration level should not exceed
15%, except for the LLOQ where it should not exceed 20%.
229-231
DC01/ 3286969.1
The requirements for validation of dilution are not clear.
Please clarify: If it is sufficient to simply demonstrate the ability to dilute from a
concentration at least as high as the highest analyte concentration found in samples down
into the standard curve range, or is it necessary to verify the ability to perform at each
dilution factor applied. In order to address these points, consideration should be given to
aligning the recommendations with those in the EMEA guidance.
Proposed change: “Dilution integrity should be demonstrated by spiking the matrix with
analyte at a concentration above the ULOQ of the assay that is at least equal to the highest
anticipated concentration in study samples and diluting this sample with blank matrix using
a dilution factor at least equal to the greatest factor to be applied to study samples.
Accuracy and precision, based on n 5 determinations, should be within ±15%. “
The current description of recovery confounds matrix effects with recovery. It is generally
recognized that response for analyte spiked into an extracted matrix blank should be used as
the comparator rather than analyte in neat solvent.
Proposed change: “The recovery of an assay is the detector response obtained from a
traditionally extracted spiked matrix sample (e.g. QC), compared to the detector response
obtained for an extracted blank matrix sample spiked post-extraction with the same amount
of analyte (or internal standard).”
Consideration should be given to determining recovery at two concentrations (Low and
High) rather than three; this would align the concentrations to be used for recovery
assessment with those used for other assessments (i.e. stability).
233-240
242
DC01/ 3286969.1
In order to address matrix effects, inclusion of the following text, which is in alignment
with other bioanalytical guidances, is suggested.
Recommendation: “Matrix effect pertains to the signal suppression and/or enhancement
for the detection of the analyte and/or internal standard via the intended bioanalytical
method within the limits of variability. For each analyte and the IS, the matrix factor (MF)
should be calculated for each of 6 lots of matrix from individual donors, by calculating the
ratio of the peak area in the presence of matrix (measured by analyzing extracted blank
matrix spiked with analyte post-extraction), to the peak area in absence of matrix (pure
solution of the analyte). The IS normalized MF should also be calculated by dividing the
MF of the analyte by the MF of the IS. The CV of the IS-normalized MF calculated from
the 6 lots of matrix should not be greater than 15 %. This determination should be done at a
low and at a high level of concentration.”
3. Calibration
Curve
All references to QCs in this section should either be removed, with reference made to the
Method Use section (preferred), or clarified as pertaining to validation runs only.
244-256
Line 252 – Recommendation: For clarity, this section should say that "Each run should
contain at least 6 non-zero standards in matrix (with internal standard where applicable).
The run should also include a blank (matrix sample processed without analyte or internal
standard) and a zero sample (matrix sample processed with internal standard but without
analyte)”
The latter 2 are not part of the standard curve, so should not be in the same sentence.
Priority
258-260
DC01/ 3286969.1
Line 258 - The requirement for 6 validation runs over 3 days is unnecessary for
chromatographic methods in most cases, and it is unclear how this should be implemented
as described.
Line 259-260 - The reference to “4 concentrations… analyzed in duplicate” in lines 259260 mixes the requirements for accuracy and precision (A&P) during validation (4
concentration levels used) with Method Use run acceptance QC’s (run in duplicate).
Proposed Change: Remove paragraph. Include the following statement after line 207 at
the beginning of the A&P section. “A validation should contain a minimum of 3 precision
and accuracy runs conducted over at least 2 days, followed by as many additional runs as
are necessary to establish the required validation criteria.”
a. Lower Limit of
Quantification (LLOQ)
Line 267- The response of the LLOQ and blank should be qualified in a separate specificity
experiment using individual lots of plasma, where the response in the blanks should be
≤20% of the LLOQ responses. This can be assessed during one of the A&P runs and is
independent of the response in the LLOQ calibration standard.
Line 269- Please clarify: that the CV and accuracy referred to in this section pertains to the
inter-run performance of the LLOQ calibration standard during validation runs (specifically,
this is independent of the performance of the A&P validation samples).
262-274
Line 273-274- Recommendation: Delete this sentence. This section is on calibration
curves during validation. The A&P of the LLOQ is already addressed in the earlier A&P
section where n5 is indicated.
Definitions of accuracy and precision criteria should be clear and consistent, avoiding terms
like “at least” or “does not exceed”. The following terms are recommended to be used
consistently throughout the guidance:
Precision: ≤15 %CV, ≤20 %CV for the LLOQ
where %CV = (STDDEV / MEAN)*100% for all included replicates.
Accuracy: ± 15%, ± 20% at the LLOQ, where this is the percentage error from the
nominal concentration.
276-282
DC01/ 3286969.1
b. Upper Limit of
Quantification (ULOQ)
Please clarify: that the CV and accuracy referred to in this section pertains to the inter-run
performance of the ULOQ calibration standard during validation runs.
c. Calibration
Curve/Standard
Curve/ConcentrationResponse
284-295
DC01/ 3286969.1
We would like for FDA to provide more clarity or detail on how they want us to “justify”
regression and weighting selection. A suggestion is that the curve fit be judged by the
residual values.
Recommendation: It would be helpful to distinguish between the definition components of
the standard curve and the run acceptance criteria to be applied during validation. We
suggest removing all run acceptance criteria from the Calibration Curve section and
replacing the section below (QCs) with a section called “Validation Run Acceptance
Criteria”
Proposed chance: Include the minimum number of passing standards to define the
calibration curve and move this paragraph to the next section: “The acceptance criterion for
the standard curve is that at least 75% of non-zero standards should meet the above
acceptance criteria, including the LLOQ and the curve must consist of at least 6 acceptable
non-zero concentrations.
d. Quality Control
Samples (QCs)
Recommendation: As mentioned above, it is recommended that this section be renamed
and modified to cover run acceptance criteria during validation.
297-320
Proposed Change: Suggested text:
Run Acceptance Criteria During Validation
“Run acceptance for A&P runs should be based on the performance of the calibration
standards only. Standards/calibrators should not deviate by more than 15% of the nominal
concentrations, except at the LLOQ where the standard/calibrator should not deviate by
more than 20%. At least 75% of non-zero standards should meet the above criteria,
including the LLOQ and the curve should consist of at least 6 acceptable non-zero
calibration standards. Data for all passing A&P runs should be included in the bioanalytical
report. No run acceptance QC samples are required during validation A&P runs since the
validation samples themselves are used to assess A&P.
For non-A&P runs, both standards and run acceptance QCs should be included in each run
and the run acceptance criteria described in the Method Use section should be followed.”
Recommendation: Lines 299-310 should be removed from this section and moved to the
section on Validated Method Use (Section C), as these statements refer to sample analysis
and not the topic of Section B – Bioanalytical Method Validation.
Priority
322-325
DC01/ 3286969.1
4. Sensitivity
Lines 311-317- Proposed change: We suggest simplification to: “If duplicate stock
solutions are compared and verified (within 10% of each other), and stability has been
demonstrated, then standards and QCs may be prepared from a single stock solution.”
Recommend eliminating the “requirement” that sample runs must be performed with two
separate stock solutions.
