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14 Pharmaceutical Technology Europe BIO/PHARMA OUTSOURCING INNOVATION eBOOK 2022 P h a r mTe c h . c o m The Linearity Ratio formula is shown in Equation 1: !"#$%&'&$()*+,-./&,.&+-+*0& "#$%&'()!*!! ! ! !"#$%&"'()*%'"+,- . /0%1$2)34'"5%'$)67%2&%'"0) 8 . /0%1$2)34'"5%'$)9+4$):; 8 -):)<==! "#$%&'()!+! ,-!.%)/0!1!(2!3(405%)60!7!,-!.%)/089:;!?!*@@! !"#$%&'&$()*+,-./&,.&+-+*0& "#$%&'()!*!! ! ! !"#$%&"'()*%'"+,- . /0%1$2)34'"5%'$)67%2&%'"0) 8 . /0%1$2)34'"5%'$)9+4$):; 8 -):)<==! "#$%&'()!+! ,-!.%)/0!1!(2!3(405%)60!7!,-!.%)/089:;!?!*@@! [Eq. 1] Validity criteria: assay repeatability/analytical error In order for an analytical method to be validated, it must be shown to be repeatable at the moment of measurement (4). Be- fore evaluating repeatability, outliers should be removed from the assay run or run of the analytical method. Jackknife z is the preferred method of outlier identification within each dose. Jackknife z is calculated independently for each concentration in the dose response for both reference and sample type. Jack- knife z evaluates the influence each point is having on the curve fit where each point is removed from the model; the model is regenerated and evaluated, iteratively, for each data point. Jack- knife z = (measurement - dose mean (measurement removed)) - Standard deviation (measurement removed). CIs should be included when reporting the results for any and all test articles. CIs control for sample size, variation in the method, and risk (95%). CI range is calculated by taking the difference of the upper confidence limit of the constrained relative potency and the lower confidence limit. A narrower CI range indicates that the assay is less variable and, therefore, more repeatable. Excessive CI range may not make the assay run invalid; it demonstrates that additional assay runs are re- quired to tighten the error. Normally, the CI range is reported as a percent of tolerance (Equation 2): !"#$%&'&$()*+,-./&,.&+-+*0& ! ! ! "#$%&'()!*!! ! ! !"#$%&"'()*%'"+,- . /0%1$2)34'"5%'$)67%2&%'"0) 8 . /0%1$2)34'"5%'$)9+4$):; 8 -):)<==! ! ! ! ! ! ! ! ! "#$%&'()!+! ! ! ,-!.%)/0!1!(2!3(405%)60!7!,-!.%)/089:;!?!*@@! ! !"#$%&'&$()*+,-./&,.&+-+*0& ! ! ! "#$%&'()!*!! ! ! !"#$%&"'()*%'"+,- . /0%1$2)34'"5%'$)67%2&%'"0) 8 . /0%1$2)34'"5%'$)9+4$):; 8 -):)<==! ! ! ! ! ! ! ! ! "#$%&'()!+! ! ! ,-!.%)/0!1!(2!3(405%)60!7!,-!.%)/089:;!?!*@@! ! [Eq. 2] where USL is upper specification limit and LSL is lower spec- ification limit. The coefficient of variance (CV) is often used to evaluate repeatability of the dose response. CV is not recom- mended for use because it incorrectly scales the analytical error by concentration; high concentrations appear to have low error and low concentrations seem to have high error. Setting systems suitability and validity criteria limits When setting specification limits based on the representa- tive sample, risk must be considered. The wider the interval, !_quivalence between Sample 1 an� Reference 1.75 - 1.5 1.25 - . Q 1.00 0.75 - 0.5 0.25 - 0.00 - a= 0.05 I u C Equivalence Summary Parameter 0 UDL LDL Lower Upper Limit Level Confidence Limit Ratio Confidence Limit Exceeded Intercept 0.92958 0.955834 0.982089 Slope 0.747411 � 1.239282 Figure 3. Slope ratio. UDL is upper detection limit. LDL is lower detection limit. Analytics