Analy Meth Dev

Pharmaceutical Development with Focus on Paediatric formulations
WHO/FIP Training Workshop

Hyatt Regency Hotel Sahar Airport Road Andheri East, Mumbai, India 28 April 2008 2 May 2008
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Birgit Schmauser | April 2008
Analytical Method Development
Presented by: Birgit Schmauser, PhD

Federal Institute for Drugs and Medical Devices (BfArM) b.schmauser@bfarm.de
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In this presentation: Standards in developing analytical methods for
Originator and multisource generic FPPs Specifications Stability
Parallel development of analytical methods for cleaning validation
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Originator, First-time Generic and Multisource Generic
Originator
API quality standards Originators specifications
First-time Generic
Information from regulatory agencies (publicly available) & literature data Information from regulatory agencies (publicly available) & literature data
Multisource Generic
Pharmacopoeias
FPP quality standards
Originators specifications
Pharmacopoeias
Analytical methods
Establish identity, Derive identity, potency, Verify identity, potency, potency, purity of API purity of API and FPP by purity of API and FPP by and FPP by in house methods pharmacopoeial methods in-house methods and in-house methods
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HPLC-method to assay potency and purity risk assessment
Originator Selectively screen/detect any impurity or degradant Establish potency Identify impurities/degradants Characterise all Derive impurities/degradants from impurities/degradants Calculate Originator Response factors Characterize in-house (qualification by clinical use) impurities/degradants Calculate response factors Establish reference materials Adapt to routine use Extract (& reproduce) reference materials Adapt/modify to/for routine use Verify impurities from Pharmacopoeia Characterise in-house impurities/degradants (Response factors) Use pharmacopoeial reference materials Implement for routine use First-time Generic Multisource Generic
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Interchangeability (IC) of multisource generic FPPs (Essential similarity with Innovator FPP)
Pharmaceutical + Bioequivalence Equivalence
IC = PE + BE
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Pharmaceutical equivalence
FPPs meet the same or comparable standards by use of equivalent analytical methods Same API (chemical and physical equivalence) Same dosage form and route of administration Same strength Comparable labeling Equivalence in pharmaceutical development Equivalence in stability Equivalence in manufacture (WHO-GMP)
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Prequalification requirements
Validation of analytical methods is a prerequisite for prequalification of product dossiers
Non-compendial APIs and FPPs are tested with methods developed by the manufacturer For compendial APIs and FPPs the applicability of pharmacopoeial methods to particular products must be demonstrated (verification)
Analytical methods must be developed and validated according to TRS 823, Annex 5, Validation of analytical procedures used in the examination of pharmaceutical materials ; ICH Q2 (R1)
To be used within GLP and GMP environments
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Use of analytical methods - generics
CLINICAL PHARMACEUTICAL METHODS At initial phase of pharmaceutical development
To understand the profile of related substances and to study stability To start measuring the impact of key product and manufacturing process parameters on consistent FPP quality To determine To develop a stable and bioavailability in healthy reproducible formulation for the volunteers manufacture of bioequivalence, dissolution, stability and pilotscale validation batches
At advanced phase of pharmaceutical development

To prove bioequivalence To optimise, scale-up and transfer To be robust, transferable, accurate after critical variations to a stable and controlled and precise for specification setting, the prequalified dossier manufacturing process for the stability assessment and QC release of prequalification product prequalified product batches
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Prerequisites for analytical method validation
Six Ms
Man
qualified
Machine
Methods
characterised documented robust
calibrated
skilled Reference standards
qualified
suitable
Vibrations Temperature
Irradiations
Time
Quality of the analytical method

Analysts support
Quality
Humidity
Supplies
Material
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Milieu
Management