5. Reproducibility
327-331
Recommendation: To remove this section or change the title of this section to “Reinjection
Reproducibility”, which was defined in the previous guidance (autosampler or extract
stability). In this regard, it is believed that no additional reinjection reproducibility tests are
required if there is a good demonstration of ‘processed sample stability’ during Pre-study
validation as elucidated in lines 385-388.
With reinjection reproducibility, it is not clear whether the reproducibility refers to
reinjection of an entire assay sequence or partial assay sequence and, if so, what are the
acceptance criteria? ISR is not a part of the pre-study validation and incurred samples are
rarely available during a typical pre-study validation.
Since, an independent section (V) has already been dedicated to incurred sample reanalysis
(reproducibility) there is no need to repeatedly mention ISR in the section.
6. Stability
333
It is not clear whether there is a need for stability assessment on dry ice to satisfy the
requirements.
335-338
DC01/ 3286969.1
Recommendation: The chemical stability of an analyte of interest in a given matrix under
specific conditions or a range of storage temperature conditions (i.e. removing study
samples from deep freezer at ≤ -60C to regular freezer at ≤ -15C) for given time intervals
should be assessed. Although it might not be feasible to obtain complete stability coverage
prior to study sample collection or study sample analysis, stability evaluation should be
initiated as early as possible to ensure the coverage of the expected sample handling and
storage conditions during the conduct of the study, including conditions at the clinical site
and at all other secondary sites.
Priority
Please clarify: Does “container system” refer to container material (glass vs. plastic) or a
specific brand of container or specific dimensions of a container?
Proposed change: Remove container system from the wording of this paragraph.
340-343
It is believed that analyte “stability” has nothing to do with container (plastic or glass).
However, loss of analyte in certain matrices (e.g. urine, CSF, or ultrafiltrate) due to
container surface adsorption is indeed an issue in bioanalysis. This issue should be assessed
and addressed during method development.
Proposed change (lines 340 to 343): Drug stability in a biological fluid is a function of the
storage conditions (e.g. storage temperature) and the physicochemical properties of the drug
in the matrix. The stability of an analyte in a particular matrix under a defined storage
conditions is relevant only to that matrix and storage condition and should not be
extrapolated to other matrices and conditions.
345-351
DC01/ 3286969.1
Please clarify: Whether stability in blood (the first step of sample collection) needs to be
generated or not.
Priority
It is unclear why stock solution must be made fresh for these assessments (line 354). A
stock solution within the established stability window for the intended method should
suffice for use in matrix stability assessments. Furthermore, it appears that the stability of
analyte in matrix vs. in solvent is mixed in this paragraph. In practice, stock solution
stability and analyte stability in matrix should be discussed separately. Furthermore,
assessment of stock solution stability against nominal values is impractical and acceptance
criteria tighter than ±15% are generally applied for stock solution comparisons.
Proposal change: Remove the wording ‘freshly prepared stock solution’ and have two
separate paragraphs, one for stock solution stability and the other for biological matrix
stability.
353-359
Proposed change for lines 353-359 (having two paragraphs): The procedure should also
include an evaluation of analyte stability in stock solutions, for which new stock solutions
prepared in an appropriate solvent at known concentrations are to be compared with those
previously prepared. The measured instrument responses (peak area or instrument response
ratio of the analyte to internal standard) of the freshly prepared stock solutions and aged
stock solutions are compared using at least three replicates. The difference (%) in
instrument responses of the two stock (fresh vs. old) solutions should be within ± 10% of
each other.
Stability assessment of the analyte in biological matrix should use a set of calibration
standards prepared from valid (freshly made if stock solution stability has not been
established) stock solution(s) of the analyte using appropriate analyte-free, interference-free
biological matrix. At least three replicates of validation stability samples at each of the low
and high concentrations should be assessed. Stability sample results should be within ±15%
of nominal concentrations.
361-365
DC01/ 3286969.1
a. Freeze and Thaw
Stability
Line 361-365 on the duration of freezing and thawing as described in the previous version
of the draft guidance is missing. This can be misleading. Can we flash freeze in dry ice bath
then thaw and repeat for 3 cycles and then assay all on same day?
367-370
b. Bench-Top Stability
c. Long-Term Stability
372-375
Priority
Proposed change: We suggest changing the sentence from “the time between the date of
first sample collection and the date of last sample analysis.” to “the longest time between
sample collection and analysis for any individual study sample”.
d. Stock Solution
Stability
Please clarify: The meaning of “solid stock solution”. We note that the 6 hr room
temperature stock stability in the current Guidance has been removed from this draft. Is it
no longer required?
377-383
Stability for the stable isotope labeled internal standard (SLIS) is not critical since it is not
being quantified. SLIS should be acceptable as long as it is demonstrated that it does not
interfere with the analyte. In practice, blanks with and without the IS are included in any
single validation or sample analysis run to demonstrate the IS is not contaminated with the
analyte and that there are no interferences with the analyte channel (SRM transition), which
should be considered sufficient. In addition, in some cases the Certificate of Analysis, purity
information, etc., are missing from the internal standard document.
Priority
DC01/ 3286969.1
Clarification is needed since ‘long term stability’ should cover the longest time period of
any given sample between collection and analysis, but not for the duration of the first
sample collected and the last sample analysis.
Proposed change: To remove the requirement for internal standard stock stability,
particularly when stable isotope labeled internal standard is used showing no evidence of
isotope loss.
e. Processed Sample
Stability
385-388
Please clarify: Why is additional ‘Processed Sample Stability’ needed if ‘Reinjection
reproducibility’ has already been discussed and highlighted in line 330. If a lab has
established ‘reinjection reproducibility’ as highlighted in line 330, this reproducibility data
should be sufficient to scientifically justify the reinjection of the entire assay batch. There
is no need to re-inject with a fresh standard curve against so-called ‘processed sample
extract’ to establish stability.
Recommendation: To omit this requirement since it is never utilized in practice.
390
C. Validated Method:
Use, Data Analysis,
and Reporting
395-398
Current section indicates that SST data should be maintained with study records. This
contradicts line 930 stating these data should be stored in specific file on-site. Please
provide additional clarifications on requirement of SST.
399-408
Recommendation: Line 405-408, It is generally understood that partial run acceptance is
not appropriate. Propose to remove acceptance criteria for each processing batch within an
analytical run. For analytical runs separated in distinct batches (96-well plates), it may not
be practical to include duplicated QC of all levels on every plate. Propose to remove such
requirement or allow scientific judgment on including a limited number of QCs per
batch/plate as long as the required of numbers calibrators and QCs for the run is met (e.g.
QCs numbering  5% of unknowns)
409-410
Priority
DC01/ 3286969.1
Recommendation: This section should include a detailed description of the run
requirements and acceptance criteria to be used during study sample analysis.
“curve to cover expected study sample concentration range” is mainly for BE type of
studies. For dose ranging studies (Tox), FIH, etc, this likely will not be possible. In these
cases dilution of samples into the curve range should be allowed as long as the maximum
dilution factor has been validated.
Run requirements and acceptance criteria for Validated Method Use should be defined in
this Validated Method Use section. Example text for this section is provided below.
