International Analytical Methodology

Quality by Design Highlights
Analytical Method Guidelines, Transfer,
System Suitability, Traceability, and
Variability Minimization
By

Satendra Kumar Vishwakarma, PhD

Declaration on Outlines
The materials/ contents presented in the following slides , collected
from various sources, reflect general information and minimum
requirements for conducting analytical activities in a qualified
laboratory system for regulatory submissions.

General Outlines are not complete Reference Guidelines

The readers are further requested to consult First Corporate Method
Guidelines and then other appropriate Regulatory Web Sites for
additional details and verification of outline contents.

Ж♦Ж♦Ж♦Ж Thank You Ж♦Ж♦Ж♦Ж

Outlines of Presentation
 R&D Analytical CMC (Activity Classification and Responsibilities)
 Departmental Analytical Suitability
 Origin of Assay Method and Classifications
 Definition and Determination of Selectivity and Specificity
 International Validation Terms and Terminology
 Data Elements for Verification
 Methods for Cleaning Validation
 Chromatographic Adjustments and Changes in HPLC
 Technical Method Transfer Criteria & Concept
 International System Suitability Requirements
 Impurities/ Degradants in Drug Products/ Drug Substances
 Variability and Minimization by Quality by Design in Laboratory
 Quick Web Link References

Compliant Analytical CMC Activity
Analytical Procedure &
Method Development

API, Excipient Pre-/ Post- Compatibility In-Process Specification QA &
Evaluation & Formulation Studies Studies Safety Regulatory
Control Support Support Support Evaluation Compliance

API, Product, & Packaging: Safety, Quality & Performance Testing
 Chemistry of Drug Molecule: Characterization of Reference & Raw Material and Control*
 Chemistry of Excipients: Characterization and Variation Control* for intended functions
 Chemistry of Drug Interaction: Preformulation/ Formulation Compatibility
 Drug Analysis: Method Development, Validation, Verification, and Transfer
 Stress Degradation and Stability Studies
 Identification/ Quantitation of Known and Unknown Impurities
 Acceptance Criteria/ Specification Development
 Method Life Cycle Management
 Bioequivalence: Verification of Product Quality and Integrity Studies
 Evaluation: Packaging, Drug Substances/ Drug Product Intrinsic Stability Testing
 Process Verification: Process Validation Batches Data Development
 Regulatory and Guidance: Compliance Data/ Reports and Maintenance

* Worldwide Historical data on pharma products indicates that poor characterization and control
on raw materials accounts to Long- term Manufacturing and Product Performance Issues.

State of Process Product Analysis
The Old Way of Product / Substance Analysis – Lab:
Off-line: Current Laboratory practices are destructive and non-destructive (regulatory
disadvantage).

The New Way of Product / Substance Analysis – PAT:
 At-line: In production area, analysis performed during production close to the
manufacturing process.
 On-line: Analysis on diverted stream connected to process.
 In-line: Process stream disturbed.
 Non-invasive: Sensor not in contact with material, process not disturbed (regulatory
advantages).

Goals of both (Lab and PAT Way): Provide information about the sample of interest.

Perspective different:
Lab Way: Time dependence not critical for technical reasons.
PAT Way: Typically used to control processes through feedback in real time or to diagnose
and explain process anomalies.

This dictates differences between the Two Ways with regard to Sampling and Rate of Data
Collection

Process Product Quality
Process Analysis – Sources of Error and Variability
Current Quality By Lab Future Quality By Design
Off-Line At-Line / By-Line In-Line / On-line
Product Oriented Process Oriented

Drug Release, Impurities Blending, Homogeneity, Filling, Granulation, Drying, Coating, Screening

 PAT 

Manual Sampling Manual Sampling
Sample At Interface
Transportation Sample Preparation Sample Preparation / Minimum
Direct Analysis
Preparation
Slow Response Immediate Response
Rapid Response
Quality Not Controlled Rapid Quality Controlled
Quality Controlled
High Manufacturing Cost Low Manufacturing Cost
Low Product Cost

Excipients in Product Quality
Selection and Characterization
Physicochemical Physicochemical Manufacturing
Properties of Excipients Properties of Drug Process Requirement
 Physically Stable  Polymorphic / Forms  Direct compression
 (Polymorphic / Forms Hydrates  Wet Granulation
Hydrates)  Heat & moisture sensitive  Fluid Bed Coating/
 Hygroscopic  Poorly Soluble Granulation
 Chemically Stable  Poorly absorbable  Spray Drying
 Compatible with drug  Poorly Stable in vivo  Other novel
 Rheology Flow processes
 Direct Liquid
Formulation
 Filtration & Fillings

Route of Administration
Desired Release
 Oral Excipients Choice in
Characteristics
 Injection Dosage Forms
 Immediate release
 Pulmonary
 Sustained Release
 Transdermal
 Modified Release
 Buccal
e.g. enteric
 Rectal/Vaginal

Delivered Dose
of Drug
 High Dose
 Low Dose

Dosage Form Development Chart
Active Drug

Suspension
Solution

Suppositories Topicals Intrinsic
Dissolution

Dissociation Tonic
Constant pKa pH Effect Co-solvents Adjustment

pH?
Intrinsic
Salts Saturated
Solubility
Solubility IV Injection
PEG 400 + 5%
H2O + Glycerin
Other
Delivery Capsule
System Solution Tonic
Adjustment

Excipient Compatibility
Other
Dosage Forms Stability

Tablets

Analytical Quality-by-Design
Molecular Properties and Impurities Identification
• Spectroscopy, Hyphenated, Orthogonal Techniques (Process Tools) in DS/DP Impurities Identification.
• Molecule Properties Identification and Characterization like Polymorphs and Polarity Screening by HPLC.
• Application of Multiple or Suitable Detectors.
Method • Selectivity/ Identification and Application of On-line Analyzers.
Development • Application of Target Methods for Particular Impurities or Enantiomers.

• Physiochemical Properties Identification of Formulation Components.
• Phases Interaction and Evaluation at Rapid/ Accelerated Environmental Conditions.
• Application of on-line Laboratory based Analyses and Chemometrics Knowledge.
Formulation • Finger Print Mapping of Formulation Components.
Development • Re-optimization of Methods against Re-optimized Formulations.

• Monitor the identified Process Variable Parameters.
• Control the Impurities by Controlling the Variable Parameters.
Process • Direct Application of on-line Laboratory based Knowledge.
Understanding

• Integral to Product Specifications.
• Integral to Process Controls.
Control • Optimization for Ruggedness.
Methods

Analytical Quality-by-Design
FT-IR and HPLC Comparison

Features FT- IR HPLC

Sample Preparation Non-Destructive Pipetting, Crushing &
Dissolving
Time per Analysis Typically 30-90 seconds Typically 20-40 minutes

Solvent Required No Yes

Chemical Information Active Concentration/ Active Concentration/
Uniformity Uniformity
Physical information Yes No

Surface Analysis Yes in reflectance mode No

Moisture Content Yes No

Low Concentration Impurities No Yes

QbR/ Regulatory Compliance Yes Yes/ No

Smart Analytical NIR Spectral Range

Medium

Long
Short
X-ray

UV
Cosmic Gamma IR Micro UHF

Radio

Vis
Ultra violet Infrared

Near Mid Far

1 400 750 2500 16000 1000000 nm

Elemental Appearance Constituent Assay Functional Group Analysis

 Scan NIR spectrum range ≈ 780nm - 2526nm (12820-3959cm‾¹) non-invasively and non- intrusively. Scan in the
range of 1100nm – 2300nm @ 2nm intervals. Spectra generated is average of 100 NIR scans

