Pharmaceutical Development

Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations
Protea Hotel Victoria Junction, Waterfront Cape Town, South Africa Date: 16 to 20 April 2007
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April 2007

Pharmaceutical Development
Analytical Method Development

Presenter:

János Pogány, pharmacist, PhD
pogany.janos@chello.hu

WHO expert

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April 2007

Analytical Method Development
Outline and Objectives of presentation
 Introduction, guidelines  Dossier requirements
– Assay – Related substances – Other issues

 Main points again

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April 2007

Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations
Introduction, guidelines

Interchangeability (IC) INTERCHANGEABILITY (IC) OF MULTISOURCE FPPs = (ESSENTIAL SIMILARITY WITH INNOVATOR FPP) = PHARMACEUTICAL EQUIVALENCE (PE) + BIOEQUIVALENCE (BE) IC = PE + BE | Slide 5 of 43 April 2007 .

g.Pharmaceutical equivalence  FPPs meet same or comparable standards (e.. analytical methods) – – – – Same API (chemical and physical equivalence) Same dosage form and route of administration Same strength Comparable labeling  Pharmaceutical development equivalence  Stability equivalence  WHO-GMP (manufacturing equivalence) | Slide 6 of 43 April 2007 . marketing authorization.

and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2(R1) | Slide 7 of 43 April 2007 .Prequalification requirements  Analytical method validation is required by WHO for the prequalification of product dossiers. Non-compendial ARV APIs and FPPs were/are tested with methods developed by the manufacturer.  Analytical methods should be used within GMP and GLP environments.

11 „It is of critical importance that particular attention is paid to the validation of analytical test methods. 40th Report. (2005) http://www. 937).”  Appendix 4. http://whqlibdoc.org/LOB/media/MEDIA417.int/trs/WHO_TRS_937_eng. Analytical method validation (in WHO Expert Committee on Specifications for Pharmaceutical Preparations.ich. Geneva.Guidelines used in PQP  „WHO-GMP 4. No.pdf | Slide 8 of 43 April 2007 .who.pdf  Validation of analytical procedures: text and methodology Q2(R1) ICH Harmonized Tripartite Guidelines. 2006 (WHO Technical Report Series. WHO. automated systems and cleaning procedures.

General requirements  Qualified and calibrated instruments  Documented methods  Reliable reference standards  Qualified analysts  Sample selection and integrity  Change control | Slide 9 of 43 April 2007 .

46% | Slide 10 of 43 April 2007 .Measure of variation (spread of data) 68.26% 95.

Mean (average) chart Abnormal variation of process – special causes USL Upper specification limit average = mean Normal variation due to common causes LSL Lower specification limit Abnormal variation of process – special causes | Slide 11 of 43 April 2007 .

itl.27%.66 2. 64 ppm http://www.gov/div898/handbook/pmc/section1/pmc16.nist.Capable process Almost all the measurements of a stable process fall inside the specification limits USL – LSL Cp 6 8 10 12 1.htm | Slide 12 of 43 April 2007 .00 6 ppm 2 ppb OoS results: .00 1.33 1.

167 6.166 0.166 6.85% | 1 2 3 4 5 6 Mean STD RSD 6.NEVIRAPINE – Reference Standard Injection tR A System suitability requirement: RSD is NMT 0.06% 12466 12311 12432 12530 12457 12479 12446 74 0.172 6.160 6.004 0.59% Slide 13 of 43 April 2007 .168 6.165 6.

Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations Dossier requirements .

dissolution. stability assessment. transferable. and precise for specification setting.Use of analytical methods . and QC release of prequalified batches Slide 15 of 43 April 2007 . scale-up. and transfer a stable and controlled manufacturing process for the prequalification product To be robust.generics Clinical To determine bioavailability in healthy volunteers Pharmaceutical To develop a stable and reproducible formulation for the manufacture of bioequivalence. stability and pilot-scale validation batches Methods To understand the profile of related substances and to study stability and 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 | To optimize. accurate.

Analytical procedure characteristics Type of characteristic Accuracy Precision Specificity Identification + Impurities Quantitative Limit Assay + + + + + + + Detection limit Quantitation limit Linearity Range Robustness | + April 2007 + + + + + + + + + Slide 16 of 43 .

ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. | Slide 17 of 43 April 2007 .ISO 5725 1-6 Accuracy Trueness Precision (Random errors) Systematic errors Intra-assay variability Intra-laboratory variability Inter-laboratory variability Repeatability Intermediate precison Reproducibility Source: ISO. 1994.Accuracy . ISO. Switzerland. Geneva.

Accuracy and precision Inaccurate and imprecise Inaccurate & imprecise Inaccurate but precise Precise Accurate April 2007 Accurate but imprecise Accurate and precise | Slide 18 of 43 .