Proposed change: “Each sample analysis run should contain a calibration (standard) curve
and quality control samples. Specifically, each run should consist of a blank sample (matrix
sample processed without analyte or internal standard), a zero sample (matrix sample
processed without analyte but with internal standard), and at least six non-zero calibration
standards (matrix samples processed with analyte and internal standard) covering the
expected range, including the LLOQ. The calibration standards provide one basis for
accepting or rejecting the run. The acceptance criteria for the calibration curve are that back
calculated values for calibration standards should not deviate by more than ±15% of the
nominal concentration, except at the LLOQ where the concentration should not deviate by
more than ±20%. Furthermore, at least 75% of non-zero standards should meet the above
criteria, including the LLOQ. Excluding an individual standard should not change the
regression model used. Exclusion of calibrators for reasons other than failing to meet
acceptance criteria and assignable causes is discouraged.
411-412
DC01/ 3286969.1
Each sample analysis run should also included quality control samples. At least three
concentration levels of QCs in duplicate should be incorporated into each run as follows:
one level within three times the LLOQ concentration (low QC), one level in the midrange of
the calibration curve (middle QC), and one approaching the high end of the calibration
curve (high QC). The QCs also provide a basis for accepting or rejecting the run. At least
67% (e.g., at least four out of six) of the QC concentration results should be within ±15% of
their respective nominal (theoretical) values. At least 50% of QCs at each level should be
within ±15% of their nominal concentrations. The minimum number of QCs should be at
least 5% of the number of unknown samples or six total QCs, whichever is greater. A
confidence interval approach yielding comparable accuracy and precision in the run is an
appropriate alternative for assessing run acceptance.”
Recommendation: Line 412 requires reporting of A&P results for failed runs which
conflicts with line 450 and the standard practice that data from failed runs is normally not
reported. Runs that fail due to assignable cause (e.g. curve or QC failure) should not be
included in study A&P statistics, although the fact that the run failed and the reason should
be included in the report.
Proposed Change: “…and tabulated for all acceptable run”
Priority
413-416
The guidance calls for levels that are below the LLOQ to be reported as zero. We
recommend that samples with concentrations below the LLOQ be reported as <LLOQ or
BQL, not as zeros. The use of PK data since 2001 has changed significantly, with many
more population PK and PK/PD studies now being performed. The arbitrary setting of
concentrations <LLOQ to zero may have serious implications for modeling analyses.
Pharmacokineticists require the flexibility to use these data in a flexible but scientifically
rigorous way.
Proposed change: Change the sentence "Concentrations below the LLOQ should be
reported as zeros" to "Concentrations below the LLOQ should be reported as <LLOQ or
BLQ"
Propose to include exceptions for rare matrices.
417
418-419
Recommendation: Requirement is for stability to be generated before running samples.
This is not always possible. Requirement should be for adequate stability to be generated
before results are reported. We recommend allowing analysis prior to generation of the
stability as long as stability assessment is documented when reporting the data.
420-426
427-428
DC01/ 3286969.1
Priority
Please Clarify: It would be very helpful if industry and regulators could agree on criteria
(process and acceptance criteria) for variability of the IS response.
It looks like the reference to section III.B.2. is not correct, the correct section is III.B.3.d.
Remove sentence if above recommendation on placement of run acceptance criteria in this
section is adopted.
429-431
Recommendation: Allow both interspersing of QC samples with study samples and
bracketing study samples with QC samples.
Comment in line 427-428 applies as well.
432-436
Recommendation: Allow adding of additional QC samples without requiring validation
before continuing with the analysis. Propose to limit this requirement to BE studies.
Proposed Change: ‘If the study sample concentrations are clustered in a narrow range of
the standard curve, additional QCs should be added to cover the sample range. Samples that
have already been analyzed do not require re-analysis.’
Priority
437
438-439
No comment
Priority
442-447
DC01/ 3286969.1
Recommendation: The draft guidance indicates that “all study samples from a subject
should be analyzed in a single run”. This requirement should be only for BE studies.
Analysis of all subject samples in a single batch may not be feasible for most early-phase
studies where multiple PK samples are collected over longer sampling intervals and might
end up being shipped at different times. Also, this would not be possible in oncology trials
where PK samples may be collected over multiple cycles. This also impacts long term GLP
Tox studies, where samples are collected at different time intervals, up to 12 months.
Additionally, this is not necessary since ISR analysis verifies the methodology is consistent
across multiple analytical runs/batches.
Recommendation: The draft guidance indicates that “reassays should be performed in
triplicate”. We recommend that a single reanalysis be used in situations where the original
concentration data are considered invalid (sample prep, chromatographic issues, and other
assignable causes) and when reanalyzing samples above the validated range by dilution.
We recommend using duplicate reanalysis (when sample volumes allow) in situations
where a valid concentration is available, but where the sample is being reanalyzed for
confirmation/verification, as in approved PK repeats.
448-449
Proposed change: We agree with the guidance; however, we recommend rephrasing this
sentence for additional clarity, to read “In samples involving multiple analytes, a valid
result for a specific analyte should not be rejected due to failure of a result for another
analyte in the same run”.
452-455
This paragraph is out of context and either needs to be clarified with more detail or the
MIST guidance should simply be referenced for information pertaining to qualification of
metabolites (preferred).
456-458
Recommendation: This sentence refers to Method Validation accuracy and precision
reporting. We recommend moving it to section III B (Bioanalytical Method development
and Validation), and we recommend reporting accuracy and precision with outliers
excluded, since that is what is required for acceptance; however, data tables will contain
flagged values for all outliers.
Priority
459-463
465
IV. LIGAND BINDING
ASSAYS
467-470
Please specify or give examples for “microbiological” assays
A. Key Reagents
472
DC01/ 3286969.1
Proposed change: The draft guidance (lines 462-463) indicates that “original and
reintegration data should be reported”. We recommend revising this sentence to state that
“only final data should be reported and that the original (prior to reintegration) and final
data will be retained as raw data (as part of study records)”.
Proposed change: Propose to use “critical reagent”, which is commonly used by the large
molecule BA community.
Proposed change: The terminology “tracer” (i.e. “Labeled analytes”, as defined in line
480) is mostly used in competitive-binding assays (i.e. RIA). Most current LBA assays do
not use labeled analytes. It is recommended to replace “tracer” with “detector reagent”.
474-489
Please clarify: The requirement that “Tracer experiments above should be repeated” needs
clarification on what specific experiments are needed. The requirement of “Key crossreactivities should be checked” also needs clarification on whether this means specificity or
other experiments to be conducted.
491
B. Bioanalytical
Method Development
and Validation
506-509
Please clarify: The requirement to demonstrate “Recovery” in LBA assay’s validation, in
association with accuracy and precision, needs clarification. Most LBA assays do not
require an “extraction” step and the terminology “recovery” usually means accuracy in LBA
literatures. Also, suggest to consider removing line 557, as it's the same as accuracy (with
standard being processed the same).