Departmental Analytical Suitability
Regulatory Conditions form Method Development to Method Applicability

 Suitability of Analytical Instrument & Support System
Current status of Qualification, Calibration, and approved documentation.
 Suitability of Required Materials
Qualified reference standards, APIs, reagents, matrix/ placebo.
 Suitability of Analytical Chemist
Approved status of qualification, training, and GLP-GMP experience.
 Suitability of Method Validation Documentation
A well written analytical procedure and approved protocol with pre-established
acceptance criteria.
 Suitability of Method/ Method Transfer Documentation
A written protocol, defined responsibilities, identified & approved analytical
procedure, statistical analysis, pre-defined acceptance criteria for each
performance parameters, time frame with solutions stability time and
suitability parameters.
 Suitability of Data, Method Audit, and Deficiency Documentation
Chemist authorization, data audit trail & control, method audit trail, deviation/
Investigation, change control and records management.

Assay Method Classifications
Classification of Analytical Assay Methods & Methods to be Validated

Method Class Definition by Performance Characteristics
Class 1 Analytical methods for Quantification (Identification) of major
(Identity) components in drug substances (active ingredients), drug
products (finished products), support vehicles (preservatives
and excipients).
Class 2 Analytical methods for Determination (Identification,
(Detect & Quantification , Limit ) of known and unknown impurities in
Quantitate) bulk drug substances (active ingredients), drug products
(finished products), support vehicles (preservatives and
excipients).
Class 3 Analytical methods for Determination of Performance
(Concentration) Characteristics – Drug Release ( e.g. Dissolution of Drug
Products).
Class 4 Identification tests – Physical, Chemical limit tests, FTIR, Chiral
(Characteristics) Test, & Spectroscopy.

Evolution of Regulatory Method
Stages of Continuous Verification & Validation of Pharmaceutical HPLC Method
(from Drug Discovery to Early Drug Development to IND to NDA)

Research & Method Development (RMD) Technology
Analytical Validation Formulation Development (FD) Mfg Method
Early Drug
Transfer
Parameters (v) Development Pre-FD-Phase-1 FD-Phase-2 Final FD-Phase-3 & Submission

RMD-1 RMD-2 RMD-3 RMD-4 RMD-5

Accuracy … v v v v
Precision
 Injection Repeatability v v v v v
 Impurity Reproducibility … … v v v
 Assay Intermediate … v v v v
Specificity/ Forced Deg … v v v v
Detection Limit … … v v v
Quantitation Limit … v v v v
Linearity v v v v v
Analytical Range v v v v v
Solution Stability … … v v v
Robustness … … v v v

Validation/ Verification Terminology
Assay Parameters Basic Description of Analytical Performance Parameters
Accuracy Evaluate the closeness of “Measured” value & „True‟ value.

Imprecision Evaluate the variability in replicate measurements (intra- and
inter-assay).
Specificity Evaluate the ability of the method to measure all other
(Selectivity)* components without reacting with other related substances.
Analytical Range Establish analytical range of Active and Impurities over which the
method shows acceptable performance.
Reportable Range Establish range of reportable results over which the method
is validated (may exceed analytical range when samples are
diluted or concentrated).
Sample Stability Evaluate stability of reference standard, impurity solution, and
sample matrix under conditions that mimic the study conditions.
Quality of Standards Evaluate the quality of calibrators/ standards.

Calibration/ Standard Evaluate relationship between known quantities (concentration) of
Curve reference standards in artificial or true matrix and measured
instrument response.

* See next slide for Definition and Significance of Specificity

Definition of Specificity/ Selectivity
Specificity (Selectivity)**
 The analytical performance and ability of a method to measure accurately
and specifically the analyte in the presence of complex components (Active
Ingredients, Degradation Products, Placebo Ingredients, Impurities) that may
cause a degree of interference. This requires separation and characterization.
Separation
 Resolution (Determination of separation between peaks), Plate Count
(Determination of a systems efficiency), Tailing Factor (Calculation reference
peak shape).
Characterization (PDA/DAD)
 Peak Purity Test (angle and plots) is necessary to demonstrate that the
analyte chromatographic peak is not attributed to more than one components.

** See slide on “Stress or Forced Degradation” & its relation with Method Specificity

Determination of Specificity/ Selectivity
Qualitative Identification Tests
 Demonstrate ability to select between compounds of closely related structure
and confirm positive and negative results.
Assay
 Demonstrate that the results are unaffected by spiked impurities or excipients.
Impurities
 If the Impurities are Available
Spike the drug product/ drug substance with impurities and demonstrate
appropriate accuracy and precision. This demonstrate that assay is
unaffected by the presence of spiked materials (impurities and/or excipients).
Compare the results on unspiked assay samples.
 If the Impurities are Not Available**
Compare test results to a second well-characterized procedure. For Assay,
compare the two results. For Impurity Tests, compare impurity profiles Peak
Purity Test ( by diode array detector or by mass spectrometer). Compare the
results obtained under stress (forced degradation) conditions samples.

** See slide on “Stress or Forced Degradation” & its relation with Method Specificity

Methods Under Guidelines
Validation of New Test Methods (Also known as Complete Method Validation)
 All New Non-compendial or Alternative Test methods will be evaluated as per ICH/FDA
 Critical Raw Material and Components Tests
 Process Validation and In-Process Tests
 All GMP Lot Release Tests
1. Potency and Purity
2. Stability Assays for Approved Products
3. Other Safety Tests including Sterility and Pyrogenicity
Verification of Pharmacopeia Methods (Also known as Partial Method Validation)
 All USP/EP Compendial Tests
 In-house Test Methods already used for Approved Products and Not further Modified
 System Suitability must be Demonstrated
1. Ensure Test System Working Properly when Run Performed
2. Needs Equipment Qualification and Periodic Calibration
3. Needs Demonstration of Analyst‟s Competence
4. Needs Reliable Reference Standards and Reagents
5. Test perform for specificity, intermediate Precision and sample solution stability
Qualification of Test Methods
 Comparability and Characterization Tests
 Stability Tests, Pre-NDA
 In-Process and Final Quality Tests during Development
 Some Clinical Test depending on Clinical Phase

International Analytical Methods
Analytical Methods and their Analytical Performance Characteristics

Validation Parameters FDA ICH USP EP JP
Accuracy (Trueness) Yes Yes Yes * Yes
Precision (Repeatability / Intra-Assay) Yes Yes Yes * Yes
Precision (Reproducibility / Labs) Yes Yes Yes * Yes
Precision (Intermediate / Ruggedness) Yes Yes Yes * Yes

Specificity Yes Yes Yes * Yes
Detection Limit Yes Yes Yes * Yes
Quantitation Limit (= Detection + Concentration) Yes Yes Yes * Yes
Linearity Yes Yes Yes * Yes
Analytical Range Yes Yes Yes * Yes
Reportable Range Yes Yes Yes * Yes
Robustness Yes Yes Yes * Yes
Analytical Solution Stability Yes Yes Yes * Yes
Term Precision means 4Rs - Repeatability, Reproducibility, Ruggedness and Robustness