209 1. | Slide 19 of 43 April 2007 .917 Recovery % 99.8 99.2 99.4 99.795 1.8 RSD % 0.499 0.77 The data show that the recovery of analyte in spiked samples met the evaluation criterion for accuracy (100 ± 2.001 1.64 0.796 1.88 0.27 1.12 0.8 99.8 99.005 1.211 1.495 0. mg Added 0.9 100. % 50 70 80 100 120 130 Mean Nevirapine.31 0.296 0.918 Recovered 0.703 0.299 0.701 0.0% across 50–130% of target concentrations).38 1.Percent accuracy (hypothetical figures) Sample 1 2 3 4 5 6 LA.

Percent accuracy (hypothetical figures) 104 103 102 101 100 99 98 97 96 0 1 2 3 4 5 6 7 Number of samples % Recovery Red line: LA | Green lines: USL and LSL Slide 20 of 43 April 2007 .

) of a series of measurements. The confidence interval (CI) should be reported for each type of precision investigated. RSD%. PRECISION | Slide 21 of 43 April 2007 . or coefficient of variation (= relative standard deviation.Precision (of any process) Measured mean Real mean The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S). variance (S2).

Repeatability is also termed intra-assay precision. REPEATABILITY | Slide 22 of 43 April 2007 . variability) under the same operating conditions over a short interval of time.Repeatability (of any process) Measured mean Repeatability expresses the precision (spread of the data.

300 0.300 The repeatability precision obtained by one analyst in one laboratory was 1. STD RSD 95% CI | 257 0.301 3 4 5 6 Mean 57497 57617 57778 57231 57649 0.299 0.Repeatability (hypothetical figures) Injection 1 2 Peak area 57935 57833 Imp1 0.0013 0.301 0. meets the evaluation criterion of RSD ≤2%. therefore.4% 0.301 0.0014 Slide 23 of 43 .25% RSD for the analyte and.4% 270 April 2007 0.298 0.

#1. Reproducibility expresses the precision between laboratories #1. usually applied to standardization of methodology). etc. different (manufacturing) equipment. #2 and #3: different days. #2 and #3 (collaborative studies. (Transfer of technology) Intermediate precision or Reproducibility | Slide 24 of 43 April 2007 . different analysts.Intermediate Precision and Reproducibility (of any process) Measured means Intermediate precision expresses within-laboratories variations.

3 52.44 0.7 0.7 52.3 52.9% 0.0 52.5 52.9% 0.7 52.48 0.9 53.51 April 2007 Combined values Mean STD RSD 95% CI 52.46 Slide 25 of 43 .1 52.6 51.Intermediate precision (ruggedness) Sample 1 2 3 4 5 6 Mean STD RSD 95% CI | Assay (mg/5ml) 51.5 0.1 52.9 53.8% 0.49 0.31 52.4 0.2 53.

| Slide 26 of 43 April 2007 . which may be expected to be present. Typically these might include impurities.Specificity (selectivity)  Specificity is the ability to assess unequivocally the analyte in the presence of components.  An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2. degradants and excipients.0 minutes from all other sample components  Stability indicating analytical methods should always be specific.

2%)  Illustrative regulatory issues – Legislation – GMP  Specificity is an essential but not sufficient characteristic of identification | Slide 27 of 43 April 2007 .1 %. GC seemed preferable. The victims were mainly children. – The assay was the most relevant test (accurate within ± 0.Identification – a special case  Diethylene glycol (DEG) in paediatric dosage forms has been implicated as the causative agent in numerous deaths since 1937.  Illustrative analytical issues of investigation – IR identity test was able to detect DEG at about 20 %w/w – Testing of DEG in Glycerol (and in Propylene Glycol) was recommended with a LOD (sensitivity) of NLT 0. For detecting DEG at low levels.

(b) FPP and (c) placebo | Slide 28 of 43 April 2007 .Specificity (hypothetical figures and data) HPLC chromatograms of (a) API reference standard.

630 0.3 99.280 0.045 0.690 NA Purity threshold 0.410 1.0 99.280 0. methylparaben (MP) and propylparaben (PP) peaks. | Slide 29 of 43 April 2007 . specific and stability-indicating.610 0. The method is selective. The three ingredients can be assessed in the presence of (nonexpected) degradants.SPECIFICITY – degradants Stress A (%) * Initial Acid Peroxide 100.250 NA There were no peaks in the placebo chromatogram at the retention times of nevirapine (N).120 1.270 0.360 1. MP and PP peak areas.380 1.725 0.110 0.8 All others 100.0 Purity angle 0.105 0.040 0. *Sum of N. The peaks are homogeneous and pure.040 0.060 0.

LOD. LOQ and SNR  Limit of Quantitation (LOQ)  Limit of Detection (LOD)  Signal to Noise Ratio (SNR) Peak B LOQ Peak A LOD Baseline noise | Slide 30 of 43 April 2007 .

