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 1| Birgit Schmauser | April 2008 Analytical Method Development Presented by: Birgit Schmauser, PhD Federal Institute for Drugs and Medical Devices (BfArM) b.schmauser@bfarm.de 2| 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 3| Birgit Schmauser | April 2008 Analytical Method Development Originator, First-time Generic and Multisource Generic Originator First-time Generic Multisource Generic Originator´s specifications Information from regulatory agencies (publicly available) & literature data Pharmacopoeias FPP quality Originator´s standards specifications Information from regulatory agencies (publicly available) & literature data Pharmacopoeias Analytical 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 API quality standards 4| Establish identity, potency, purity of API and FPP by in-house methods Birgit Schmauser | April 2008 Analytical Method Development HPLC-method to assay potency and purity – risk assessment Originator First-time Generic Multisource Generic Characterise all impurities/degradants Calculate Response factors (qualification by clinical use) Derive impurities/degradants from Originator Characterize „in-house“ impurities/degradants Calculate response factors Verify impurities from Pharmacopoeia Characterise „in-house“ impurities/degradants (Response factors) Establish reference materials Extract (& reproduce) reference Use pharmacopoeial materials reference materials Adapt to routine use Adapt/modify to/for routine use „Implement“ for routine use Selectively screen/detect any impurity or degradant Establish potency Identify impurities/degradants 5| Birgit Schmauser | April 2008 Analytical Method Development Interchangeability (IC) of multisource generic FPPs (Essential similarity with Innovator FPP) Pharmaceutical + Bioequivalence Equivalence IC = PE + BE 6| 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) 7| 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 8| Birgit Schmauser | April 2008 Analytical Method Development Use of analytical methods - generics CLINICAL PHARMACEUTICAL METHODS At initial phase of pharmaceutical development To determine bioavailability in healthy volunteers To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches 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 advanced phase of pharmaceutical development To prove bioequivalence after critical variations to the prequalified dossier 9| To optimise, scale-up and transfer a stable and controlled manufacturing process for the prequalification product Birgit Schmauser | April 2008 To be robust, transferable, accurate and precise for specification setting, stability assessment and QC release of prequalified product batches Analytical Method Development Prerequisites for analytical method validation – Six “M”s Machine Man Methods calibrated qualified robust skilled Reference standards Quality Material 10 | qualified Milieu Birgit Schmauser | April 2008 documented suitable Quality of the analytical method Vibrations Temperature characterised Irradiations Humidity Time Analysts´ support Supplies Management Analytical Method Development Method development life cycle Planning Method development Development and Validation Policy Initital Method Development Pre-Validation Evaluation Objectives/Requirements of Method Development Plan – Project Information Gathering Resource Gathering Validation Experiments Method Transfer Experiments Method Optimization Robustness System Suitability Customer Evaluation Testing 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. 11 | Birgit Schmauser | April 2008 Periodically Monitoring/Review of Methods in Control Labs 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 12 | 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 13 | Birgit Schmauser | April 2008 Analytical Method Development Innovator versus Generics Innovator Generics R & D on API + - Preclinical trials + - Clinical trials phase I and II Method validation summary - Clinical trials phase III Method validation completed - Post marketing phase IV Validated methods - Entering of Generics; Pharmaceutical development, Comparability with Innovator Validated methods Validated methods: GMP and GLP 14 | Birgit Schmauser | April 2008 Analytical Method Development Validation Characteristics Identification Impurities Assay quantitative limit Accuracy - + - + Precision - + - + Specificity + + + + Detection Limit - - + - Quantitation Limit - + - - Linearity - + - + Range - + - + Robustness + + + + 15 | Birgit Schmauser | April 2008 Analytical Method Development Accuracy and precision Accurate & precise 16 | Birgit Schmauser | April 2008 Accurate & imprecise Inaccurate & precise Inaccurate & imprecise 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) 17 | Birgit Schmauser | April 2008 Analytical Method Development Normal