Data Elements for Validation
Table I: Data Elements required for Validation in a given Method Class
Analytical Class 1 Class 2 Class 3 Class 4
Performance
Characteristics Quantitative Limit Tests

Accuracy Yes Yes MBR MBR No

Precision Yes Yes No Yes No

Specificity (Selectivity) Yes Yes Yes MBR Yes

Detection Limit No No Yes MBR No

Quantitation Limit No Yes No MBR No

Linearity Yes Yes No MBR No

Range Yes Yes MBR MBR No

Ruggedness Yes Yes Yes Yes Yes

MBR = May Be Required, depending upon nature of the specific tests

Data Elements for Verification of
DRUG SUBSTANCES
Table II: Data Elements required for Verification of Drug Substances and Excipients

Techniques
Quantitative Limit Tests

HPLC/GC Precision Precision Specificity - Specificity
Specificity Detection -
Quantitation -
Spectrophotometric/ Precision Precision Specificity - Specificity
Colorimetric Quantitation Detection
Titrimetric Precision Precision - - -

TLC - Specificity Specificity - Specificity
Quantitation Detection
Electrophoresis - Specificity Specificity - Specificity

Data Elements for Verification of
DRUG PRODUCTS
Table III: Data Elements required for Verification of Dosage Forms (Products)
Techniques
Quantitative Limit Tests
HPLC/GC Precision Precision Specificity Precision Specificity
Specificity Specificity Detection
Linearity Quantitation
Range
Spectrophotometric/ Precision Precision Specificity Precision Specificity
Colorimetric Specificity Quantitation Detection
Range
Titrimetric Precision Precision - - -
Linearity
Range
TLC - Specificity Specificity - Specificity
Electrophoresis - Specificity Specificity - Specificity

International Validation Parameters
Common Analytical Methods and their Assay Validation Parameters
M E T H O D S
Validation Parameters
ID Impurities Cleaning Assay Specific
(Analytical Characteristics)
Test
Quantitation Limits
Accuracy (Trueness) N Y N (Y) Y Y Y1
Precision – Repeatability N Y N Y Y Y1
Precision – Intermediate N Y3 N N Y3 Y1
Precision – Reproducibility Y Y N Y Y Y1
Specificity (Selectivity/ Sensitivity) Y2 Y Y Y Y Y
Detection Limit N N (Y) Y Y N N
Quantitation Limit N Y N Y N N
Linearity N Y N Y Y N
Range N Y N (Y) Y Y N
Robustness Y Y N N (Y) Y Y1
Surface Recovery N N N Y N N
Stability Indicating N Y N N Y N
Solution Stability (Standard/Sample) N Y Y Y Y Y
Reference Standard Evaluation Y Y Y Y Y Y
N = Signifies that this characteristics is not normally evaluated. Y = Signifies that this characteristics is normally
evaluated. N(Y) = May be needed in some cases. 1 = May not be needed in some cases. 2 = Lack of specificity for
an analytical procedure may be compensated for by addition of second analytical procedure. 3 = In cases where
reproducibility has been performed, intermediate precision is not needed.

Method Re/Validation Parameters
Partial/ Revalidation Required by ICH Q2A under Circumstances
Changes in the synthesis of the drug substance
Changes in the composition of the finished product
Changes in the analytical procedure or method is modified and modifications
are outside original scope of the method. (e.g. robustness)
Change in analytical instrument conditions for which the method has been
validated (e.g. Instrument with different characteristics)
* Revalidation is necessary, if ranges covered during validation of API-methods
are different from those of the FPP-methods (different test concentrations).

Requirement for Revalidation of Analytical Methods
Accuracy Influence of formulation components
Precision Influence of formulation and sample preparation
Specificity Presence of new API(s) and impurities / degradants /
formulation components
LOD/LOQ Test concentrations of API(s) versus FPP)
Range* Test concentrations of API(s) versus FPP
Robustness Change of column material, column parameters, solvents

Methods for Cleaning Validation
Procedure Assay and Related Substances used in Stability
Studies of API and FPP
Specificity Samples taken from a cleaning assessment

Linearity Response (from 50% of the cleaning limit to 10x
this concentration; R2 ≥ 0.9900)
Precision Repeatability Precision (RSD ≤ 5%)
Intermediate Precision [Ruggedness (USP)]
Reproducibility Precision
Limits of Detection N(Y) Desirable to set in some cases

Limit of Quantitation Acceptance Criteria

Accuracy or Rinsate (≥ 80%), Swabs (≥ 90%), and process
Recovery surface (≥ 70%)
Range Lowest level is at least 2x higher than LOQ

USP Adjustments/Changes in LC
Multiple Adjustments / Changes? Regulation may require Additional-/Re-Validation
HPLC Parameters Specification Comments
pH of Aq. Mobile Phase Within ± 0.2 units (value or range) …
Buffer Salt Concentration ± 10% Provide pH variation is met
Ratio of Mobile Phase Components specified at 50% or Whichever is larger. Change in any
Components less: ± 30% or ± 2% component can not exceed ± 10% Absolute,
nor any reduced to Zero.
UV-Visible Detector No deviations A validated procedure must be used to verify
Wavelength that error in the detector wavelength setting
is, at most, ± 3.0 nm.
Column Length ± 70% …
Column Inner Diameter ± 25% …
Flow Rate ± 50% …
Injection Volume Reduced as far as consistent with Increased to as much as 2X volume specified
accepted Precision and Detection as long as there are no adverse
Limits chromatographic effects.
Particle Size Reduced by as much as 50% …
Column Temperature ± 10% °C Recommended to improve control and
reproducibility of RT

(Login Required) http://www.uspnf.com/uspnf/pub/index?usp=31&nf=26&s=0

EP Adjustments/Changes in LC
Multiple Adjustments / Changes? Regulation may require Additional-/Re-Validation
HPLC Parameters Specification and Comments
pH of Aqueous part of May vary by ± 0.2 pH unless otherwise stated in the monograph, or ± 1.0 pH for
Mobile Phase neutral substances.

Column Temperature Can be adjusted by ± 5 °C.

Ratio of Mobile Phase The amount of minor solvent component can be adjusted by ± 30% relative (or ±
Components 2% absolute).

UV-Visible Detector No Adjustment is permitted.
Wavelength

Buffer Salt Concentration Concentration of salts in the buffer component of mobile phase: ± 10%.

Flow Rate ± 50%

Injection Volume May be decreased provided detection & repeatability are satisfactory.

Stationary phase Column Length: ± 70%, Column Int. Diameter: ± 25%, Particle Size: max - 50%, no
increase permitted. Flow Rate correction required.

Gradient Elution Mobile Phase composition not permitted

The percentage variation of Ion Pair Reagent were not found in either USP or EP

Technical Method Transfer
 R&D Analytical Method Transfer to Regulated Quality Control Laboratory
Procedure and Acceptance Criteria Driven Technical Protocol to fulfill proficiency/
reliability checks for Precision, Accuracy and Ruggedness parameters
 Do not change Validated Chromatographic Variable Parameters
Ionic strength in mobile phase
Solvent strength or ratio in mobile phase
Final pH of mobile phase
Temperature (column, vial, mobile phase)
Flow rate (isocratic / gradient ratio and time)
Sample diluent / extraction /sonication time
Test solution stability
Injection volume
Specified analytical column (No equivalent terminology)
Mode of detection or Wavelength of detection
 Measure Chromatographic Parameters
Analytical system suitability parameters and injection frequency
Response (area/amount) for repeatability & reproducibility
Retention time
Selectivity and/or resolution
 Method Transfer Acknowledgement/ Documentation/ Return with Feedback
Document the acceptance criteria with pertinent observations/ Return to
originating laboratory on failure for further optimization.

