PHARM 462 PART /31

2009 2/31 Good Manufacturing Practices (GMP) VALIDATION of ANALYTICAL TEST METHODS

GMP Analytical methods, computers and cleaning procedures  „It is of critical importance that particular attention is paid to the validation of analytical test methods, automated systems and cleaning procedures.”  Validation of analytical procedures used in the examination of pharmaceutical materials (WHO Expert Committee on Specifications for Pharmaceutical Preparations. 32nd Report. Geneva, WHO, 1992 (WHO Technical Report Series, No. 823).  Text on Validation of Analytical Procedures Q2A (1994) Validation of Analytical Procedures: Methodology Q2B (1996) ICH Harmonized Tripartite Guidelines /32

Characteristics of methods  Accuracy (also termed trueness)  Precision  repeatability  intermediate precision (intra-laboratory variation)  reproducibility (inter-laboratory variation)  Robustness, ruggedness  Stability /31

Accuracy and precision Accurate and precise Precise Accurate Inaccurate and imprecise /31

Accuracy (of any process) The accuracy of an analytical procedure 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 (individual observation or mean of measurements).  application of an analytical procedure to an analyte of known purity (e.g. reference material);  comparison of the results of the proposed analytical procedure with those of a second well- characterized procedure, the accuracy of which is stated and/or defined  accuracy may be inferred once precision, linearity and specificity have been established /31

Precision (of any process) The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S), variance (S 2 ), or coefficient of variation (= relative standard deviation, R.S.D.) of a series of measurements. The confidence interval should be reported for each type of precision investigated. Measured mean Real mean PRECISION /31

Repeatability (of any process) Repeatability expresses the precision (spread of the data, variability) under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. REPEATABILITY Measured mean /31

Intermediate Precision and Reproducibility Intermediate precision expresses within-laboratories variations. #1, #2 and #3: different days, different analysts, different (manufacturing) equipment, etc. Reproducibility expresses the precision between laboratories #1, #2 and #3 (collaborative studies, usually applied to standardization of methodology). (Transfer of technology) Measured means Intermediate precision or Reproducibility /31

Sensitivity and robustness Input-output relationship /31

Stability Stability (of the analytical solution) expresses variation of the measured mean as a function of time. (Manufacturing machines) #1 … First mesurements #2 … Second mesurements of the same sample within a relatively short period of time. Stability Measured means /31

Characteristics of analytical procedures  Linearity  Range  Specificity (selectivity)  Sensitivity (versus robustness)  Limit of detection  Limit of quantitation /31

Linearity Linearity expresses differences in precision at different points of a given range. “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.” Measured Real mean mean Precision /31

Range (minimum requirements)  Assay of an API or a FPP: ± 20% of the test concentration.  Content uniformity: ± 30% of the test concentration (unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified).  Dissolution testing: ± 20 % over the specified range.  Impurity: from the reporting level of an impurity to 120% of the specification. (Unusually potent or toxic impurities, LOD and LOQ should be commensurate with ICH requirement.)  If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification /31

Specificity (selectivity)  Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants, excipients, etc.  Stability indicating analytical methods should always be specific /31

 Limit of Quantitation (LOQ)  Limit of Detection (LOD)  Signal to Noise Ratio (SNR) noise Peak A LOD Peak B LOQ Baseline LOD, LOQ and SNR /31

Classes of analytical tests  „The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intented purpose.”  Class A: To establish identity  Class B: To detect (B d ) and quantitate (B q ) impurities  Class C: To determine quantitatively the concentration, or assay  Class D: To assess characteristics  Other classes not covered in the guides /31

Criteria for analytical classes CriteriaABqBq BdBd CD AccuracyXXX PrecisionXXX RobustnessXXXXX Linearity and rangeXXX SpecificityX ?XXXX Limit of detectionX Limit of quantitationX /31

General requirements  Qualified and calibrated instruments  Documented methods  Reliable reference standards  Qualified analysts  Sample integrity  Change control (e.g., synthesis, FPP composition) /31

HPLC Method Development and Validation for Pharmaceutical Analysis Pharmaceutical Technology Europe, 1 March /34

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, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2A and Q2B /31

HPLC system /31

Linearity and range /31

ICH requirements  Concentration range 0.025–0.15 mg/mL (25–150% of the theoretical concentration in the test preparation, n=3)  Regression equation was found by plotting the peak area (y) versus the analyte concentration (x) expressed in mg/mL: y = x (r 2 = 1.000).  The regression coefficient demonstrates the excellent relationship between peak area and concentration of analyte.  The analyte response is linear across % of the target progesterone concentration /31

Accuracy The data show that the recovery of analyte in spiked samples met the evaluation criterion for accuracy (100 ± 2.0% across 80–120% of target concentrations) /31

Specificity /31

Specificity  An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 minutes from all other sample components.  In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution. Former slide demonstrates specificity /31

Repeatability The repeatability precision obtained by one analyst in one laboratory was 1.25% RSD for the analyte and, therefore, meets the evaluation criterion of RSD ≤2% /31

Intermediate precision /31

Limit of detection  The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal:noise ratio (SNR), usually between 3 and 2:1.  In this study, the LOD was determined to be 10ng/mL with a signal:noise ratio of 2.9: /31

Limit of quantitation  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. The ICH has recommended a signal : noise ratio (SNR) of 10:1.  The LOQ was 20 ng/mL with a signal:noise ratio of The RSD for six injections of the LOQ solution was ≤2% /31

Analytical solution stability Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period /31

Main Points Again  Validation of analytical procedures is a critical requirement in risk assessment and management:  establishment of product-specific acceptance criteria, and  stability of APIs and FPPs.  Validation should demonstrate that the analytical procedure is suitable for its intented purpose.  HPLC systems and method validation deserves special attention during the inspection of QC laboratories /31