511
Recommendation: Specificity and Selectivity should be more clearly defined in context of
LBA assay. The specificity of an antibody refers to its ability to bind to the antigen of
interest. Selectivity, a concept related to specificity, is the ability of an assay to measure the
analyte of interest in the presence of other constituents in the sample.
513-515
1. Selectivity
DC01/ 3286969.1
Recommendation: Some things in this section are completed in method development but
not repeated in validation. Since method development is not a regulated activity, it would
be helpful to see the recommendations for development and validation separately. It is
preferred that all references to method development to be left out of this guidance, which is
intended for guiding BA validation.
Recommendation: It is not clear why this section references Section III on
chromatographic methods. The selectivity and specificity in LBA (see above comments)
has two separate definitions. Suggest defining clearly these terms for the different
technologies.
Recommendation: Propose that Section a. under Selectivity be removed due to following
considerations:
517-524
(1) It is not feasible to assess interferences by physiochemically similar substances in the
case of large molecules, as concomitant medications and protein "metabolites" are not/ need
not be physiochemically similar to macromolecular analytes. In addition, the mechanism for
analytical interference between protein drugs and small molecules are different, thus the
recommended testing is not warranted;
Priority
526-534
Line 530-531: Direct comparison of performances for standard curves prepared in buffer vs.
that in diluted matrix is not commonly done for PK assays. The guidance already states
that standard curves should be prepared in the relevant biological matrix (Line 571).
Proposed change: Either remove this paragraph, or clarify that buffer standard curve
evaluation may be considered where appropriate, e.g. for biomarker type tests. Applicable
criteria could be established to ensure the (pooled) matrix does not have cross-reactive or
inhibitory elements that may bias sample results. It is recommended such test, if needed,
should be conducted in method development.
Evaluating curves with at least 10 sources of blank matrix is not a common practice.
Recommendation: Propose to evaluate 10 individual lots, unspiked matrix & spiked QCs
(e.g. LLOQ, HQC), against a curve prepared in pooled matrix.
Priority
DC01/ 3286969.1
(2) It is rare that two assay methods using separate platforms (e.g. LBA and LC/MS) are
available. While two methods that provide the same result is indicative of accuracy,
disagreement between two methods (i.e. LBA and LCMS) does not indicate which method
is more or less accurate. Even if a LC/MS assay is available, it is not recommended to
consider it as a reference method by default.
Please clarify: Line 532 – 533- Parallelism evaluation needs clarifications on samples
selection (concentration, number of samples, etc.), acceptance criteria, timing and frequency
of such test. Also, Line 534: Suggest to clarify what “non-specific binding” (in context
of matrix effect) is referring to, and what specific experiments are needed.
536
2. Accuracy,
Precision and
Recovery
Recommendation: The guidance states that a minimum of 3 concentrations in the range of
expected study sample concentrations is recommended for accuracy and precision. For
LBAs accuracy and precision should be conducted with QCs that span the assay range. The
guidance should also clarify as the minimum number of determinations (“a minimum five
determinations per concentration”?) and should apply the same acceptance criteria to ULOQ
as to LLOQ (25%).
Proposed change: We suggest changing the sentences to: Accuracy should be measured
with a minimum of 5 replicates of QCs that span the assay range (including LLOQ, LQC,
MQC, HQC and ULOQ). The mean value should be within 20% of the nominal value
except at LLOQ and ULOQ, where they should not deviate by more than 25%.
538-542
Similar changes as above should be made to this section (see comment for lines 538-542).
544-552
561
563-573
DC01/ 3286969.1
The guidance defines accuracy, precision, and other related terms in various sections.
Suggest defining these terms in one section of the guidance and referring to these terms
when applicable.
Furthermore, as was the case for Chromatographic assays, we suggest placing the
requirements and acceptance criteria for Method Validation and Method Use (run
acceptance) in appropriate sections.
3. Calibration
Curve
Recommendation: Because of the non-linear nature of LBAs, anchor points below and
above the quantitative range of the assay (from LLOQ to ULOQ) are often included in the
calibration curve to aid in curve fitting. We suggest including the anchors to the calibration
curve and the acceptance criteria for anchors.
575-577
Please clarify: As mentioned earlier please clarify the requirements for both validation and
Method Use calibration curves, specifically the “ minimum of six runs”, “conducted over
several days”, “with at least six concentrations”, and “analyzed in duplicate in each run.”
Proposed change: We suggest changing the sentence to: “Accuracy and precision
experiments should include a minimum of six runs conducted over three days, with at least
six calibration concentrations from LLOQ level to ULOQ level analyzed in each run.”
579-588
590-596
598-612
DC01/ 3286969.1
a. Lower
Limit of
Quantification
(LLOQ)
Recommendation: The guidance states that the LLOQ is the lowest concentration on the
calibration curve. For most LBAs the lowest concentration of the calibration curve is an
anchor point. We suggest revising to include the anchor point.
b. Upper
Limit of
Quantification
(ULOQ)
Recommendation: The guidance states that the ULOQ is the highest concentration on the
calibration curve. For most LBAs the highest concentration of the calibration curve is an
anchor point. We suggest revising to include the anchor point.
c. Calibration
Curve/Standar
d
Curve/Concen
trationResponse
Proposed change: Total error is not applicable for the calibration curve. Suggest deleting
the calculation of total error for calibration from this section and adding it as part of the
acceptance criteria for QCs (section IV.B.3.d). Also allowing for a total error of up to 40%
for QCs at LLOQ level.
Proposed change: We also suggest deleting the phrase “Analyte peak (response) should be
identifiable, discrete, and reproducible” since that seems to be language copied from small
molecule chromatography based assays.
Proposed change: The guidance applies different acceptance criteria to LLOQ vs. ULOQ.
However, since LBAs are typically having sigmoidal concentration – response relationship,
thus same acceptance criteria should be applied to ULOQ as to LLOQ. We suggest
changing Line 595 to read: “…not exceed 25% CV and accuracy within 25% of”.
Proposed change: Suggest deleting sentence of “Selection of weighting and use of a
complex regression equation should be justified”. Using complex regression and weighting
is standard for LBAs, therefore there should be no justification required.
d. Quality
Control
Samples
(QCs)
Recommendation: We recommend that the emphasis on QCs within the range of expected
study sample concentrations should only apply to BE/BA studies but not for all studies.
Firstly, it is not always known “a priori” what the concentrations will be especially in FHD
studies, DDI studies, special populations and blinded Phase II and III studies. Secondly, the
calibration curve range of an LBA may be limited by the assay format and different from
the expected study sample concentrations.
Please clarify: For LBA assay, overall assay performance including all runs is also
assessed. Are there recommended acceptance criteria for overall assay performance?
614-634
Lines 626-627 refers to the minimum number of QCs to be used for sample testing. This
sentence needs clarification as it references unknown samples which are not tested during
method validation. We recommend removing this sentence since it belongs to the sample
testing section and the same sentence is also present in lines 723-724 under validated
method use.
From lines 628-634, it is not clear if one stock or spiking solution may be used for
calibration standards and QCs. We recommend that this section be clarified and the terms
"stock solution" and "spiking stock solution" should be defined. Furthermore, a separate
stock solution may not be practical for LBAs since a reference material may be provided as
a solution and a separate stock would just be a different aliquot form the same solution. We
recommend that the requirement for separate stock solutions be removed or at least
exempted for reference materials in solution form.