Method development life cycle
Planning
Development and Validation Policy Objectives/Requirements of Method Information Gathering Resource Gathering Validation Experiments Customer Evaluation Testing
Development Plan Project
Method development
Initital Method Development Pre-Validation Evaluation Method Optimization Robustness System Suitability
Method Transfer Experiments
Filed Method in Use
. From: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN 0-471-31431-5, Wiley & Sons Inc
Periodically Monitoring/Review of Methods in Control Labs
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Validation should verify the suitability of an analytical method for its intended purpose Validation should be founded on method development performed beforehand that suggest the suitability and robustness of the method Validation may be performed in different ways (individual purpose) according to common standards
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Validation protocol
Method principle / objective Listing of responsibilities Laboratories involved and their role in the validation Method categorization List of reagents (including test lots) and standards Test procedures to evaluate each validation parameter and proposed acceptance criteria Plan or procedure when acceptance criteria are not met Requirements for the final report
The validation process cannot proceed until the protocol and all parties involved approve the acceptance criteria
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Innovator versus Generics
Innovator R & D on API Preclinical trials Clinical trials phase I and II Clinical trials phase III Post marketing phase IV Entering of Generics; Pharmaceutical development, Comparability with Innovator + + Method validation summary Method validation completed Validated methods Validated methods Generics Validated methods: GMP and GLP
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Validation Characteristics
Identification
Accuracy Precision Specificity Detection Limit Quantitation Limit Linearity Range Robustness + +
Impurities
quantitative limit
Assay
+ + + + + + + + +
+ + + + + + +
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Accuracy and precision
Accurate & precise
Accurate & imprecise
&Inaccurate precise
Inaccurate & imprecise
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Precision
Expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample Is usually expressed as the standard deviation (S), variance (S2) or coefficient of variation (RSD) of a series of measurements Precision may be considered at three levels Repeatability (intra-assay precision) Intermediate Precision (variability within a laboratory) Reproducibility (precision between laboratories)
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Normal distribution, probability function [P(x)] and confidence interval [CI]
Probability (P), that measurements from a normal distribution fall within [-xn, +xn]
Number of times each value occurs
for xn = n is described by the erf-function ( = mean): xn P 0.6826895 2 0.9544997 3 0.9973002 4 0.9999366 5 0.9999994
An interval of 3 covers 99.73% of values

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Values

Normal distribution, probability function [P(x)] and confidence interval [CI] Probability-P Confidence interval [CI] centered around the mean [] in units of sigma [ ] described by inverse erf-function: A CI of 95% includes values 1.95 around the mean
P 0.800 0.900 0.950 0.995 0.999
xp 1.28155 1.64485 1.95996 2.57583 3.29053
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Relationship of variability, probability and reliability of data
High variability of data (large ) generate large confidence intervals and thus lower the reliability of the mean Low variability of data (small ) generate small confidence intervals and thus increase the reliability of the mean
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Repeatability
Six replicate sample preparation steps from a homogenously prepared tablet mixture (nominal value of API 150 mg)
Injection 1 2 3 4 5 6 Mean SD () RSD Peak area Assay 173865 174926 172933 175011 179557 176425 175453 2329 1.32% 147.10 mg/98.06% 148.00 mg/98.66% 146.32 mg/97.54% 148.08 mg/98.72% 151.95 mg/101.30% 149.28 mg/99.52% 148.45 mg/98.96% 1.98 mg/1.32% 1.32%
Mean 3 SD = Confidence interval of 99.73% 3x1.32% = 95% - 102.92% 98.96
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Intermediate precision
Expresses within-laboratories variations (different days, different analysts, different equipment etc.)
Injection 1 2 3 4 5 6 Mean SD () RSD Peak area analyst 1 173865 174926 172933 175011 179557 176425 175453 2329 1.32% Peak area analyst 2 175656 175878 176004 176344 175332 174959 175695 495 0.28% Peak area analyst 3 177965 178556 177342 178011 179466 179688 178504 918 0.51%
)Mean 3 SD: (177252 100%

Analyst 1: 98.96% 3 x 1.32% Analyst 2: 99.12% 3 x 0.28 Analyst 3: 100.70% 3 x 0.51
Average of 3 analysts 3SD: 95% - 102.23%
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Reproducibility
Expresses the precision between laboratories Collaborative studies, usually applied to standardisation of methodology Transfer of technology Compendial methods
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Accuracy
Expresses 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 Sometimes referred to as TRUENESS
mean
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true

To find out whether a method is accurate:
Drug substance (assay)
Application of the method to an analyte of known purity (e.g. reference substance) Comparison of the results of one method with those of a second wellcharacterised method (accuracy known)
Drug product (assay)

Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added Drug product may exceptionally be used as matrix
Drug substance/Drug product (Impurities)

