Analytical Method Validation in The Drug Development Process

1
Analytical Method Validation in The Drug
Development Process
Kim HyunSung Ph.D. Berna Biotech KoreaQC
Chemistry lab
2
Contents

• Introduction
• The importance of Analytical method in drug
  development
• Drug development and GMP
• Requirements of analytical method in drug
  development Validation
• What is validation?
• Analytical method validation
• Analytical performance characteristics
• Definitions
• How to calculate characteristics?
• Summary of validation protocol report
• When do you perform revalidation?
• Conclusion

3
Introduction
Pharmaceutical? A pharmaceutical product
manufactured by biotechnology methods (involving
live organisms bioprocessing)
4
Introduction
Scope of Pharmaceuticals
5
The Importance of Analytical Methodology in the
Drug Development Process

• During the drug development, the evaluation of
• Determining optimum crystalline and salt form of
  the drug substance, testing support to process
  development and cleaning validation
• The dosage forms bioavailability
• Determining optimum formulation
• The setting of specifications for the drug
  substance, intermediates, and drug product
• Identification, quantitation, and qualification
  of impurities and degradants
• The shelf-life of the product (stability)
• Product safety
• Support to preclinical and clinical studies
  (safety and efficacy)

6
The Importance of Analytical Methods in the Drug
Development Process
The chemistry, manufacturing, and controls
section of a US regulatory filing is required to
contain the following directly related to
analytical testing 1) Method
validation 2) Physical description
and characterization of the drug
substance and drug product-proof of the chemical
structure 3) In-process controls
4) Characterization of reference
standards 5) Specifications-descriptio
n of analytical methods 6) Evaluation
of container/closure system for storage
7) Justification of drug product development
8) References to compendial test methods
for inactive ingredients 9)
Bioequivalence 10) Pharmaceutical
development report
7
The Importance of Analytical Methodology in the
Drug Development Process
Physicochemical Characterization (1) 1)
UV/visible or mass spectrometry 2) Amino acid
analysis 3) Amino acid or Nucleic acid
sequencing 4) Carbohydrate analysis and, if
appropriate, sequencing 5) Peptide mapping 6)
Determination of disulfide linkage 7) SDS-PAGE
8) Isoelectric focusing (1D or 2D) 9) HPLC,
GC, LC, or thin layer chromatography 10) Nuclear
magnetic resonance spectroscopy
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The Importance of Analytical Methodology in the
Drug Development Process
Physicochemical Characterization (2) 11) Assay
to detect related proteins including deamidated,
oxidated, processed, and aggregated forms 12)
other variants, such as amino acid substitutions
and adducts/derivatives 13) other process
contaminants such as sulfhydryl reagetns, urea,
residual host proteins, residual DNA, and
endotoxin ??? 3?? ???? ?????? Stability
indicating method? ????? ??.
9
The Importance of Analytical Methodology in the
Drug Development Process
Biological Characterization 1) Specific identity
testing such as Western blot analysis or
ELISA 2) Cytometric analysis 3) Neutrovirulence
testing, if appropriate 4) Serotyping 5)
Electrophretic typing 6) Inactivation studies
Irreversibility 7) Neutralization assays 8)
Titration 9) in-vivo assay 10) in-vitro assay
(ELISA) 11) Specific toxicity 12) Abnormal
toxicity
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The Importance of Analytical Methodology in the
Drug Development Process
After the registration of drug product to
regulatory authority, the test methods are used
in the pharmaceutical quality control laboratory
to do the following. 1) Identify the drug
substance and product 2) Quantitate the
pharmaceutical active ingredient 3) Determine
level of purity 4) Guarantee the overall quality
of the product
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Question Answer
Q 1 Quality? ??? ????? Ans.) 1) ???? Lot? ??
(??, ???? ?) 2) ?????? ??? ??? ??? Why?
?? lot? ???? ???? ???, ???, ???? ??? ??? ??
?? ????.
12
Question Answer
Q 2 Quality?? ?? ??? ???? Ans.) 1) ???
(Safety) 2) ??? (Stability) 3) ??? (Efficacy,
Potency, Strength) Q 3 Quality? ??? ?????
???? Ans.) ????
???? 1) ??? 2) ???
3) ???
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Stakeholders in Quality Analytical Testing
During Drug Development
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Drug Product Development and GMP
Validated Method
Pre-qualified method (Standard screening methods)
15
Question Answer
Q 4 ???? ???? ??? ????? ???? Ans.) ?? 2? ????
Why? ????? ???? ??? ?? 2??? ???
???? ??? ????? ???? ????? ??.
16
Question Answer
Q 5 ?? 2? ??? ????? ????? fix ??? ?? ???
????? Ans.) ??? ????? ??? ??? ??? ??? ???? ??
??? ?? ????? ???? ???? ??? ??? ???? ????
?? ??? ????.
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Question Answer
Q 6 Process development? ?? Product quality?
upgrade? ?? ??? ???? Ans.) ?? lot? ???
comparability test (?????)? ???? ????? 1) ??? ??
??????? ????? ?? ?? ? ??????? 2) ??? ?? ??,
??????? ? ????? ?? Q 7 Process development? ??
Product quality? upgrade? ?? ??? ???? Q 8 ????
??? ????
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Requirements of Analytical methods During the
Drug Development Process