636-637
Proposed change: Lines 636-637 refer to the acceptance/re-injection criteria. While this
makes perfect sense for exogenous analytes, it is more difficult to comply with the “nominal
concentration” concept when analyzing endogenous analytes, like biomarkers (which are
supposed to be covered by this guidance as mentioned in line 22). We recommend to start
the sentence with: “for exogenous analytes …” to be aligned with section VI A.
Endogenous compounds
639-642
DC01/ 3286969.1
4. Sensitivity
Please clarify: If the assay sensitivity is LLOQ or LOD. Also, other assays (i.e. kits)
define “Sensitivity” in the cut point manner (Mean + nSD of 20 or 30 blank measurements).
This may bring confusion in terminology.
644-648
5. Reproducibility
Proposed change: Line 647 refers to “reinjection reproducibility” which is not applicable
to LBAs. We recommend either to delete this sentence or to change the wordings from
“reinjection reproducibility” to “reanalysis reproducibility”.
6. Stability
650
652-655
DC01/ 3286969.1
Proposed change: Line 652 refers to chemical stability which is not applicable to LBAs.
We recommend changing the wording from “chemical” to “biochemical”.
Please clarify: Line 657 indicates that stability samples should be compared to freshly
made calibrators and QCs. Clarification is needed to define “fresh” for LBA. We
recommend defining it as freshly made frozen calibrators and QCs that are made within 3
days for the stability evaluation.
657-666
Proposed change: Lines 660 and 665 mentions stock solution that may not be applicable
for LBA since the reference material, for LBAs, is often provided in solution form and the
making of stock solution is not needed. We recommend removing the “stock solution” term
from this section...
Please clarify: Line 662 refers to both “bench top” and “room temperature” storage
stability. The difference between “bench top” and “room temperature” storage stabilities is
not clear. We recommend that the difference between “bench top” and “room temperature”
stabilities should be clarified or addressed by removing one of the tests.
Priority
668-672
674-677
DC01/ 3286969.1
a. Freeze and
Thaw Stability
b. Bench-Top
Stability
Proposed change: Line 666 proposes that “stability sample results should be within 15% of
nominal concentrations”. We recommend changing this sentence to “Stability sample
results should be within the acceptance criteria of the assays”, for consideration of the LBA
assay acceptance criteria.
679-682
684-690
d. Stock
Solution
Stability
692-695
e. Processed
Sample
Stability
697
703-704
DC01/ 3286969.1
c. Long-Term
Stability
C. Validated Method:
Use, Data Analysis,
and Reporting
Proposed change: This reads as though a 5 year clinical study would require 5 year LTS to
be demonstrated. We recommend changing the wording to “The storage time in a long-term
stability evaluation shall equal or exceed the duration from time of sample collection to
analysis for any given sample”.
Recommendation: It appears this section was cut/pasted from the chromatography section.
Certain aspects of which do not apply to LBAs. Recommend deleting portions that don’t
apply and expanding on LBA expectations.
Recommendation: Currently it is proposed that “calibration (standard) curve should cover
the expected study sample concentration range”. Because typical biologic drugs are often
administered at high doses and a typical LBA method dynamic range (quantification range)
is limited, samples are often tested at high dilutions. It is not practical to expect that the
assay calibration curve will cover the entire range of concentrations observed in incurred
study samples. It is suggested that clarification be added to allow dilutions to within the
range. A reasonable expectation for LBAs is to ensure that the dynamic range of the assay
including the LLOQ as well as a demonstration of dilution integrity are appropriate to
support study sample analysis.
Please clarify: Line 705: This section states that Inter- and intra-assay is discussed for both
accuracy and precision as being reported in MVR but in section IV.B.2 (Lines 536-552)
intra- and inter- assay is only described for precision assessment. Suggest clarification of
wording in Section IV.B.2 to include inter- and intra-assay assessment of accuracy to
provide consistency throughout the guidance.
705-706
Proposed change: The rationale for including data from failed runs in accuracy and
precision calculations during method use is unclear. It also contradicts line 742. Consider
the following re-wording, “Accuracy and precision as outlined in Section IV.B.2 should be
provided for both the inter-run and intra-run experiments and tabulated for all acceptable
runs”. (This rewording assumes that section IV.B.2 is also clarified regarding
precisions/accuracy.)
Line 710: The guidance calls for levels that are below the LLOQ to be reported as zero. We
recommend that samples with concentrations below the LLOQ be reported as <LLOQ or
BQL, not as zeros. The use of PK data since 2001 has changed significantly, with many
more population PK and PK/PD studies now being performed. The arbitrary setting of
concentrations <LLOQ to zero may have serious implications for modeling analyses.
707-711
Pharmacokineticists require the flexibility to use these data in a flexible but scientifically
rigorous way.
Proposed change: Change the sentence "Concentrations below the LLOQ should be
reported as zeros" to "Concentrations below the LLOQ should be reported as <LLOQ or
BLQ"
Proposed Change: Line 708-709: Extension of the curve for LBAs is not always possible
and it is suggested that this language regarding extension or revalidation be removed
allowing for dilution of samples into the validated range of the existing method.
DC01/ 3286969.1
714-719
Proposed Change: Lines 714-719: If “response function” = curve fitting, the entire
paragraph can be deleted.
722-724
Proposed Change: Interspersed QCs are not used for LBAs and Positional affects are
addressed during validation. Clarify or remove this language from this section.
725-729
Please clarify: Line 725-726 Define a curve range beyond which "clustering" of samples
"in a narrow range" is meaningful. For LBA with narrow dynamic ranges (e.g. <100-fold),
is 3 nominal QCs spanning the range (LQC, MQC and HQC) is sufficient? Due to limited
applicability in LBA, remove or define for assays with assay ranges > 100 fold.
Additionally, if this happens for LBAs it is usually because the sample dilution scheme did
a good job of targeting the midrange of the assay. There is no added value in adding more
QCs in the middle of curve.
Recommendation: It is not always practical to run all study samples from a subject in a
single run. Examples of this include; long-term studies where the collection period may
exceed sample storage stability, testing of early time points for dose escalation, repeat
analysis, etc…. This is a best practice should not be a requirement.
730
731-732
DC01/ 3286969.1
Proposed change: Additionally, this is not needed if the method has demonstrated
acceptable precision and accuracy during validation. Reword to state, "All study samples
from a subject should be analyzed in a single run whenever possible"
Please clarify: For LBA methods carryover is not an issue and this appears to be duplicated
from the small molecule section. Please clarify if this pertains to the use of liquid handling
during LBAs.
740-741
Proposed change: Line 740 There is no possibility of reinjecting samples in LBA. Please
remove.
744-746
Not applicable for LBA, please remove.
750
V. INCURRED
SAMPLE
REANALYSIS
Proposed change: The draft guidance indicates that “ISR is conducted by repeating the
analysis of a subset of subject samples from a given study in separate runs on different days
to critically support the precision and accuracy measurements established with spiked QCs”.