Application of the method to samples spiked with known amounts of impurities
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Accuracy: Application of the method to synthetic mixtures of the drug product components to which known quantities of the analyte have been added Recovery reduced by ~10 15%
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN 0-471-25953-5, Wiley & Sons
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When to expect Accuracy problems
Insufficient selectivity of the method Impurity peaks are not resolved and account for assay value Recovery is < 100% Irreversible adsorption of analyte to surfaces of the system Incorrect assay value of a reference standard Due to decomposition of reference standard Incorrect assay value due to change in matrix Analytical laboratory still uses the preceding matrix as standard
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Specificity
Is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix)
Identity testing
To ensure the identity of an analyte
Purity testing
To ensure accurate statement on the content of impurities of an analyte
Assay
To allow an accurate statement on the content of an analyte in a sample
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Specificity: Overlay chromatogram of an impurity solution with a
sample solution
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Specificity and stability
Stress stability testing to ensure the stability indicating potential of an analytical method
Apply diverse stress factors to the API Apply diverse stress factors to the FPP Stress conditions: e.g. Supplement 2 of Generic Guideline; TRS 929, Annex 5
Assure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impurities Assure that known impurities/degradants can be specifically assessed in the presence of further degradants By peak purity assessment and (overlay of) chromatograms
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Stress stability studies versus forced degradation studies
Stress parameter Acid Base H2O2 / Oxygen Heat Heat UV or Light Humidity Forced degradation 0.2 ml 1N HCl / 5 ml API-solution / 3h, 6h, 12h, 24h7d (RT & 60C) 0.2 ml 1N NaOH / 5 ml API-solution / 3h, 6h, 12h, 24h7d (RT & 60C) 0.2 ml 5% or 35% H2O2 / 5 ml APIsolution (RT, to 7d & 60C, 3h) 60C / 5 ml solution (3h, 6h7d) 105 C / solid API (1d and 7d) 365 nm or white fluorescent light / solid API (1d and 7d) 50C / 80% RH (4 weeks) Stress stability (5 15% decomposition) pH 2 (2 weeks) pH 10 (2 weeks) 1 g/ml oxygen bubbled through (8 hours) 0.1 2% H2O2 (24 hours) 60C (4 weeks)
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Limit of Detection (LOD, DL)
The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value
Determination is usually based on

Signal to noise ratio (~3:1) (baseline noise)
or
Standard deviation of response () and Slope (S) 3.3 /S
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Limit of Quantitation (LOQ, QL)
The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy The quantitation limit is used particularly for the determination of impurities and/or degradation products
Determination is usually based on

Signal to noise ratio (~10:1) (baseline noise) or Standard deviation of response () and Slope (S) 10 /S
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) LOD, LOQ and Signal to Noise Ratio (SNR
LOQ
Signal to Noise = 10:1

LOD Noise
Signal to Noise = 3:1
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LOQ
Quantitation by SNR is accepted Quantitation by Standard deviation of response () and Slope (S) (10 /S) is more adequate as it involves the response of the actual analyte Best to calculate in the region close to y-intercept
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LOQ and impurities
In determination of impurities in APIs and FPPs the LOQ should be determined in the presence of API LOQ should be NMT reporting level LOQ should be given relative to the test concentration of API Specificity of impurity determination should always be demonstrated in the presence of API at API specification levels Spiking of test concentration (API/FPP) with impurities at levels of their specification range
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Spiking
API test concentration (normalised) 0.1 mg/ml (100%) Impurity spiking concentrations 0.001 mg/ml (1%) specification limit 0.0001 mg/ml (0.1%) limit of quantitation (minimum requirement)
API at test concentrations API below test concentrations
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Linearity
of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample
If there is a linear relationship test results should be evaluated by appropriate statistical methods
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Correlation coefficient (r) Y-intercept Slope of regression line Residual sum of squares PLOT OF THE DATA

Usual acceptance criteria for a linear calibration curve
r > 0.999; y-intercept a < 0 to 5% of target concentration RSD (wrt calibration curve) < 1.5-2%
r > 0.997
r < 0.997
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Range
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity
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Range
Assay 80 to 120% of test concentration Content uniformity 70 to 130% of test concentration Dissolution Q-20% to 120% Impurities Reporting level 120% of specification limit (with respect to test concentration of API) Assay & Impurities Reporting level to 120% of assay specification
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Linearity is limited to 150%of shelf life specification of impurities
Test concentration can be used to determine impurities To determine drug substance (assay) the test concentration must be diluted The range is 0 ~ 150% of impurity specification
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Robustness
Robustness of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters The evaluation of robustness should be considered during the development phase If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure
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Influence of buffer pH and buffer concentration in mobile phase on retention times of API and impurities
API As is buffer pH 5.9 buffer pH 6.9 Buffer conc. 83% Buffer conc. 87% 10.46 10.45 10.46 7.84 15.26 Impurity A 3.86 3.94 3.94 3.43 4.77 Impurity B 7.43 7.51 7.49 6.16 9.61 Impurity C 8.26 8.38 8.34 6.66 11.18
Conclusion: The buffer composition should be maintained in a range of 85 0.5%

