The presentation discusses the complexities of bioanalytical method validation from the FDA's perspective, highlighting ambiguities in guidance documents and common pitfalls that can lead to regulatory scrutiny. It emphasizes the need for clear directives on validation processes and documentation while critiquing existing guidelines for their vagueness. The conclusion advocates for a harmonized validation program that incorporates FDA guidance and external accreditation to mitigate risks associated with compliance failures.

1. Bioanalytical Method
Validation: FDA perspective
by Debanjan Das
This presentation focuses on guidance
documents, 483s, dockets aiming to construct
a robust bioanlaytical validation program
{DISCLAIMER: Certain details used here, pertaining to proprietorship of the project
had been altered. This exercise is solely for enhancement of knowledge-base and
not for any “for-profit” venture}

2. Interesting Observations:
 Guidance for Industry:Bioanalytical Methods
Validation for Human Studies,Dec’98
 Guidance for Industry:Bioanalytical Method
Validation,May’01
 Warning Letters & Dockets
 Views of private agencies of US & Europe, and
on ICH guidelines on current trends of
Bioanalytical validation.

3. Do guidance documents actually
misguide you?
 There had been a plethora of instances
where the guidance documents had been
vague and such were those conceptions
which called in for 483s
 Was that deliberate and vaguely
conceptualized?
 Or was that for you to read between the
lines?

4. Reading between the lines; the
safest way to keep a 483 away
 Guidance documents should ideally
instruct what needs to be done, not how.
 However, when FDA issues one,it’s best
taken care of if followed to the maximum.
 Inspections, almost always, look for
compliance based on the outlines given in
guidance documents.

5. Thinking the FDA way: dissecting
the guidance documents.
 The following exercise (read anatomy lessons!)
had been primarily on finding out the glitches in
the FDA guidance sheets.
 The idea is if we know where the trap is, we
know a way to go round it!
 The following sections will take each part (and
subpart) of such documents (both the Dec’98
and May’01) and analyze the ambiguities blow
by blow.

6. Introduction Section
 Non-clinical studies are left out,although basic
pharmacology and underlying principles are the
same.
 Use of ICH definitions are seriously restricted
 Immunochemical assays are too vaguely
defined,& are classically driven(based on GC &
HPLC),rather than biologically.
 Statements related to analytes of tissue samples
and matrix requirements are unclear.

7. Background Section
 “The acceptability of analytical data corresponds directly
to the criteria used to validate the method”.The object of
any validation exercise should be to assess the
performance of the assay,not the other way round.
 Linearity term is poorly dealt with.There is virtually no
room for methods which doesn't conform a linearity in
concentration vs. response curve.
 Phrases like “predictive mathematical function” are
missing which will cover any type of curve fit integral for
assay development activities.

8. Reference Standard
 It is unclear as to what frequency the reference
materials have to checked prior to use.
 The use of the term “interfering peak” is not
relevant for immunoassays.
 Due to limited bulk supplies it is difficult to
maintain a master standard for comparison with
future lots.
 Any guidance on on the degree of
characterization required for internal standards
are missing.

9. Pre-Study Validation,In-study Validation and
Method Development section(which includes
chemical,microbial & ligand binding assays)
 Since validation is a continued and an ongoing process,
and that all validation must be complete prior to the
onset of sample analysis, the guidelines of pre-study
validation is ambiguous.
 For In-study validation, reporting a bioanalytical data to
regulatory agencies assumes that a method has
undergone validation & that a written procedure for the
conduct of analysis & reporting data exists. How correct
is this?

10.  Method development, on the contrary, is research
oriented,& might not be included as a component of
validation at all!
 Limit of Quantification(LOQ):”The results should be
compared to those obtained from an extracted sample of
analyses in matrix, at a concentration near LOQ”. The
response from an aqueous solution of analyze may be
different from that of analyte in matrix, due to possibility
of ion suppression or matrix effect.
 Unclear extraction procedures for compounds which
interfere chromatographically, and which can be eluted
by other procedures.

11.  Regression equation is ambiguous,and
there’s a need for each method for each
process used. The all-encompassing power
model, a more objective tool to determine the
best fit, is missing.
 The criteria for LOQ & ULQ is difficult in
immunochemistry methods. The calculations
for 6 non-zero standards including LOQ &
the calibration std at the highest conc. (ULQ)
does not seem feasible.

12.  Precision,Accuracy,Recovery:Use of confusing
terminologies are used.There is a difference
between intraday & intrabatch.A lab may
analyze several batches in one day by using
several instruments(usual practice for large
scale studies)
 “Although recoveries close to 100 are
desirable,a lower recovery of an analyte and or
IS is acceptable”.The focus of assay
development is the establishment of an
accurate,precise and sensitive method with
good selectivity & not necessarily high recovery!

13.  Quality control samples:Differences in response
to QCs due to matrix effect is evident when
each set of QCS are prepared from a separate
source of matrix lot.
 LOQ QC sample:This is run during a validation
to establish the precision & accuracy at the LOQ
& not to evaluate the error.Is there any degree
of variation allowed?Is the tolerance of LOQ QC
concentration be 20/0 ?

14. Documentation section
 “all SOP, raw data, calculations of concentration &
necessary sample sets are to be documented”. Inclusion
of all these documentation puts an additional burden on
any analytical lab. Inclusion of SOPs will add hundreds
of pages of documents in each study report, even if
submitted electronically,& a redundancy of effort of each
submission are inevitable.
 Which raw data is required? Does it refer to instrument
response values?
 Which SOPs are required? Do they refer to assay
validation reports?

15. Doubts,Doubts,Doubts! Lets bring in Z540,ISO/IEC
17025 & Mil-Std 45662A!
 What's this Z540? Why its important?
 American National Standards Institute and the
National Conference of Standards published the
ANSI/NCSL Z540 Std dealing with calibration of
laboratories, measuring equipment(part 1) &
guidance on control of measuring and test
equipment(part 2), which is the American
National Std equivalent of the ISO guide for the
determination of measurement uncertainty.

16. ISO/IEC 17025 impact.
 Although its not mandatory to get an
accreditation unless it’s a regulatory or a
customer requirement, major customers,
governmental bodies(even FDA), look into
the use of accredited laboratories to
perform calibration & testing. This is
specially important to comply with in the
European Union.

17. Mil-Std 45662A
 Standard for calibration systems utilized
by the military, aerospace & later by other
industries. It’s a guidance having useful
information regarding what is necessary in
terms of establishing & documenting a
calibration system which forms an
essential part of bioanalytical validation
protocol.

18. Conclusions
 A harmonized validation program,
encompassing all the finer prints of the FDA
guidance documents, along with accreditation
from other agencies, seems a plausible rationale
to run a bioanalytical validation program
smoothly without the risk of 483s.
 It’s the matter of ends justifying the means, and
never the other way round!