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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Originator, First-time Generic and Multisource Generic
Originator
API quality standards Originators specifications

First-time Generic Multisource Generic

Information from regulatory agencies (publicly available) & literature data Information from regulatory agencies (publicly available) & literature data Pharmacopoeias

FPP quality Originators standards specifications

Pharmacopoeias

Analytical methods

Establish identity, potency, purity of API and FPP by in-house methods

Derive identity, potency, Verify identity, potency, purity of API and FPP purity of API and FPP by by in house methods pharmacopoeial methods and in-house methods

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Birgit Schmauser | April 2008

Analytical Method Development

HPLC-method to assay potency and purity risk assessment
Originator First-time Generic Multisource Generic

Selectively screen/detect any impurity or degradant Establish potency

Identify impurities/degradants Characterise all impurities/degradants Calculate Response factors (qualification by clinical use) Establish reference materials Adapt to routine use
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Birgit Schmauser | April 2008

Derive impurities/degradants from Originator Characterize in-house impurities/degradants Calculate response factors

Verify impurities from Pharmacopoeia Characterise in-house impurities/degradants (Response factors)

Extract (& reproduce) reference Use pharmacopoeial materials reference materials Adapt/modify to/for routine use Implement for routine use

Analytical Method Development

Interchangeability (IC) of multisource generic FPPs (Essential similarity with Innovator FPP)

Pharmaceutical + Bioequivalence Equivalence

IC = PE + BE
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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Use of analytical methods - generics
CLINICAL
To determine bioavailability in healthy volunteers

PHARMACEUTICAL
To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches

METHODS
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

At initial phase of pharmaceutical development

At advanced phase of pharmaceutical development

To prove bioequivalence after critical variations to the prequalified dossier
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To optimise, scale-up and transfer a stable and controlled manufacturing process for the prequalification product

To be robust, transferable, accurate and precise for specification setting, stability assessment and QC release of prequalified product batches

Birgit Schmauser | April 2008

Analytical Method Development

Prerequisites for analytical method validation
Six Ms

Man
qualified

Machine

Methods
characterised documented suitable

calibrated

robust skilled qualified

Reference standards

Vibrations Temperature Irradiations

Quality of the analytical method

Time Analysts support

Quality

Humidity

Supplies

Material
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Milieu

Management

Birgit Schmauser | April 2008

Analytical Method Development

Method development life cycle
Planning
Development and Validation Policy Objectives/Requirements of Method Information Gathering Resource Gathering Customer Evaluation Testing
Development Plan Project

Method development
Initital Method Development Pre-Validation Evaluation Method Optimization Robustness System Suitability

Validation Experiments

Method Transfer Experiments

Filed Method in Use

Periodically Monitoring/Review of Methods in Control Labs

From: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN 0-471-31431-5, Wiley & Sons Inc.

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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

 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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Validation Characteristics
Identification
Accuracy Precision -

Impurities
quantitative limit

Assay
+ +

+ + + +

Specificity
Detection Limit Quantitation Limit Linearity Range Robustness

+
+

+ + -

+
+ + +

+
+ +

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Birgit Schmauser | April 2008

Analytical Method Development

Accuracy and precision

Accurate & precise

Accurate & imprecise

Inaccurate & precise

Inaccurate & imprecise

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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Normal distribution, probability function [P(x)] and confidence interval [CI]
Probability (P), that measurements from a normal distribution fall within [-xn, +xn] for xn = ns is described by the erf-function ( = mean): xn s P 0.6826895
Number of times each value occurs

2s 0.9544997

3s 0.9973002
4s 0.9999366 5s 0.9999994

An interval of 3 s covers 99.73% of values

3s

2s

s Values s

2s

3s

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Birgit Schmauser | April 2008

Analytical Method Development

Normal distribution, probability function [P(x)] and confidence interval [CI] Probability-P Confidence interval [CI] centered around the mean [] in units of sigma [s] described by inverse erf-function:

P 0.800 0.900 0.950 0.995 0.999

xp 1.28155s 1.64485s 1.95996s 2.57583s 3.29053s

A CI of 95% includes values 1.95 s around the mean

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Birgit Schmauser | April 2008

Analytical Method Development

Relationship of variability, probability and reliability of data
High variability of data (large s) generate large confidence intervals and thus lower the reliability of the mean Low variability of data (small s) generate small confidence intervals and thus increase the reliability of the mean

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Birgit Schmauser | April 2008

Analytical Method Development

Repeatability
Six replicate sample preparation steps from a homogenously prepared tablet mixture (nominal value of API 150 mg)
Injection Peak area Assay 1 2 3 4 5 173865 174926 172933 175011 179557 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%

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

6
Mean SD (s) RSD
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176425
175453 2329 1.32%

149.28 mg/99.52%
148.45 mg/98.96% 1.98 mg/1.32% 1.32%

Birgit Schmauser | April 2008

Analytical Method Development

Intermediate precision
Expresses within-laboratories variations (different days, different analysts, different equipment etc.)
Injection 1 2 3 4 5 6 Mean SD (s) 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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Birgit Schmauser | April 2008

Analytical Method Development

Reproducibility
Expresses the precision between laboratories Collaborative studies, usually applied to standardisation of methodology Transfer of technology Compendial methods

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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

true

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Specificity: Overlay chromatogram of an impurity solution with a
sample solution

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

Stress stability studies versus forced degradation studies
Stress parameter Acid Base H2O2 / Oxygen Heat Heat UV or Light 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) 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)

Humidity
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50C / 80% RH (4 weeks)

Birgit Schmauser | April 2008

Analytical Method Development

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 (s) and Slope (S) 3.3 s/S

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Birgit Schmauser | April 2008

Analytical Method Development

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 (s) and Slope (S) 10 s/S

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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

LOQ
Quantitation by SNR is accepted Quantitation by Standard deviation of response (s) and Slope (S) (10 s/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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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

Birgit Schmauser | April 2008

Analytical Method Development

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

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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 FPP-stability degradation and sample solution degradation

Should be analysed immediately

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Birgit Schmauser | April 2008

Analytical Method Development

When to be surprised about validation data:
Precision of impurity determination Precision of API determination Method precision of released API (dissolution)
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

Acceptance criterion % RSD 10.0

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Birgit Schmauser | April 2008

Analytical Method Development

Specification range (USL-LSL)
Process variability (usually 2 SD) Analytical variability ( 3s) ~ 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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

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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Birgit Schmauser | April 2008

Analytical Method Development

THANK YOU

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Birgit Schmauser | April 2008