• Depending on the stage of development, these
  analytical methods are standard screening methods
  at the start of the development process, which
  over time are gradually upgraded to thoroughly
  validated methods for NDA application.
• The filed methods must be simple, robust, and
  reliable- that is, easy to use and perform
  without deviations when appropriately applied in
  a qualified laboratory.
• Note
• Screening methods are typically not optimized for
  speed and robustness.
• The filed methods referred to as VTR2AP methods
• (validated, transferable, robust, rapid,
  accurate, and precise)
• James M. Miller and Jonathan B. Crowther,
  Analytical Chemistry in a GMP envirinment A
  practical guide, John Wiely Sons,New York
  (2000)

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Laboratory Analytical Methods Flow During the
Drug Development Process
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Question Answer
Q 9 ??? ??? ?? ???? ?? ???? Ans.) ????? ???
??? ??? ??? ???? ???? ???? ?? ??. (RD or QC)
??? ?????? RD?? ??? ??? ???? ??? ???? QC?
???? ?? ?? ??.
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Whats validation?
The FDA defines the term as Established
documented evidence which provides a high degree
of assurance that a specific process will
consistently produce a product meeting its
pre-determined specifications and quality
attributes. General Principles of Validation
(1987)
ICH guideline defines the term as A documented
program that provides a high degree of assurance
that a specific process, method, or system will
consistently produce a result meeting
pre-determined acceptance criteria. Q7A-GMP for
active phamarceutical ingredients (2000)
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Specification
A specification is defined as A list of tests,
references to analytical procedures, and
appropriate acceptance criteria that are
numerical limits, ranges, or other criteria for
the tests described. It establishes the set of
criteria to which a drug substance or drug
product should confirm to be considered
acceptable for its intended use Test
procedures and acceptance criteria for New drug
substances and New drug products Chemical
products (1997)
A list of detailed requirements with which the
products or materials used or obtained during
manufacture have to conform. They serve as a
basis for quality evaluation. GMPs for
pharmaceutical products main principles WHO
TRS 908 Annex 4 (2003)
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Question Answer
Q 10 Quality? Specification? ???? Ans.) Specifi
cation? Quality? ?????. Specification? ???? ?
??? ???? ??? ??? ?? Product Quality? ????.
????? Product Quality? ????? ???? ????.
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Acceptance criteria
ICH guideline define the term as Numerical
limits, ranges, or other suitable measures for
acceptance of test results. Q7A-GMP for active
phamarceutical ingredients (2000)
Acceptance criteria means The product
specifications and acceptance / rejection
criteria, such as acceptable quality and
unacceptable quality level, with an associated
sampling plan, that are necessary for making a
decision to accept or reject a lot or batch (or
any other convenient subgroups of manufactured
units) CFR Part 210.3 Definitions (2000)
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Analytical Method Validation