Evolution of Stability Indicating Method
Implementation

Re-validation
Re-validation
QC/ Stability Method Transfer Formulation
Problem Problem

Validation

Pre-validation

DOE for Qualification
Optimization

DOE for Development
Screening

Characterization Information Identification

Purity & Stability Indicating Method
 A stability method transfer must be Stability-Indicating Assay
Method is a validated quantitative analytical procedure that can
detect the changes with time the pertinent properties of the drug
substance and drug product.
 A stability-indicating assay accurately measures the active
ingredients, without interference from degradation products, process
impurities, excipients, or other potential impurities.
 A stability indicating methods are developed, optimized and adapted
according to the ICH principles for forced degradation studies.
 A non-stability indicating analytical procedure can be used for
release testing, then an analytical procedure capable of qualitatively
and quantitatively monitoring the impurities, including degradation
products, is required to complement it.
 Assay analytical procedures for stability studies should be purity and
stability-indicating, unless scientifically justified.

Method Transfer Testing Criteria
Method Transfer Testing Criteria involves either for
Verification of Method (under actual conditions – Validation requirements),
Qualification of Method (performing tests– Specification requirements) or
Comparison of Method (evaluation of test solutions – Confirmation requirements)

Best Laboratory Practices/ Guidelines for Qualification Transfer Criteria

Method Class Class 1 Class 2 Class 3 Class 4
Assay Performance ID Test
Quantitative Limit
Parameters
Accuracy Y Y N N N
Precision Y Y N Y N
Specificity Y, 1 Y1 Y, 2 N Y, 2
Quantitation NR Y N N N
Linearity Y Y N N N
1 Tested with respect to Sample Diluent.
2 Positive response for analyte presence. No response required for Blank.
Y Verifying laboratory needs to perform tests.
NR Not required.
N Testing not suggested.

Method Transfer Testing Criteria
Method Transfer Testing Criteria involves either for
Verification of Method (under actual conditions – Validation requirements),
Qualification of Method (performing tests– Specification requirements) or
Comparison of Method (evaluation of test solutions – Confirmation requirements)
Best Laboratory Practices/ Current Validation / Revalidation Guidelines for
Comparative Testing Criteria: Originating (O) & Receiving (R) Laboratories
Method Class Class 1 Class 2 Class 3 Class 4
Assay Performance ID Test
Quantitative Limit
Comparative
O R O R O R O R O R
Laboratory
Parameters
Accuracy N N N N N N
Precision Y Y Y Y N (Y) N (Y)
Specificity N Y N Y N Y
Currently Currently
Detection N N N N N N Not Not
Required Required
Quantitation N N N Y N N
Linearity N Y N Y N N
Range N N N N N N
Results Y Y N(Y)

Method Transfer Design Text
Typical Method Transfer Experimental Design and Acceptance criteria Example
Method Type Chemists Product Acceptance Criteria Comments
Assay 2 3 Batches in A two one-sided t- Each chemist should use different
Triplicate test with intersite instrument & columns, independent
differences of ≤ 2% at preparations. System suitability
95% CI criteria must be met.
Impurities and 2 3 Batches in For high levels, a two All samples age, homogeneity
Degradation Duplicate one-sided t-test with packaging and storage should be the
(Triplicate if intersite differences same. All system suitability criteria
done of 10% at 95% CI. For must be met. The LOQ should be
together low levels, criteria confirmed and chromatograms should
with assay are based on the be compared with impurity profile. If
absolute difference samples do not contain impurities
of mean ±25% above reporting limit, then spiked
sample are recommended
Identification 1 3 Batches RT/ Spectral/
chemical results
Cleaning 1 Two spiked Spiked levels should All system suitability criteria must be
Validation samples – not deviate from by met.. Cleaning is a limit test. Low and
one above an amount 3 x the high samples to confirm both positive
and one validated standard and negative information is required
below limit/ deviation of method,
spec or 10% of the spec,
which ever is greater

Regulatory Analytical Suitability

System Suitability
Validation
of

Pump Module Injector Module

Instrument Calibration & Computer System Validation

Detector Module Temperature Module

Chemist & Method

Testing & Criteria

Regulatory System Suitability
What‟s in System Suitability Criteria (Testing)
 System suitability testing is an integral part of many analytical procedures.
The test concept constitute equipment, electronics, analytical operations,
standards, and samples.
 System suitability criteria are Not The Same as method performance criteria.
They usually provide a surrogate measure of the method performance criteria
(Retention Characteristics, Resolution, Tailing, etc.)
 System suitability are limits to various tests and are designed to ensure the
adequate performance of analytical procedure (Repeatability of Injections).
 System suitability test parameters to be established for a particular procedure
depend on the type of procedure being validated.
 System suitability requirements should met before samples are analyzed.
 System suitability must be performed for each study at the beginning and at the
end by each analyst, preferably at the middle of standard loop.
 System Suitability may be needed to demonstrate / determine carryover and
confirm reagent purity by injecting blank (diluent or matrix).
 Compliance with the system suitability criteria (sensitivity and selectivity) is
required throughout the chromatographic procedure.

USP & ICH System Suitability
USP<1225>System Suitability Parameters Requirements
Capacity factor (K‟) : ≥ 2.0
Peak Resolution (Rs) : > 2.0
Symmetry/ Tailing Factor (T) : ≤ 2.0
Theoretical Plates (N) : > 2000
Repeatability (Reproducibility), RSD : ≤ 2.0% (n≥5)
Repeatability (Reproducibility), RSD : ≥ 2.0% (n≥6)
Separation Factor (Relative Retention): Set Retention Time Window
ICH <Q2(R1)>Go to Respective Pharmacopoeias
Definition: Evaluation of equipment, electronic, analytical
operations and samples as a whole.
Determination: Repeatability, tailing factor (T), capacity factor (k‟),
resolution (R), and theoretical Plates (N).

USP is the only document that comes the closest to
specific guidelines on System Suitability.

EP System Suitability Criteria
Suitability in terms of System Performance
 System Suitability Criteria are limits applied to various tests
designed to ensure the adequate performance of analytical
procedure.
 Compliance with the system suitability criteria is required
throughout the chromatographic procedure.

Suitability in terms of Selectivity
 Resolution of two closely eluting peaks (critical pair): preferably
peaks of similar size or at least not saturating).
 Peak-to-valley ratio (incomplete separation, peaks of very different
size)
 “Similarity” or “Concordance” with a chromatogram supplied.
 “Limit” Percent of individual impurity
 “System Suitability” Signal-to-noise minimum 3 for the peak due
to impurity in reference solution.