(%w/w) | 0.019 0.9% Conc.9% 7950 8166 7847 8415 7952 402 5.033 0.039 Slide 31 of 43 April 2007 .171 0.3% 8292 8050 8368 8284 8113 238 2.017 0. (μg/ml) Conc.1% 3275 3464 4008 4702 3867 551 14.086 0.107 0.214 0.LOD and LOQ (hypothetical figures) Injection 1 2 Impurity 1 LOD 4176 3608 LOQ 7235 8099 Impurity 2 LOD 3497 4258 LOQ 7892 7791 3 4 5 6 Mean STD RSD 4196 4303 3932 5238 4242 548 12.

It is expressed as a concentration at a specified signal : noise ratio (SNR).6 : 1  The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. not quantified.086 μg/ml (Impurity 1) with a signal : noise ratio of 3. usually between 3 and 2 : 1.LOD and LOQ  The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected. The ICH has recommended a signal : noise ratio (SNR) of 10:1. The RSD for six injections of the LOQ solution was ≤2%. the LOD was determined to be 0.  In this study.  The LOQ was 0.3.171 μg/ml (Impurity 1) with a signal:noise ratio of 11. | Slide 32 of 43 April 2007 .

„The 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.” | Slide 33 of 43 April 2007 .Linearity Measured mean Real mean Precision Linearity expresses differences in precision at different points of a given range.

7. % Acceptance criterion: correlation coefficient should not be less than 0.124x .9990 | Slide 34 of 43 April 2007 .2984 R = 0.9998 2 Assay mean 3000 2000 1000 0 0 20 40 60 80 100 120 140 Concentration.Linearity and range 5000 4000 y = 36.

 The regression coefficient demonstrates an excellent relationship between peak area and concentration of analyte.  The analyte response is linear across 10-130% of the target nevirapine concentration. | Slide 35 of 43 April 2007 . n=3)  Regression equation was found by plotting the means of peak area (y) against the analyte concentration (x) expressed in %: y = 36.7.Linearity and range  Concentration range 1.0–1.3 mg/ml (10–130% of the theoretical concentration in the test preparation.124x .9998).2984 (R2 = 0.

)  If assay and purity are performed together as one test and only a 100% standard is used.Range (minimum requirements)  Assay of an API or a FPP: ± 20% of the test concentration..g. linearity should cover the range from the reporting level of the impurities to 120% of the assay specification | Slide 36 of 43 April 2007 . (Unusually potent or toxic impurities. is justified).  Dissolution testing: ± 20 % over the specified range. LOD and LOQ should be commensurate with ICH requirement. based on the nature of the dosage form (e.  Impurity: from the reporting level of an impurity to 120% of the specification. metered dose inhalers).  Content uniformity: ± 30% of the test concentration (unless a wider more appropriate range.

| Slide 37 of 43 April 2007 .Stability of analytical solution Stability (of the analytical solution) expresses variation of the measured mean as a function of time. …n Series of measurements of the same sample within a relatively short period of time. #3. Stability Measured means #1 … First measurements #2. #4.

8% Slide 38 of 43 . Conclusion: the stability of the -0.3% An analytical solution prepared from Nevirapine 50mg/5ml Oral Suspension was spiked with Impurity-1 at specification level and stored in a capped volumetric flask on a laboratory bench at uncontrolled room temperature under normal lighting conditions for 25 hours.3% analytical solution of Impurity-1 is -0.3% not a source of variation.7% -0. -0.Stability of test analytical solution Time in hours 0 1 2 Impurity-1 Area 72079 71574 71740 0.4% 0.5% Difference 3 4 5 10 15 20 25 | 71960 72352 71573 72322 72310 72312 72670 April 2007 0.7% 0.2% -0.

Sensitivity and robustness | Slide 39 of 43 April 2007 .

Robustness Method parameter STP Flow Wavelength Variation of mobile phase Variation -10% 10% -5nm +5nm -2% +2% -5oC +5oC -0.83 0.83 Impurity 2 1.80 Column temperature pH | Slide 40 of 43 April 2007 .84 0.80 1.83 0.82 0.89 1.81 1.83 0.80 0.82 0.84 0.76 1.83 0.83 0.81 1.81 1.81 1.82 1.80 1.3 tR Impurity 1 0.3 +0.81 1.

R2 ≥ 0. • intermediate precision [ruggedness (USP)].9900. swabs (≥ 90%). and process surface (≥ 70%) – Range (lowest level is at least 2x higher than LOQ) | Slide 41 of 43 April 2007 . ) – Precision • Repeatability (RSD ≤ 5%) .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. and • reproducibility – Limits of detection and quantitation – Accuracy or recovery from rinsate (≥ 80%).

 Validation should demonstrate that the analytical procedure is suitable for its intented purpose.Main Points Again  Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment / management: – establishment of product-specific acceptance criteria.  HPLC systems and method validation deserves special attention during the assessment of dossiers for prequalification. and – stability of APIs and FPPs. | Slide 42 of 43 April 2007 .

THANK YOU | Slide 43 of 43 April 2007 .