• Initial Method Validation Guidance Issued in 1987
• Guideline for submitting samples and analytical
  data for methods validation. Food and Drug
  Administration, February 1987. US Government
  Printing Office1990-281-79420818.
• Updated in August 2000 (Draft Guidance!)
• Analytical Procedures and Method Validation.
  Fed. Reg. 65(169), 52,776-52,777, 30 August 2000
• CFR Part 211.165 Testing and release for
  distribution (e)
• The accuracy, sensitivity, specificity, and
  reproducibility of test methods employed by the
  firm shall be established and documented.
• ICH guideline Q7
• GMP guide for active pharmaceutical
  ingredients
• ICH guideline Q2(R1)
• Validation of analytical Procedure Text
  and Methodology

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ICH USP Method ValidationAnalytical
Performance Characteristics
Validation parameter Validation parameter ICH USP
Accuracy Accuracy O O
Precision Repeatibility O O
Precision Interm. precision O -
Precision Reproducibility O -
Specificity of Selectivity Specificity of Selectivity O O
Detection limit Detection limit O O
Quantitation limit Quantitation limit O O
Linearity Linearity O O
Range Range O O
Robustness Robustness O O
Ruggedness Ruggedness - O
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Definitions

• Specificity
• The ability to access unequivocally the analyte
  in the presence of components which may be
  expected to be present. Typically these might
  include impurities, degradants, matrix, etc.
• Linearity
• Its ability to obtain test results which are
  directly proportional to the concentration
  (mount) of analyte in sample within given range.
• Range
• The Interval between the upper and lower
  concentration (amounts) of analyte in the sample
  ( including these concentrations) for which it
  has been demonstrated that the analytical
  procedure has a suitable level of precision,
  accuracy and linearity.

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Definitions

• Precision
• The closeness of agreement (degree of scatter)
  between a series of measurements obtained from
  multiple sampling of the same homogeneous sample
  under the prescribed conditions.
• Accuracy
• The closeness of agreement between the value
  which is accepted either as a conventional true
  value or an accepted reference value and the
  value found.

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Definitions
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Definitions

• Limit of detection
• The lowest amount of analyte in a sample which
  can be detected but necessarily quantitated as an
  exact value.
• Limit of quantitation
• The lowest amount of analyte in a sample which
  can be quantitatively determined with suitable
  precision and accuracy.
• Ruggedness
• The degree of reproducibility obtained under a
  variety of conditions, such as different
  laboratory, different analysts, different
  instruments, environmental conditions, operators
  and materials. (USP)
• Robustness
• A measure of its capacity to remain unaffected by
  small but deliverate variations in method
  parameters and provides an indication of its
  reliability during normal use. (ICH)

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Comparison of ICH USP Analytical Performance
Characteristics
Characteristic Refer. Man Equip. or Cond. Material Inter-lab Intra-lab.
Precision ICH same same same - O
Interm. Precision ICH different different same - O
Reproducibility ICH different different different O -
Robustness ICH same same / different different - O
Ruggedness USP different different different O -
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1. How to measure the Specificity?

• Method Impurity challenge and Recovery test
• Impurity Hydrolyte of HBG by hydrogen peroxide
  
• Challenge half volume of hydrolyte
• Calculation
• Measured con.
  of challenge
• Recovery yield ----------------------------
  --------------- X 100
• Calculated
  con. of challenge

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2. How to measure the Linearity?

• The correlation coefficient(r2 or r), slope of
  regression line and residual sum of squares
  should be submitted.
• A minimum of 5 concentrations is recommended.
• Used the standards (5 con.) and samples (4 con.)

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3. How to set the Range?

• The specified range is normally derived from
  linearity studies and depends on the intended
  application.
• Acceptable degree of linearity, accuracy and
  precision.
• The following minimum specified ranges should be
  considered.
• For assay of active substance or a finished
  product, 80120 of concentration.

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4. How to calculate the Accuracy?