ΣMethod = ∫Stability & Selectivity
Method Stability:
 System Suitability and Repeatiability Over Time
 Stability of Analytical Solutions
 Sample Solution Stability
 Impurity-spiked Sample Solution Stability
Method Selectivity (Specificity & Sensitivity):
 The Sensitivity for an impurity can change depending on detector, lamp condition, mobile
phase composition and purity, column efficiency, etc.
 Wavelength selection is often optimized for active ingredient but also should be considered
of impurities. Wavelength sensitivity should be demonstrated during stress testing studies.
 Detection Limits (is estimated as 0.02% w/w by visual inspection of chromatogram) should
be evaluated for all significant potential impurities and degradation products.
 Limit of Quantitation is estimated as 0.05% w/w and sample at this concentration included in
linearity (0.05% to 0.5% w/w) and in accuracy.
 Significant impurities and degradation products include those that are specifically monitored
as part of Release Testing and Stability Testing.
Method Adaptability:
 Meets acceptance criteria of Specifications (limits), Robustness and Ruggedness
performance characteristics of intended applicability of analytical method.

Organic Impurity and Degradant
Specified Impurity
 An impurity that is individually listed and qualified in product with a
specific acceptance criterion (can be identified or unidentified).
Impurity Definition (API Impurities)
 An impurity is any substance that is in the Drug Substance……..
that is not that Chemical Entity.
Conclusion: It may be: Known, Unknown, Specified, or Unspecified.
Degradant Definition (FPP Impurities)
 A degradant is an Impurity resulting from a Chemical Change of the
Drug Substance brought on by Manufacturing or Storage of a Drug
Product.
Conclusion: It may be: Known, Unknown, Specified, or Unspecified.
Reference Identification Procedure (API & FPP)
 An analytical procedure to differentiate and separate potential
impurities from matrix under a given conditions and specification.
Conclusion: It may be: Specificity, Sensitivity, Stability and Purity.

Quick Link: http://www.fda.gov/cber/gdlns/ichq3a.htm#att1
http://69.20.19.211/cder/guidance/2452fnl.htm#ATTACHMENT%20I

Stress or Forced Degradation
Method Specificity through Forced Degradation Activities
ICH Q2 (R1)
Stress studies (e.g. products of acid and base hydrolysis, thermal degradation,
photolysis, oxidation) for the drug substance and for the active ingredient in the
drug product should be provided to demonstrate the specificity of the assay and
analytical procedures for Impurities.
Goals
 To understand the drug substance stability.
 To establish degradation pathways.
 To validate the stability indicating power of the analytical procedures used.
 To support the severe conditions that may be encountered during
distribution.
To generate typical degradation products which may be expected on stability
at sufficient levels to allow identification.
 To avoid secondary degradation.
 To get the target range is 5-20 % loss of active as judged by assay relative to
an un-degraded sample.
 To look for purity and mass balance.

Stress or Forced Degradation
Stress Testing – Forced Degradation (API)
Stress studies elucidate intrinsic stability of the API and is part of the
development strategy and is normally carried out under more severe
conditions than those used for accelerated testing.
Stress Testing – Forced Degradation (FPP)
Studies undertaken to assess the effect of severe conditions on the FPP.
Such studies include photostability testing and compatibility testing on APIs
with each other in FDCs and API(s) with excipients during formulation
development.
ICH Guidelines on Stress Testing
http://www.ich.org/cache/compo/276-254-1.html
Reference Subject Title
ICH Q1A(R2) Stability Testing of New Drug Substances and Products
ICH Q1B Stability Testing: Photostability Testing of New Drug Substances
and Products
ICH Q2(R2) Validation of Analytical Procedures: Text and Methodology
ICH Q3A(R2) Impurities in New Drug Substances
ICH Q3B(R2) Impurities in New Drug Products

Impurities / Degradants From Stress
Stress conditions for Exploratory and Drug Substance/ Impurity Peak Purity
Definitive studies* Product, %** Identification*** Match****
Control Y Y Y
0.1 M HCl/ Temp/ Hrs Y Y Y
0.1M NaOH/ Temp/ Hrs Y Y Y
3% H2O2 / Temp/ Hrs Y Y Y
Humidity/ % RH/ Temp/Days Y Y Y
UV (Short and Long wavelength / Temp/ Y Y Y
Days
Light/ Lux/ Days Y Y Y
Thermal/ Temp/ Hrs+ Y Y Y
Transitional ion, Fe / Cu (Optional) … … …
Visit ICH Guidelines Website for Stress Stability Section for further Information
* Variable environmental conditions may be needed based on stability of molecule.
** Desirable to achieve realistic impurities/ degradation level 10 % to 30 % (NMT 50%) based
on active component mass balance.
*** Desirable to monitor impurity/ degradants at various wavelengths.
**** Drug Peak Purity Match is highly desirable (to indicate Peak Purity Indicating Method).
+ Induced temperature should be below the melting point by 10°C to 20°C.

Source of Impurities / Degradants
Potential Impurities (degradants) Source: May be affecting Drug Stability
 Impurities in starting materials
 Products of over reaction or decomposition
 Products of incomplete reaction and synthesis precursors
 Impurities from the solvents of the reaction
 Impurities from catalysts
 Products of side reactions (synthesis by-products)
 Degradation products (metabolites)
 Residual solvents
 Inorganic impurities
 Impurities in excipients or reaction products of API with excipient(s) in formulation
 Process Related impurities (e.g. Thermal Sterilization, Oxidative Environment)
 Reaction products of API / Drug Product with immediate container / closure system
 Enantiomeric forms as impurities*
 Polymorph forms as impurities*
*(basically these are not impurities)

Impurities: Identification & Characterization
Impurities Identification: (Not exhaustive List)
 Organic Impurity
 Each specified identified impurity
 Each specified unidentified impurity
 Any unspecified impurity with an acceptance criterion of not more than ()
the figure in the identification threshold [ICH Q3A/B(R)]
 Total impurities (including extractable and leachable impurities)
 Residual Solvents (By GC/GC-MS: Not covered in this presentation)
 Inorganic Impurities (By compendial methods: Not covered in this presentation)
Impurities Characterization:
Impurity ID Chemical Name Molecular Structure Source/ Origin

Impurity A Chemical Name A Structure A Degradation and process
impurity
Impurity B Chemical Name B Structure B Process impurity

Impurity C Unknown Structure unknown Process impurity (levels do not
(RRT by HPLC) increase on stability / forced
stress testing)
 Summarize all potential and actual impurities arising from the synthesis.
 Identify impurities by Names, Structures, or RRT/HPLC.
 Specify impurities as process impurities and/or degradants

Impurities: Acceptance Criteria
Release and Stability Specifications: Justification are based on
 Pharmaceutical Development Studies Data
 Analytical Data from Batches
 Compendial Specifications (USP/EP Monographs)
 Scientific Literature including EP, JP, etc
 Safety Data on Qualification

Impurity specifications: Justification are based on
 ICH Q3B and ANDA Guidance: Impurities in Drug Products
 Level of Impurity observed in an FDA-Approved Drug Product (RLD),
batch analysis of the RLD close to expiry should be included
 Significant Metabolite of Drug Substance
 Scientific Literature including EP, JP, etc
 Toxicology Studies
 Compendial Specifications (Unspecified Impurities can not be
justified based on USP specifications)
 Negotiation with FDA

Defining Impurities Safety: DS/ DP
ICH Q3A/B (R2) Threshold Terminology
Listing, Reporting, Identification and Qualification of Degradation Products /
Drug Substances.
Threshold for degradation products are expressed either
Threshold
as % of drug substance or Total Daily Intake (TDI) of the
Drug Product.
Reporting A limit above (>) which a degradation product should be
Threshold reported.
A limit above (>) which a degradation product should be
Identification
identified.
Threshold
 Achieving of the structural characterization or
qualitative parameter e.g. retention time in HPLC.
A limit above (>) which a Impurity/ degradation product
Qualification found to be toxicologically qualified.
Threshold  Process of acquiring and evaluating data that
establishes the biological safety of an individual
degradation product or a given degradation profile.
See Next Decision Tree for Threshold Safety Studies

Start Here

Decrease impurity
level below threshold
Yes
Above threshold?
No
Qualified
Decision Tree
for
Yes

No
Threshold
Structure elucidated?
Safety Studies
Yes

Yes
Toxicity documented
and sufficient?