distribution, probability function [P(x)] and confidence interval [CI] xn P s 0.6826895 2s 0.9544997 3s 0.9973002 4s 0.9999366 5s 0.9999994 An interval of ± 3 s covers 99.73% of values 18 | Birgit Schmauser | April 2008 Number of times each value occurs – Probability (P), that measurements from a normal distribution fall within [µ-xn, µ+xn] for xn = ns is described by the “erf-function” (µ = mean): 3s 2s s Values s 2s 3s Analytical Method Development Normal distribution, probability function [P(x)] and confidence interval [CI] – Probability-P Confidence interval [CI] centered around the mean [µ] in units of sigma [s] described by “inverse erf-function”: – A CI of 95% includes values ± 1.95 s around the mean 19 | Birgit Schmauser | April 2008 P xp 0.800 1.28155s 0.900 1.64485s 0.950 1.95996s 0.995 2.57583s 0.999 3.29053s 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 20 | 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 173865 147.10 mg/98.06% 2 174926 148.00 mg/98.66% 3 172933 146.32 mg/97.54% 4 175011 148.08 mg/98.72% 5 179557 151.95 mg/101.30% 6 176425 149.28 mg/99.52% Mean 175453 148.45 mg/98.96% SD (s) 2329 1.98 mg/1.32% RSD 1.32% 1.32% 21 | Birgit Schmauser | April 2008 Mean ± 3 SD = Confidence interval of 99.73% 98.96 ± 3x1.32% = 95% - 102.92% 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 22 | Peak area analyst 1 173865 174926 172933 175011 179557 176425 175453 2329 1.32% Birgit Schmauser | April 2008 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% Analytical Method Development Reproducibility – Expresses the precision between laboratories • Collaborative studies, usually applied to standardisation of methodology – Transfer of technology – Compendial methods 23 | 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 24 | 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 25 | 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 26 | 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 27 | 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 28 | 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 29 | 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 30 | Birgit Schmauser | April 2008 Analytical Method Development Stress stability studies versus forced degradation studies Stress parameter Forced degradation Stress stability (5 – 15% decomposition) Acid 0.2 ml 1N HCl / 5 ml API-solution / 3h, 6h, 12h, 24h…7d (RT & 60°C) pH ± 2 (2 weeks) Base 0.2 ml 1N NaOH / 5 ml API-solution / 3h, 6h, 12h, 24h…7d (RT & 60°C) pH ± 10 (2 weeks) H2O2 / Oxygen 0.2 ml 5% or 35% H2O2 / 5 ml APIsolution (RT, to 7d & 60°C, 3h) 1 g/ml oxygen bubbled through (8 hours) 0.1 – 2% H2O2 (24 hours) Heat 60°C / 5 ml solution (3h, 6h…7d) - Heat 105° C / solid API (1d and 7d) 60°C (4 weeks) UV or Light 365 nm or white fluorescent light / solid API (1d and 7d) Humidity - 31 | Birgit Schmauser | April 2008 50°C / 80% RH (4 weeks) 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 32 | 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 33 | Birgit Schmauser | April 2008 Analytical Method Development LOD, LOQ and Signal to Noise Ratio (SNR) LOQ Signal to Noise = 10:1 Signal to Noise = 3:1 LOD Noise 34 | 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 35 | 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 36 | 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 37 | 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 • • • • • 38 | 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 39 | 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 40 | 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 41 | 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 42 | 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 43 | 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 Impurity A Impurity B Impurity C As is 10.46 3.86 7.43 8.26 buffer pH 5.9 10.45 3.94 7.51 8.38 buffer pH 6.9 10.46 3.94 7.49 8.34 Buffer conc. 83% 7.84 3.43 6.16 6.66 Buffer conc. 87% 15.26 4.77 9.61 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 44 | 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 45 | 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 46 | Birgit Schmauser | April 2008 Analytical Method Development When to be „surprised“ about validation data: 47 | – Precision of impurity determination System precision % RSD 0.33 – 2.25 Method precision % RSD 0.0 – Precision of API determination Average peak area % RSD 0.08 – Method precision of released API (dissolution) Average peak area Birgit Schmauser | April 2008 Acceptance criterion % RSD ≤ 2.0 % RSD 0.4 Acceptance criterion % RSD ≤ 10.0 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) 48 | 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) 49 | 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 50 | Birgit Schmauser | April 2008 Analytical Method Development THANK YOU 51 | Birgit Schmauser | April 2008