Recommend that the requirement “on different days” be deleted since analysis in separate
runs should be sufficient.
752-760
Priority
Proposed change: The draft guidance indicates that “ISR samples should be compared to
freshly prepared calibrators”. Recommend this sentence be deleted since ISR samples
should be analyzed using the identical procedure used for the original analysis and fresh
calibrators are for stability tests. ISR is not a stability measurement.
Please clarify: The draft guidance indicates that ISR is required for pivotal PD studies.
Clarification is required as to whether this refers to PD endpoints (e.g. biomarkers) or PK
measurements obtained during pivotal PD studies (e.g. Phase III trials). It is noted that not
all biomarker assays lend themselves to ISR.
DC01/ 3286969.1
762-766
Recommendation: The draft guidance indicates that “For regulatory submissions
containing only a few studies, it may be advantageous to incorporate ISR into the method
development and validation stage by conducting a pilot study prior to the pivotal study.”
Recommend that this sentence be deleted since this proposal is not practical for most
clinical programs and a clinical pilot study may have ethical implications.
Priority
771
Proposed change: The draft guidance indicates “in selecting samples for reanalysis,
adequate coverage of the PK profile in its entirety should be provided and should include
assessments around Cmax and in the elimination phase for all study subjects. Recommend
clarification. Suggested wording “in selecting samples for reanalysis, include samples
across the PK profile and across subjects.
772-774
784
786
DC01/ 3286969.1
Proposed change: The draft guidance indicates “the total number of ISR samples should be
7% of the study sample size.” This amount is neither consistent with the GBC
harmonization team recommendation (5%), nor the EMEA guidance. Recommend
reverting to 10% of the samples should be reanalyzed for ISR in case the number of samples
is less than 1000 samples and 5% of the number of samples exceeding 1000 samples. This is
the current industry standard which was influenced by the AAPS/FDA workshop in 2008, in
addition to being harmonized with the EMEA guidance.
VI. ADDITIONAL
ISSUES
A. Endogenous
Compounds
794-803
Recommendation: Line 794-797 More often than not it is extremely difficult to use the
same matrix as the study samples to prepare calibration standards due to the levels of
endogenous drug. Therefore, to get concentrations of relative accuracies, use of a surrogate
matrix or a buffer to make the calibration standards should be allowed. It is recommend
revision be made to enable the use of surrogate or buffer matrix to prepare calibration
standards for the analysis of endogenous and biomarker analysis when endogenous levels
would interfere.
Proposed change: Line 800 To ensure standards, QCs and study samples have the same
matrix effect, it is suggested changing from “have no matrix effect” to “have no difference
in matrix effect”.
804-809
Recommendation: Line 804: The draft guidance notes that “the QC should be prepared by
spiking known quantities of the analyte(s) in the same biological matrix as the study
samples”. Often there are not fully accepted reference standards for endogenous analytes,
thus there are differences (purity, activity, etc.) between reported results in laboratories
using different sources of the analyte. It is suggested that the guidance could provide some
suggestions to the community on how to resolve this issue.
B. Biomarkers
811
Priority
819-826
828-830
DC01/ 3286969.1
Please clarify: Line 826: There are a number of questions regarding fully validated and the
regulations that are intended to apply to the laboratory activities, such as compliance with
GLP, GCP CAP and CLIA. It is recommended that the new guidance clarify the
expectations of the laboratory conditions and practices.
Recommendations:
Line 833 Due to the presence of endogenous analyte, it is recommend reference be made to
the recognized challenge of demonstrating assay selectivity
If ISR is expected for biomarker methods, it is recommended that explicit language be
included (e.g., in cases where a full advanced validation is warranted).
832-836
It is recommend language allowing establishing accuracy and precision acceptance criteria
based on a statistical approach based on the anticipated biomarker changes be added (e.g.,
detecting small differences requires greater accuracy and precision, where as changes that
are an order of magnitude larger or smaller may require less accuracy and precision. It is
further recommended that the language indicate that sponsors should work with statisticians
in such cases.
Proposed change: The guidance insufficiently addresses different study population
reference ranges and the expected magnitude of change. Propose changing the language to:
“The accuracy, precision, selectivity, reference range of study population(s),
reproducibility…”
Proposed change: With regard to defining the practices for the validation, it is suggested
language similar to ‘The purpose of the biomarker data and the context of biology should
guide the validation, incorporating as many principles of a PK validation as applicable and
practical within the context of the assay technology and biomarker biology.’ be used.
C. Diagnostic Kits
838
DC01/ 3286969.1
Recommendation: Line 838-883 There are many varieties of diagnostic kits, that include
Research Use Only (RUO) assays and CLIA assays. This section while providing some
details, does not address major components of using the diagnostic kits. It is recommended
that the FDA consider using diagnostics approved for use by another sections of the FDA,
including the use of 510K approved cleared, PMA approved or LDT diagnostics kits for
drug development.
840-847
Recommendation: Comment: 845 – 847 - It is recommended that the guidance address
the use of CLIA approved assays for a pivotal biomarker; since some biomarkers are well
validated and used for global trials.
863-865
Recommendation: It is suggested that the glossary addresses the term "specificity" and it is
distinguished from selectivity. And in the context here, it is suggested to add clarification on
whether both parameters need to be validated for biomarker assays.
872-875
Please clarify: Line 872. It is suggested that the guidance address the situation where many
kits use standards prepared in a different matrix than study samples, specifically is the FDA
truly going to require kit methods be changed to include matrix for the reasons expressed
previously.
876-878
Please clarify: Line 877 – 878 - Activity of reference standards from different sources may
differ and this is a potential significant problem. It is suggested that the guidance address
how the community approaches to correcting the activity, including where the endogenous
analyte source may not be available and surrogates analyte are used where the difference
might be evaluated indirectly (e.g. parallelism).
881-883
DC01/ 3286969.1
Please clarify: With regard to “plate” and multiplate acceptance criteria, it is suggested that
further details regarding the expected use of such acceptance criteria be added (i.e., accept
failure of one plate, but accept others?)
D. New Technologies
Priority
Recommendation from IQ Microsampling Working Group:
We would like the Agency to reconsider or clarify the request for data from new
technologies to always be supported with those from established technology. This had not
been done when the industry moved from UV to MS for detection, from HPLC to UHPLC
for separation, ligand binding to MS for the detection of large molecules, etc. This
requirement would essentially stifle the growth and adoption of new technologies.
Proposed change: So long as a method using the new technology can be effectively
validated to precision and accuracy requirements, a comparison between techniques is
unwarranted.
885-899
Recommendation from IQ Microsampling Working Group:
While the industry has been looking for guidance around DBS, direct feedback from the
agency to sponsors on new technologies, including DBS, has been working appropriately;
sponsors are providing the data to the agency in the appropriate manner. Microsampling
technology is evolving rapidly; therefore this section and the requirements stated around
DBS will be outdated quickly. It is, therefore, premature to include specific guidance
around DBS in this document, also inappropriate to specify other specific technologies.