Missing: Acceptance criterion for maximal deviation of retention time should be defined unless justified
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System suitability testing
Based on the concept that equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such Suitability parameters are established for each analytical procedure individually Depend on the type of analytical procedure
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Method stability System suitability over time Sample solution stability
A solution of stavudine is stable for ~ 2 h, then it starts to degrade to thymine
Impurity-spiked sample solution stability
A solution containing stavudine spiked with its impurity thymine does not allow to clearly distinguish between degradation and spike A solution containing stavudine of a FPP-stability sample solution does not allow to clearly distinguish between FPPstability degradation and sample solution degradation Should be analysed immediately
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When to be surprised about validation data:
Precision of impurity determination Precision of API determination
System precision Method precision Average peak area % RSD 0.33 2.25 % RSD 0.0 % RSD 0.08
Acceptance criterion % RSD 2.0
Average peak area % RSD 0.4 Method precision of released API (dissolution) Acceptance criterion % RSD 10.0
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Specification range (USL-LSL)
Process variability (usually 2 SD) Analytical variability ( 3 ) ~ NMT 30% of total specification range Analytical variability Process variability
Reliability of evaluation of major process variables by analytical procedures depends on analytical variability Impurities LOQ and specification limit (e.g. qualification limits NMT 0.15%) Response factors (LOQ modified by response factor)
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Methods for cleaning validation
Method for assay and related substances used in stability studies of API and FPP Specificity (in samples taken from a cleaning assessment) Linearity of response (from 50% of the cleaning limit to 10x this concentration; R2 0.9900) Precision Repeatability (RSD 5%) intermediate precision [ruggedness (USP)] Reproducibility Limits of detection and quantitation Accuracy or recovery from rinsate ( 80%), swabs ( 90%), and process surface ( 70%) Range (lowest level is at least 2x higher than LOQ)
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Summary
Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/management
Establishment of product-specific acceptance criteria Assessment of stability of APIs and FPPs
Validation of analytical procedures should demonstrate that they are suitable for their intended use Validation of analytical procedures deserves special attention during assessment of dossiers for prequalification
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THANK YOU
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Pharmaceutical Development with Focus on Paediatric formulations

WHO/FIP Training Workshop

Birgit Schmauser | April 2008

Analytical Method Development

Presented by: Birgit Schmauser, PhD

Birgit Schmauser | April 2008

Analytical Method Development

Parallel development of analytical methods for cleaning validation

Birgit Schmauser | April 2008

Analytical Method Development

FPP quality standards

Birgit Schmauser | April 2008

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

Pharmaceutical + Bioequivalence Equivalence

Analytical Method Development

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

At advanced phase of pharmaceutical development

Birgit Schmauser | April 2008

Analytical Method Development

skilled Reference standards

Quality of the analytical method

Birgit Schmauser | April 2008

Analytical Method Development

Method Transfer Experiments

Filed Method in Use

Periodically Monitoring/Review of Methods in Control Labs

Birgit Schmauser | April 2008

Analytical Method Development

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

Accurate & precise

Accurate & imprecise

Inaccurate & imprecise

Birgit Schmauser | April 2008

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

An interval of 3 covers 99.73% of values

Analytical Method Development

P 0.800 0.900 0.950 0.995 0.999

xp 1.28155 1.64485 1.95996 2.57583 3.29053

Birgit Schmauser | April 2008

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

Mean 3 SD = Confidence interval of 99.73% 3x1.32% = 95% - 102.92% 98.96

Birgit Schmauser | April 2008

Analytical Method Development

)Mean 3 SD: (177252 100%

Average of 3 analysts 3SD: 95% - 102.23%

Birgit Schmauser | April 2008

Analytical Method Development

Birgit Schmauser | April 2008

Analytical Method Development

Analytical Method Development

Drug product (assay)