• Accuracy should be assessed on samples spiked
  with known amounts.
• Minimum 9 determinations over a minimum 3
  concentration level covering the specified
  range(e.g. 3 concentrations/3 replicate)
• Should be reported as percent recovery by the
  assay of known added amount of analyte in the
  sample or as the difference between the mean and
  the accepted true value together with the
  confidence intervals.

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Question Answer
Q 11 ???? accuracy? ???? ?? Spike test ? ?????
???? Ans.) accuracy? ???? ??? ???? ?? ??? ? ???
????? ???? ??? ???? ??? ??. ? ? ?? ? ?? ????? ??
??? ?? ????? ??? ??? ??? ??? ?? ???? ???? ?? ??.
????? 280nm??? ???? ???? ?????? ? ?? ?????
Lowry ?? ???? ??? ??? ??? ? ??. ?? Bradford???
???? Lowry?? ??? ??? ?? ??.
37
Question Answer
Q 12 accuracy? specificity? ???? ?? Spike test?
????? ???? ????? Ans.) challenge?? ??? ???.
accuracy? ????? challenge?? specificity?
????? ????? challenge??.
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5. How to calculate the Precision?

• Repeatability
• Minimum 9 determinations covering the specified
  range(e.g. 3 concentrations/3 replicate) or
• A minimum of 6 determinations at 100 of the test
  concentration.
• Intermediate precision
• Depends on the circumstance under which the
  procedure is intended to be used.
• Establish the effects of random events on the
  precision.
• Typical deviations to be studied include
  individually days, analyst, equipment.

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5. How to calculate the Precision?

• Reproducibility
• Assessed by means of an inter-laboratory trial.
• Should be considered in case of standardization
  of an analytical procedure.
• This data is not part of the marketing
  authorization dossier
• Recommended data
• Standard deviation, relative standard deviation
  (coefficient of variation) and confidence
  interval should be reported for each type of
  precision investigated.
• t-test analysis

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6. Detection limit

• Based on visual evaluation
• Mostly for non-instrumental methods
• Based on signal-to-noise
• Analytical procedures which exhibit baseline
  noise
• Compare measured signals from samples with known
  low concentration of analyte with those of blank
  samples.
• A sinal-to-noise ratio 3 or 21 is acceptable.
• Based on the Standard Deviation of the Response
  and the Slope
• The quantitation limit (QL) may be expressed as
• DL 3.3s/S
• where s the standard deviation of blank
• S the slope of the calibration
  curve
• The slope S may be estimated from the calibration
  curve of the standard.

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7. Quantitation limit

• Based on visual evaluation
• The quantitation limit is generally determined by
  the analysis of samples with known concentrations
  of analyte and by establishing the minimum level
  at which the analyte can be quantified with
  acceptable accuracy and precision.
• Based on signal-to-noise
• A typical signal-to-noise ratio is 101.
• Based on the Standard Deviation of the Response
  and the Slope
• The quantitation limit (QL) may be expressed as
• QL 10s/S
• where s the standard deviation of blank
• S the slope of the calibration
  curve
• The slope S may be estimated from the calibration
  curve of the standard.

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8. How to measure the Robustness?

• The evaluation of robustness should be considered
  during the development phase and depends on the
  type of procedure under study. It should show the
  reliability of an analysis with respect to
  deliberate variations in method parameters.
• Lowry reaction time (30min vs. 40 min)
• Measurement confidential variance,
  standard deviation, T-test