Priority
Proposed change: Remove specific reference to DBS in the document. This will allow
these new technologies to evolve further in conjunction with robust industry and agency
interactions. Feedback around microsampling comparisons should continue to be provided
directly from the agency to the sponsor as expectations may vary based on individual
situations (stage of development, patient population, historical data, etc.).
Recommendation: It is suggested that the agency indicate when correlative studies are
needed and when they could end. For example the language around correlative clinical
studies (bridging) should be changed to “be conducted in the intended patient population
and once demonstrated, there is no need to perform dual sampling and analysis in further
studies”. Additional Guidance is also requested on the statistical criteria that demonstrate
correlation of DBS and original wet matrix, including numbers of patients in the disease
population.
DC01/ 3286969.1
902
VII.
DOCUMENTATION
904-926
Lines 909- 911: The draft guidance is requesting Method Development data and reports.
Methods are validated based on guidelines and parameters to ensure performance and fit for
purpose characteristics. Critical parameters of such methods are tested during the validation
and if the method is modified the "change history" maintained. It is the outcome of
validations, not development; that determines whether a method is suitable for production.
Recommendation: remove requirement for method development data and reports.
Line 913: It is not clear how to define and document “limitation to use” of a method.
Recommendation: remove the requirement to include “limitation to use”.
Line 917: The draft guidance requires documentation of correspondence records between
involved parties. It is not clear whether it is enough for the CRO to show correspondence or
it implied that the sponsor must show all correspondence.
Please clarify: since the majority of company policies limit maintaining e-mail records.
DC01/ 3286969.1
A. System
Suitability/Equilibrati
on
Line 930: System suitability for LBA instrumentation such as plate readers is assessed
periodically (based on technology and manufacturer's recommendations), but not before
each analytical run as can be performed for chromatographic methods.
Recommendation: please provide definition, clarification and differentiation of system
suitability between LBA and chromatographic methods. Consider deleting requirement for
LBA as it is not applicable in the Guidance.
928-934
Line 930: also states “System suitability is routinely assessed before an analytical run.”,
which implies that it should be done routinely, while Line 395 states “… if system
suitability is assessed…”, which implies it may or may not be done.
Please clarify: the expectation regarding system suitability and reword lines for
consistency.
Line 931: System suitability checks are “maintained in a specific file on-site”. This
statement conflicts with line 398 stating system suitability should be stored with study
records.
Please clarify: Line 931 or 398 for consistency.
Lines 936-950: It appears that these items are related to the submission package such as
IND and NDA and not necessarily relevant to a specific method validation or sample
analysis study. There is no clarification as to what this document is, where this information
is to be submitted to and if effective dates for all method versions is being requested. Need
to understand if management approval of method in advance of signed report (i.e.,
authorization to use) is acceptable or must the signed report be in place before use?
Proposed change: remove this section or clarify its intent.
940-942
DC01/ 3286969.1
936-938
B. Summary
Information
Lines 432-436: Requires partial validation run for adding QCs to match sample
concentrations. If dilution QC beyond that previously tested, may make sense, otherwise
there is no value within established range.
Proposed change: Remove
943-947
Lines 944-947: table needs to include reasons for the new method or additional validation
& dates of final reports. Comment: It is the interpretation that is this is a form of the CTD
table.
Recommendation: That clarification be made if filing practices are not within the scope of
this guidance.
948-950
The items listed required in the summary table are related to filing and should not appear as
part of study data.
Proposed change: Remove the summary table.
952-954
C. Documentation for
Method Validation
959-960
Lines 959 – 960: Clarification is needed as to whether this is a description of the
preparation of standards and QCs OR a table of all prepared standards/QC batches and the
dates of preparation. As this information is already present in the raw data and method it is
Recommendation: Limit this requirement to a table of preparation dates.
Lines 961-964: Section is primarily chromatography focused.
Recommendation: broaden the language to include LBA.
Line 961: Only suitability of internal standard should be required.
Proposed change: Removal of the requirement for evidence of purity and identity of IS
961-964
DC01/ 3286969.1
Line 962: States that the chromatogram of the analyte should be interference-free. No
chromatogram can be totally interference-free. The current validation criteria state that
interferences, such as carryover and non-selective signal are allowed to the degree of 20%
of the LLOQ.
Proposed change: reword this requirement as “The chromatogram of the analyte should
show interference that is below that set forth in the validation criteria.”
Lines 977-978: “All validation experiments with analysis dates, whether the experiments
passed or failed and the reason for the failure.”
Recommendation: That this be a simple table of all runs and contained experiments with
no expectation that individual experimental data be presented.
Lines 979-980: Please clarify: if the data should include results of calibration standards for
failed runs due to QC performance or non-assignable causes.
976-987
Lines 986-987: This section states that dilution integrity data should be reported in the
method validation report. However, dilution integrity is not listed or discussed as a
validation parameter in this guidance.
Recommendation: add a section describing dilution integrity as a validation parameter in
this guidance. Dilution integrity is a pivotal component in LBA validation and should be
included as a validation parameter.
Please clarify: Line 987- what “anticoagulant effect” means. This is the only place it is
mentioned in the document.
989-990
The draft guidance requires inclusion of the results of “individual calculated
concentrations.” For LBA, some sponsors have samples run in duplicate
Please clarify: whether individual measurements in such cases need to be reported.
992
994-995
DC01/ 3286969.1
D. Documentation for
Bioanalytical Report
Please clarify: It is not clear whether “documentation” means for all points from 996-1020
that they have to be included in the report.
996-997
The IS CoA may not be subjected to the same purity/identify requirement as analyte, as
long as it is demonstrated in the run that it is suitable.
Proposed change: Change text to read “Purity and identity of reference standards of
analytes” and remove the requirement for purity and identity of internal standard to follow
the 2007 White Paper for IS requirement.
998
The “Step-by-step description of procedures for preparation of QCs & calibrators” is
already presented in the detailed method which is part of the validation report. There is
minimal benefit to have this in the bioanalytical study report.
Recommendation: removing this requirement as part of the sample analysis report.
999-1001
Line 999: It is not practical to include collection dates/times of all the samples in the
bioanalytical report. This information can be too excessive, especially for Ph2/3 clinical
studies. In addition, this information typically resides with the clinical study
documentation/data base.
Recommendation: reporting of when the first sample is collected and the time span of the
collection period to ensure proper stability coverage.
1002-1003
Currently only deviations with impact to the study are included in the bioanalytical study
report.
Proposed change: suggest that this only be limited to list major (significant) deviations.
Minor deviations are to be kept with the raw data.
DC01/ 3286969.1
Lines 1005-1010: "Complete serial chromatograms" are not applicable to LBA.
Recommendation: include LBA relevant requirements.
1005-1010
Lines 1008-1010. This section states that the 5% chromatograms to be submitted should be
from subjects determined a priori. This is simply not practical for many clinical studies
where subjects change, leave or fail to enter a study.
Proposed change: Modify this requirement to specify that profiles be selected from a
specific series of doses or a specific portion of the trial, chosen a priori rather than specific
subjects. If a specific portion of trial is selected a priori, then randomness will be assured,
but the uncertainty of losing patients will be removed.