No

Yes Yes
Related to others Acceptable
with known toxicity? Justification?

No No

Qualified
Consider patient
population and duration
of use

Consider need for:
1. Genotoxicity studies (point mutation, chromosomal aberration)
2. General toxicity studies (one species, min 14 days, max 90 days
3. Other specific toxicity end point as appropriate

Consider additional Yes No
testing or removal of Adverse Effects? Qualified
impurity

Acceptance Criteria: Drug Substances
Drug Substances Acceptance Criteria : ICH Q3A (R2)
Determine the Maximum Daily Dose (MDD)
Use MDD to calculate the ICH Thresholds for drug related impurities [ICH Q3A (R2)]
Maximum Daily Reporting Identification Qualification
Dose (MDD) 1 Threshold (RT) 2,3 Threshold (IT) 3 Threshold (QT) 3,*
≤ 2g/day 0.05% 0.10% or 1mg per day 0.15% or 1mg per day intake
intake (whichever is lower)
(whichever is lower)
≥ 2g/day 0.03% 0.05% 0.05%

1 The amount of drug substance, in the form of dose, administered per day.
2 Higher reporting thresholds should be scientifically justified.
3 Lower thresholds can be appropriate if the impurity is unusually toxic………….
That means Impurities below this level need not to be identified but Identification is
recommended, if it is acute toxic in nature.
* Impurities exceeding ICH Qualification Thresholds (QT)...... Try first if it is possible to
reduce the Level....If not possible, then Qualify – a process of evaluation of data which
establishes the Biological Safety of an individual impurity or an impurity profile..............
That means In all cases impurity At ICH Qualification Threshold must be QUALIFIED.
4 Ensure the Analytical Method is adequate to determine LOQ is equal or below RT.
5 Establish limits for “Individual Unspecified Impurity” to equal or below the IT.
6 Establish limits for each “Identified and/or Specified Impurity” to equal or below the QT.

ICH Q3A(R2)
Is impurity greater
Start Here than identification
threshold?

Yes
No No
action

Any Yes
Structure Yes
known human Reduce to
identified? relevant risks? safe level

No
No
Reduce
to not more than Yes
No further Greater No
() identification action
threshold? than qualification No action
threshold?
Yes
No
Yes
Is the impurity
observed in an approved Reduce
drug substance and at a similar level No to not more than Yes
or is it adequately () qualification No action
qualified by other threshold?
approaches?

No

Consider patient population and duration of use and Decision Tree for Identification
consider conducting:
 Genotoxicity studies (point mutation, chromosomal
aberration)a
and Qualification of Impurities in
 General toxicity studies (one species, usually 14 to 90
days)b Drug Substances (ICH Q3A(R2)
 Other specific toxicity endpoints, as appropriate

Any No
Quick Link for more details:
Reduce to Yes
safe level
clinically relevant Qualified http://www.emea.europa.eu/pdfs/human/ich/273799en.pdf
adverse effects?

DS: Identification & Qualification/1
Reporting Example 1 for 0.5 g Maximum Daily Dose
Reporting threshold = 0.05%, Identification threshold = 0.10%, and
Qualification threshold = 0.15%

Reporting Impurity Results for Identification and Qualification of Drug Substances
"Raw" Reported Result Calculated Total Action
Result (%) Reporting Daily Intake (TDI) Identification Qualification
(%) Threshold (mg) of impurity (Threshold (Threshold
= 0.05% (rounded result in 0.10% 0.15%
mg) exceeded?) exceeded?)
0.044 Not Reported 0.2 None None

0.0963 0.10 0.5 None None
0.12 0.12 + 0.6 Yes None +
0.1649 0.16 + 0.8 Yes Yes +
+ After identification, if the response factor is determined to differ significantly from the original
assumptions, it may be appropriate to re-measure the actual amount of the impurity present and
reevaluate against the qualification threshold (See Acceptance Criteria: Drug Substances Slide).

DS: Identification & Qualification/2
Qualification threshold = 1.0 mg TDI
Reporting Impurity Results for Identification and Qualification of Drug Substances
"Raw" Reported Result Calculated Total Action
Result (%) Reporting Daily Intake (TDI)
Identification Qualification
(%) Threshold (mg) of impurity
(Threshold 0.10% (Threshold 1.0 mg
= 0.05% (rounded result in mg)
exceeded?) TDI exceeded?)

0.066 0.07 0.6 None None
0.124 0.12 1.0 Yes None +, *
0.143 0.14 1.1 Yes Yes +
+ See Identification & Qualification/1Slide
* To verify if a threshold is exceeded, a reported result has to be evaluated against the
thresholds as follows: when the threshold is described in %, the reported result rounded to the
same decimal place as the threshold should be compared directly to the threshold. When the
threshold is described in TDI, the reported result should be converted to TDI, rounded to the
same decimal place as the threshold and compared to the threshold. For example the amount of
impurity at 0.12% level corresponds to a TDI of 0.96 mg (absolute amount) which is then
rounded up to 1.0 mg; so the qualification threshold expressed in TDI (1.0 mg) is not exceeded.

Acceptance Criteria: Drug Products
Identify Impurity Identified Unidentified
Impurity Impurity
 Unspecified Impurity has limited acceptance
Determine (ICH Q3B – see next slide) Specified Impurity
 Maximum Daily Dose (MDD)
 Reporting Threshold (RT) Degradation Product
 Identification Threshold (IT) (Degradation Impurity)

 Qualification Threshold (QT) Unspecified Impurity
Ensure
 Analytical Method is Adequate, LOQ is < Reporting Threshold
Establish
 Limits for “Individual Unspecified Impurity” < Identification Threshold
 Limits for “Specified Identified Impurity” < Qualification Threshold
 Limits for “Specified Unidentified Impurity” < Qualification Threshold
 Total Impurities
Qualify / Identify
 Any impurity if a limit is greater than Qualification / Identification
Threshold

Is Degradation
Start Here Product greater
than identification
threshold? ICH Q3B(R2)
Yes
No No
action

Any Yes
Structure Yes
known human Reduce to
identified? relevant risks? safe level

No
No
Reduce
to not more than Yes
No further Greater No
() identification action
threshold? than qualification No action
threshold?
Yes
No
Yes
Is the degradation
observed in an approved Reduce
drug product and at a similar level or No to not more than Yes
is it adequately () qualification No action
qualified by other threshold?
approaches?