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Summary of Method Validation Protocol
Parameter Sample Testing No Specification Acceptance Criteria
Accuracy Low, Medium, High spiked L, M, H 6 Spiked recovery 80120
Repeatability 20, 50, 70, 90ug/mL 6 CV lt20
Intermediate precision Low, Medium, High 6 t-test P gt a
Linearity 5 Std 20, 50, 70, 90ug/mL 6 Correlation coefficient gt0.99
Specificity HBG bulk treated with H2O2 5 Recovery 80120
Range Low, Medium, High 5 Accuracy, Precision Linearity pass
LOQ Blank 6 (10SD)/slope Calculated value
Robustness Medium 6 (2 person) t-test P gt a
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Question Answer
Q 13 Protocol?? parameter??? LOD? ??
???? Ans.) ????? ??? ???? ????? ????????
?? ?? ??? ?? ????? ????? ???? ??. ??? ???? ??
??? ??? ?? ????? ?? ????? ??? ? ??? ??.
?? ?? ?????? ????? ????? ? ?? ?? ?? ??? ?? ???
???? ???? ??????? ????? ? ??? parameter? ??.
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Question Answer
Q 14 ???? ????? ??? ?????? Ans.) ??? ??? ?????
?????? ??, ????(SD)? ???? 2SD, 3SD? ??? ???
???? ??? ? ????. ???? ??? 200ug/mL, SD
13.3ug/mL? ?? 2SD? ???? ????? ?? ?? ???? ???
??. Acceptance criteria 200
(213.3) / 200 100 86.7 (LL)
200 (213.3) /
200 100 113.3 (UL) 3SD ?? Bioassay
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Summary of Method Validation Report
Parameter Specification Acceptance Criteria Result
Accuracy Spiked recovery 80120 ave. 96.02
Repeatability CV lt20 1.92
Intermediate precision t-test P gt a(0.05) L 0.768,M 0.293, H 0.293
Linearity Correlation coefficient gt0.99 gt0.995
Specificity Recovery 80120 ave. 106.18
Range Accuracy, Precision Linearity pass 25110ug/ml
LOQ (10SD)/slope Calculated value 2.11ug/ml
Robustness t-test P gt a 0.652 gt 0.05
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Question Answer
Q 15 ?? validation? ??? ?? protocol? ?? ?? ???
??? ???? ?? ??? ???? Ans.) 1) ??? (??, ??, ????
??) review 2) ??? ??? ?????? ?? 3) ??? ?? ??
??? ??? ??? ???? ?????? 4) ??? ?? ?? - ???
????? ?? ????? ???? ?? acceptance criteria? ????
? ?? ??? (?? ??? ??? ????.) Why? ??? ????? worst
case? ??? ???? ?? ???? acceptance criteria? ???
???? ?? worst case? ???? acceptance criteria?
??? ??? ??.
48
Question Answer
Q 16 ?? ? ???? ??? ???? ??? ?????? Ans.)
System suitability test ?? 1) Negative control
(Blank) 2) Positive control (Standard
material) 3) Reference (Normal product or
Clinical product) 4) Calibration curve
linearity regression coefficient (r or r2)
49
Q 17 When do you perform revalidation?
Question Answer

• Ans.)
• Changes in the synthesis of the drug substance
• Changes in the composition of the finished
  product
• Changes in the analytical procedure
• ICH Q2(R1) Validation of analytical procedures
  Text and Methodology (2005)

In other case, some companies perform the method
validation regular interval.
50
Sample Size 293
51
Q 18 483?? ?????
Question Answer
Ans.) Warning letter?? inspection ? GMP? ??? ??
??? ??? ????
52
Q 18 483?? ????? (??)
Question Answer
The inspection revealed 1. Analytical results
were reported to redacted stating that a sample
met specifications when either out-of-specificatio
ns (OOS) results were obtained on the sample
analysis or on the quality control samples used
to determine the validity of the analytical
results. These OOS results were not
investigated/documented properly to assure
results reported to redacted were accurate and
valid. 21 CFR 211.165(a) 2. Inadequate method
validation in that OOS findings were discarded
without investigating the cause of the OOS
results and analytical data was selectively
reported to support the validation. 21 CFR
211.165(e)3. Use of reference standards and
reagent solutions for extended periods of time
without data in the analytical records supporting
time of use. 21 CFR 211.194(c)
53
Q 19 21 CFR 211.165(e)?
Question Answer
e) The accuracy, sensitivity, specificity, and
reproducibility of test methods employed by the
firm shall be established and documented. Such
validation and documentation may be accomplished
in accordance with CFR 211.194(a)(2)
54
Conclusion
GMP is Good Manufacturing Practice. GMP is based
on documentation. Time is money. Sometimes,
Time is more valuable than money in especially
drug development process.