1011
While for GLP studies this information might be readily available, in clinical trials the
Bioanalytical labs generally do not have information regarding missing samples. This
information is usually captured clinical study files.
Proposed change: remove requirement of “reasons for missing samples” or clarify the
scope to which this is to be applied (i.e. all study types or only to BE) and the format in
which it should be presented.
1012-1015
DC01/ 3286969.1
Recommendation: Lines 1006- 1007: It seems that the "20%" cap of chromatograms even
for BE studies are excessive. Has consideration been given to reduce this 5% threshold to
limited representative study subject chromatograms (e.g., predose and Cmax)?
Lines 1013-1014: As repeat analyses (e.g., ULOQ, analyte in placebo, poor
chromatography, IS acceptance criteria failure and other assignable cause re-analysis, etc)
of a bioanalytical nature are SOP driven, they should not require manager approval.
Recommendation: limit manager authorization only to PK repeats. Documentation of
manager approval should be maintained in the study file, not the report. Clarity should be
provided on which manager should authorize reanalysis. In some instances, the sponsor
will provide justification for reanalysis. In other instances, the TPO bioanalytical lab
manager may.
1016-1020
Lines 1016-1020: Does not apply to LBA.
Recommendation: add “as applicable to the technology used”.
Proposed change: data for concentration data generated using initial integration and from
re-integrated chromatograms to be maintained in the study file and not included in the BA
report. Also, clarification should be added to the guidance defining re-integration as those
chromatograms integrated using different integration parameters than others in the same
batch.
1024-1025
Proposed change: remove line 1025 on requirement to report QC graphs and trend
analysis. This is more a GMP requirement and has no added value in regulated BA since
each batch is accepted using clear criteria on accuracy and precision using independent QC
and calibration samples.
1026-1029
Lines 1027-1029: Inclusion of listing of subjects analyzed in each run and tables with
individual back-calculated results for all study samples does not add value to the
bioanalytical report and increases the risk of transcription errors. These are maintained in
the study raw data and LIMS databases.
Proposed change: remove this requirement or limit to BE studies only. Also, specify that
method included only if multiple methods used during course of the study since usually a
single method is used the majority of the time.
Proposed change: change text in lines 1028-1029 to read: “Tables with the individual
results for all study samples should be submitted”, as the study samples do not have known
concentrations and therefore do not have “back-calculated” results.”
1030-1031
Please Clarify: Whether the tabular listings are meant for both validation and bioanalysis
reports or just needed for bioanalysis reports to summarize the conducted validation
assessments.
VIII. GLOSSARY
1033
DC01/ 3286969.1
Proposed change: Add a classification named “Validation Samples” that include proposed
validation (QC) concentration levels assessed in P&A experiments (e.g. LLOQ, LQC,
MQC, HQC and ULOQ) (Line 539).
1081-1082
There are several types of Reproducibility: between lab, re-injection, incurred sample,
between instrument, etc.
Recommendation: that a more generic definition be provided or concisely define each type
of reproducibility.
1084-1086
Recommendation: define selectivity and specificity separately for LBA in line 1084.
1089-1090
Recommendation: change “chemical” to “biochemical” in line 1089.
1100-1103
DC01/ 3286969.1
IX. APPENDIX
Priority
Recommendation: Use of hyperlinks for eCTD submissions sounds good but is fraught
with issues in practice. The process needs someone with a full overview of study/program to
manage submission/report/data links.
Table 1
Priority
New and extensive hyperlinking required between non-clinical and clinical
biopharmaceutical sections and specific portions or validation or study reports. This would
require nonspecialist dossier managers to link between the tables and specific portions of
reports. It is unlikely that this would be consistently performed.
Recommendation: It is recommended that the hyperlinking be to the reports table of contents or
to reports that are adequately bookmarked.
If table is to remain as is:
Table 1: ISR here is being referenced in validation report, not consistent with text in Lines
782-783 which indicates that ISR be included in the final study report.
Recommendation: ISR should be removed from this table.
1105-1110
Recommendation: New and extensive hyperlinking required between non-clinical and
clinical biopharmaceutical sections and specific portions or validation or study reports a
significant logistical challenge and resource burden. Recommend deleting or limiting scope
to critical items.
For most of the parameters, there are columns for results and Hyperlinks, respectively.
What is the reason for only including hyperlinks but not results for recovery and matrix
effects in the summary table?
Recommendation: That recovery and matrix effects be made consistent in format with
other parameters in the table.
Table 1: It appears that Within or Intra-Run precision as well as LLOQ and ULOQ are
absent from Method Validation statistics (Inter-run accuracy and precision). Dilution
integrity: no matrix specified although required according to the first cell.
Please clarify: as to whether or not these parameters are needed in this table.
Please clarify: what information is expected for “matrix that were evaluated” under
Dilution integrity
Line 1109: No example is being provided as to how failed experiments should be listed.
Recommendation: Remove the requirement of “Listing failed experiment”. This
information is typically reported in the validation report, but not necessarily in the summary
section.
DC01/ 3286969.1
Table 2
Recommendation: Lines 1112-1113: add species/anticoagulant to the title of the table.
1112-1120
Lines 1115-1116 and 1119-1120: References are made that an itemized list is required
linking each reference standard to every validation experiment and the date of analysis.
This implies that when a reference standard (powder) expires, no further testing with
samples prepared from it can be conducted.
Please clarify: The reasoning behind this requirement.
Proposed change: remove the internal standard from this table.
Table 3
Comment: Not clear why a combined table for method validation and clinical study is given
in the guideline.
Recommendation: clarification be added to explain the purpose of this table.
Please clarify: What is expected in the next to last row in the table (“Duration from
time...”).
1122-1126
Line 1125: Within or Intra-Run precision are absent from the table. LLOQ and ULOQ are
absent from Method Validation statistics (Inter-run accuracy and precision)?
Please clarify: as being/not being required in the table.
Line 1125: The rows for Inter-run and inter-run accuracy – are these for summarizing the
performance of the assay in clinical study? If yes, suggest that they should be in the “Study
Information” section of the table.
Please clarify: what data is being requested.
Line 1126: Is this requesting a listing of the sample storage temp from, for example, every
clinical site in a study? This information is outside the control of the BA lab but would be
part of the clinical/site raw data and is not available to bioanalytical labs. Also, there can be
a large number of sites plus a central lab involved in phase 2-3.
Recommendation: remove this requirement or limiting it to the BA lab storage.
DC01/ 3286969.1
Table 4
1128-1139
DC01/ 3286969.1
Table 4: Explain or clarify the Hyperlinking. Standards or QCs are generally summarized
in 1 report table (for each sample type, per analyte, per matrix) for all acceptable batches. Is
there a need for every analytical run to be listed and hyperlinked to the same Standards or
QC table? A similar question is about the hyperlink to “Report text”. What does the
hyperlink “Raw Data” refer to since this is a bioanalytical report (the raw data (RLU) is not
really useful)? Finally, if a batch is rejected, does that need to be hyperlinked to a table
showing all failed run data for standards/QCs/report text and “raw” data?
Please clarify: Please provide an explanation as to what exactly is being hyperlinked in
more detail.