No

Consider patient population and duration of use and Decision Tree for Identification
consider conducting:
 Genotoxicity studies (point mutation, chromosomal
aberration)a
and Qualification of Degradants
 General toxicity studies (one species, usually 14 to 90
days)b in Drug Product (ICH Q3B(R2)
 Other specific toxicity endpoints, as appropriate

Reduce to Yes
Any No Quick Link for more details:
clinically relevant Qualified
safe level
adverse effects? http://www.emea.europa.eu/pdfs/human/ich/273899en.pdf

Drug Substances Acceptance Criteria : ICH Q3B (R2)
Determine the Maximum Daily Dose (MDD)
Use MDD to calculate the ICH Thresholds for drug related impurities [ICH Q3B (R2)]
Reporting Thresholds (RT)
Maximum Daily Dose (MDD) of API in Drug Product 1 Threshold Limit Based on TDI 2, 3
≤1 g 0.1% TDI
>1g 0.05% TDI
Identification Thresholds (IT)
Maximum Daily Dose of API in Drug Product 1 Threshold Limit Based on TDI 2, 3
< 1 mg 1.0% TDI or 5 μg, whichever is lower
1 mg – 10 mg 0.5% TDI or 20 μg, whichever is lower
> 10 mg – 2 g 0.2% TDI or 2 mg, whichever is lower
>2g 0.10% TDI
Qualification Thresholds (QT)
Maximum Daily Dose of API in Drug Product 1 Threshold Limit Based on TDI 2, 3
< 10 mg 1.0% TDI or 50 μg, whichever is lower
10 mg – 100 mg 0.5% TDI or 200 μg, whichever is lower
> 100 mg – 2 g 0.2% TDI or 3 mg, whichever is lower
>2g 0.15%
1 The amount of drug substance, in the form of dose, administered per day.
2 Higher reporting thresholds should be scientifically justified.
3 Threshold for degradation products are expressed either as a percentage of drug substance or as Total Daily
Intake (TDI) of the degradation product. Lower thresholds can be appropriate if the impurity is unusually toxic.

DP: Identification & Qualification/1
Reporting Example 1 for 50 mg Maximum Daily Dose
Qualification threshold = 200 ug

Reporting Degradation Results for Identification and Qualification of Drug Products
"Raw" Reported Result Total Daily Intake Action
Result (%) Reporting (TDI) of the Identification Qualification
(%) Threshold Degradation (Threshold (Threshold
= 0.1% Product (rounded 0.2% 200 ug TDI
result in ug) exceeded?) exceeded?)
0.04 Not Reported 20 None None

0.2143 0.2 100 None None
0.349 0.3+ 150 Yes None +
0.550 0.6+ 300 Yes Yes +
+ After identification, if the response factor is determined to differ significantly from the original
assumptions, it may be appropriate to re-measure the actual amount of the degradation product present
and re-evaluate against the qualification threshold (See Acceptance Criteria: Drug Product Slide).

Quick Link for more details: http://www.emea.europa.eu/pdfs/human/ich/273899en.pdf

DP: Identification & Qualification/2
Reporting threshold = 0.05%, Identification threshold = 2 mg, and
Qualification threshold = 3 mg
Reporting Degradation Results for Identification and Qualification of Drug Products
"Raw" Reported Result (%) Total Daily Intake Action
Result (%) Reporting Threshold (TDI) of Degradation
Identification Qualification
= 0.05% Product (rounded
(Threshold 2 mg (Threshold 3 mg
result in mg)
TDI exceeded?) TDI exceeded?)
0.049 Not Reported 1 None None
0.079 0.08 2 None None
0.183 0.18 + 3 Yes None +, *
0.192 0.19 + 4 Yes Yes +
+ See Identification & Qualification/1Slide
* To verify if a threshold is exceeded, a reported result has to be evaluated against the thresholds as
follows: when threshold is described in %, reported result rounded to the same decimal place as the
threshold should be compared directly to the threshold. When threshold is described in TDI, the reported
result should be converted to TDI, rounded to the same decimal place as threshold and compared to the
threshold . For Example an amount of 0.18% degradation level corresponds to a TDI of 3.4 mg impurity
(absolute amount) which is then rounded down to 3 mg; so the qualification threshold expressed in TDI (3
mg) is not exceeded.
Quick Link for more derails: http://www.emea.europa.eu/pdfs/human/ich/273899en.pdf

Example for Justification of Impurity Limits
Name Current RLD Lot Proposed Proposed Justification
Lot Release Stability
Limits Limits
Impurity A 0.80% 2.5% NMT 1.5% NMT 2.5% Metabolite

Impurity E 0.40% 1.0% NMT 1.0% NMT 1.0% Equivalent to
RLD
Any < 0.07% < 0.05% NMT 0.20% NMT ICH Q3B
Unknown 0.20% Identification
Impurity Threshold ++
Total 1.3% 3.6% NMT 2.5% NMT 3.5% Below RLD
Impurities +
+ Process-related impurities B, C, D, and F are excluded from the calculation of impurities
in the drug product.
++ The maximum daily dose of RLD is 64 mg/day and recommended Identification
Threshold is 0.2% or 2 mg TDI (Therapeutic Daily Intake/ Total Daily Intake) and
Qualification Threshold is 0.5% or 200μg TDI.

 Testing should be conducted on various samples of marketed drug
product over the span of its shelf life (one sample near its expiration date)

 Individual Unspecified Impurities in the drug product at release and for
stability should not exceed ICH Q3B “IT” based on MDD.

 The acceptance criteria for Individual Specified Impurities for release
and stability should be the same as the drug substance unless the
impurities are shown to increase during manufacture or over time. Any
impurities that increase over time and are allowed > QT will need to be
qualified for safety. If the accelerated stability data fail tightened limits full
term long term data should be provided.

Example: MDD (Maximum Daily Dose) for the DP is 24 mg
Drug Product QT = 0.5%
Impurity A: NMT 0.15% (drug substance)
NMT 0.8% (drug product release)
NMT 1.0% (shelf-life).

Minimization
Variability, Traceability and Uncertainty
Objectives of Measurement in Health Care Industries
1. To Ascertain:
The Quality of a Product and the Control of Process
2. To Demonstrate:
Potency of Product, Compliance with Specifications, and Conformity with
Regulations.
3. To Fulfill Requirements:
Pharmacopoeias and Guidelines
Goals of Determination of a Parameters
1. Testing: 2. Measurement
Test Results in Arbitrary Units Analytical Results in SI units
Method Dependent Method independent
Accepted Method Traceability
Reproducibility Uncertainty

Variability & Minimization
No Matter What….Corporate must shows that
 Analytical Instrument is Qualified
 Computer System and Software is Validated
 Audit Trail and Data Access is Limited
 Data Storage (Electronic and Paper) and Traceability
 Method is Validated & Purity-Stability Profile Indicator
 SOP is Adequately Defined and Approved
Potency Assays for Generic Pharmaceutical
 Method Feasibility
 Method DOE and Development
 Method Verification for Variables
 Method Validation
 Method Transfer for Re/Verification of Variables
 Additional Validation / Revalidation
 Monitor / Access Method Life Cycle
Quality Measurements via Technical Communication & Evaluation
 Evaluate potential uncertainty of quantitative and qualitative results and variables
 Reduce variables as much as possible
Identified and optimize key variables using cause and effects diagram

