STDs and VSs were stored at ?60C to ?80C for a minimum of 12?h and up to 3? days prior to analysis. analysis of the biocomparability study. This study was sufficiently powered using a parallel design. The bioequivalence acceptance criteria for small molecule drugs were adopted. The pharmacokinetic parameters of the subjects dosed with both formulation lots were found to be comparable. Electronic supplementary material The online version of this article (doi:10.1208/s12248-012-9414-x) contains supplementary material, which is available to authorized users. biological equivalence of two proprietary preparation of a drug. Bioequivalence (BE) studies are required by regulatory agencies to insure therapeutic equivalence. If two products are said to be bioequivalent, it means they would be expected to behave similarly for their intended use. The current industry BE criteria applies to small molecule therapeutics. For small molecule chemical entities, the Cav1.3 BRD73954 chemical structures are well defined which allow certainty of their molecular equivalence. The United States Food and Drug Administration (FDA) has defined bioequivalence as, the absence of a significant difference in the rate and extend to which the active ingredients or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administrated at the same molar dose under similar condition in an appropriately designed study (FDA 2003) (2). Most protein biotherapeutics have higher-order structure with posttranslational heterogeneity. Sometimes, in addition to analytical comparison of the drug, BRD73954 it is necessary to carry out comparability studies (3C5). Regulatory guidance on comparability has been stated in International Committee on Harmonization Q5E and Q6B (3), the European Medicines Agency 2007 guideline (4), and Guidance to industry by the FDA (5). Due to the lack of established industry guidance for biocomparability of large molecule therapeutics, we applied small molecule guidelines on BE criteria, set by FDA, during our denosumab biocomparability studies. FDA considers two products to be bioequivalent if the 90% confidence intervals (CI) of the ratio of geometric mean concentration relationship was fitted with a four-parameter logistic auto-estimate regression model with a weighting factor of 1/Y (1/OD response). Kit Method The microtiter wells precoated with RANKL were used BRD73954 to capture denosumab in the samples. STDs and QCs were prepared in human serum-derived diluent (HD6Y). Seventy-five microliters of assay buffer was added to the plate followed by 25?L of STDs, QCs, samples, and blanks. All shaking and incubation steps were carried out at a nominal BRD73954 temperature of 25C to 28C. The plate was covered with a plate sealer and shaken for 5??1?min on a shaker (300C400?rpm), and then incubated for 2?h??5?min. After washing, 150?L/well of OPGL conjugate was added. The plate was covered, mixed by shaking for 5??1?min on a shaker, and incubated for 2?h??5?min. After another wash step, 150?L/well of twofold dilution of substrate solution was added and incubated for 20??5?min. To stop the reaction, 50?L/well of the acid stop solution was added. The optical density (OD) was measured at 450?nm with reference to 650?nm. The absorbance (OD) concentration relationship was fitted with a logClog (power) equation. A summary of differences between the two methods is listed in Table?I. Table I Summary of Differences Between the Methods validation sample, quality control, upper limit of quantification, high quality control, middle quality control, low quality control, lower limit of quantification Preparation of Standards, Validation Samples, or Quality Controls In-house ELISAThe STDs, VSs, and QCs were prepared by spiking denosumab formulation lots A and B into 100% human serum. The nominal concentrations for STDs in human serum were 20, 40, 100, 250, 500, 800, 1,500, and 2,000?ng/mL. Two anchor points outside of the quantitative range at 10 and 3,000?ng/mL were included to facilitate curve fitting. The nominal concentrations of VSs were 20 (LLOQ), 60 (LQC), 400 (MQC), 1,200 (HQC), and 2,000 (ULOQ)?ng/mL. STDs and VSs were stored at ?60C to ?80C for a minimum of 12?h and up to 3?days prior to analysis. The VSs were used for the accuracy and precision experiments to determine assay acceptance criteria based on the total error (6). The low, mid, and high QCs (LQC, MQC, and HQC) at concentrations of 60, 400, 1,200?ng/mL were used to accept or reject analytical runs after the accuracy and precision experiments. IN-HOUSE ELISA METHOD VALIDATION Accuracy and Precision Eight accuracy and precision.