Variability & Traceability in HPLC
Potential Source of Method Variability: Ishikawa Diagnostic Methodology
Environment Materials Method
Lab temperature Stability and Purity Ruggedness
Lab humidity Storage and Handling Complexicity of SOP / Protocols
Light exposure Sampling of test materials Suitability of mobile phase
Air borne contamination Standard contamination Accuracy of preparation of standards
Laboratory layout Sample contamination Accuracy of preparation of samples
Glassware contamination Matrix interference
Wrong glassware selection
P R
Wrong reagents selection
R E
O S
»»»»»»»» Root Cause & Effect F r a m e W o r k Analysis »»»»»»»» B U
L L
E T
Training Time since certification Column degradation, M S
Failure to understand protocol Time since sampling / dilution temperature fluctuations
Work precision and accuracy Repeatability interference UV detector lamp with
Individual interpretation of SOP Noisy baseline, carryovers variable performance, air bubbles
Lack of Communication Density difference in solutions Pump, flow rate, blockage, leakage,
Poor housekeeping Improper correction factor entry trapped air, improper mixing,
Injector reproducibility

People Measurement LC Equipment
Develop Yourself - Knowledge Based Methods and Tools from ICH Q9 Quality Risk Management

Assay & Imp: Variability & Traceability
Potential Source of Variability in Assay and Impurities Method
Environment Materials Method
Lab temperature-N Reagents-source , grade, lot-N Diluent and Mobile Phase
Lab humidity-N Column and Vials-N Standard/ Sample Prep /
Light exposure-N Volumetric glasswares-N Resolution Prep/ Sensitivity Check
Air borne contamination-N Transfer Pipettes-N Runtime/ re-equilibration-X
Filters & Syringe-N Filtrate discard volume-X
Filtrate & glasswares-N Pipette Techniques-N
Weighing boat for Buffer-N Weighing Techniques-N A
Product Type-N P S
Reference Material-C R S
O A
»»»»»»»» Root Cause & Effect F r a m e W o r k Analysis »»»»»»»» B Y
L &
E I
Weighing Techniques -C Impurity calculation-C HPLC Parameters-N M
M P
Integration Techniques-C Data Reporting-C Sonication Time-N
Equilibration Techniques-C Suppress Integration Time-C Mechanical Shaker Action-N
Variation in Extraction-C Noisy Baseline-C Physical Balance Environment-N
Incorrect flask/Pipettes-C Software Quality-C Balance Accuracy-C
Improper Correction Factor –C pH Meter Accuracy/Calibration-C
Sampling Rate-X Run Time/ Equilibrium Time-X

People Measurement Equipment
N= Noise factor for Ruggedness, C= Control, eXperimental parameters for Robustness

Federal CFR Guidance
Most Frequent referred Code of Federal Regulations
http://www.gpoaccess.gov/cfr/index.html
http://www.betterchem.com/title21.htm
:
Analytical Method Validation
21CFR 211.165
21 CFR 211.165accuracy, sensitivity, specificity and reproducibility of test method employed
The (a) 3 :
(e) by the firm shall be established and documented. Such validation and document
may be accomplished in accordance with § 211.194 (a) (2).

Stability Indicating Analytical Method
21 CFR 211.166 There shall be a written testing program designed to assess the stability
(a) (3) characteristics of drug products. The results of stability testing shall be used in
determining appropriate storage conditions and expiration dates

Regulatory Guidance on Method Transfer
Methods used in the testing of the sample meet proper standards of accuracy
21 CFR 211.194
and reliability as applied to product tested.
(a) (2)
The suitability of all testing methods used shall be verified under actual
conditions of use.
Regulatory Guidance on GLP
21 CFR 58.190
All protocol, raw data documentation, generated reports shall be retained,
(a, b, c, d)
archives, individual shall be indentified to archives. etc.
21 CFR 211.160 Laboratory Control Systems
(a, b) Deviations from test procedure and written specifications.

Quick International References
ICH Quick Link: Http://www.emea.europa.eu/htms/human/ich/ichquality.htm
Analytical Validation:
ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and
Methodology
http://www.emea.europa.eu/pdfs/human/ich/038195en.pdf
Impurities:
ICH Topic Q 3 A (R2) Impurities in new Drug Substances
ICH Topic Q 3 B (R2) Impurities in New Drug Products
ICH Topic Q 3 C (R3) Impurities in Residual Solvents
Specifications:
ICH Topic Q 6 A Specifications: Test Procedures and Acceptance Criteria
for New Drug Substances and New Drug Products: Chemical Substances
ICH Q6A Decision Trees
http://www.ich.org/LOB/media/MEDIA431.pdf

Stability:
ICH Topic Q 1 A (R2) Stability Testing of new Drug Substances and
Products
ICH Topic Q1B Photostability Testing of New Active Substances and
Medicinal Products
ICH Topic Q1C Stability Testing: Requirements for New Dosage Forms
ICH Topic Q 1 D Bracketing and Matrixing designs for Stability Testing of
Drug Substances and Drug Products
ICH Topic Q 1 E Evaluation of Stability Data
ICH Topic Q 1 F Stability Data Package for Registration Applications
in Climatic Zones III and IV


Regulatory Acceptance:
ICH Topic Q 4 B Regulatory Acceptance of Analytical Procedures and/or
Acceptance Criteria (RAAPAC)
ICH Topic Q 4 B Annex Annex to Regulatory Acceptance of Analytical
Procedures and/or Acceptance Criteria (RAAPAC)
Pharmaceutical Development:
ICH Topic Q 8 Pharmaceutical Development
ICH Topic Q 8 Annex Pharmaceutical Development
ICH Q9 document on Quality Risk Management
http://www.emea.europa.eu/Inspections/docs/ICHQ9Step4QRM.pdf
ICH Topic Q 10 Pharmaceutical Quality System

USA Guidelines Links
CDER Master Index Page
http://www.fda.gov/cder/guidance/index.htm
Analytical Procedures and Methods Validation Chemistry, Manufacturing,
and Controls Documentation
http://www.fda.gov/cder/guidance/2396dft.htm
Reviewer Guidance Validation of Chromatographic Methods
http://www.fda.gov/Cder/guidance/cmc3.pdf
Guidance for Industry, Bioanalytical Method Validation
http://www.fda.gov/cder/guidance/4252fnl.pdf
National Archives and Records Administration (CFR)
http://www.gpoaccess.gov/cfr/index.html
USP<1225> Validation of Compendial Procedures * Login Required
http://www.uspnf.com/uspnf/pub/index?usp=30&nf=25&s=2
USP<1226> Verification of Compendial Procedures * Login Required
http://www.uspnf.com/uspnf/pub/index?usp=30&nf=25&s=2

CTD and eCTD Informational Links
M4: Organization of the CTD
http://www.fda.gov/cder/guidance/4539O.PDF
eCTD Submissions
http://www.fda.gov/cder/guidance/7087rev.pdf
Drug Substance
http://www.fda.gov/cder/guidance/3969DFT.pdf
Drug Product
http://www.fda.gov/cder/guidance/1215dft.pdf
Q8: Pharmaceutical Development
www.fda.gov/cder/guidance/6746fnl.pdf
CTD-Efficacy
http://www.fda.gov/cder/guidance/4539E.pdf
CTD-Quality
http://www.fda.gov/cder/guidance/4539Q.PDF
QbR-Quality Overall Summary Outline
http://www.fda.gov/cder/ogd/

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