At larger concentrations both 8A3 and 2B3 mAbs inhibit ocrelizumab from binding to CD20 on WIL2-S cells. data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters. Keywords: CD20 interference, PK assay, Polyclonal anti-drug CDR (PAC) assay, free or total drug, monoclonal anti-Drug CDR (MAC) assay, pharmacodynamic (PD), pharmacokinetic (PK) Introduction Monoclonal antibodies (mAbs) are a promising and rapidly growing class of targeted therapeutics for treating variety of indications including oncology, autoimmune, cardiovascular, and infectious diseases. To ensure the safety and efficacy of these molecules, robust bioanalytical pharmacokinetic (PK) assays that can reliably quantify drug concentrations are required. This data are used to assess drug exposure and safety as well as to characterize PK/pharmacodynamic (PD) relationships. There is an ongoing debate, however, as to what form of the mAb therapeutic (free or total) is most relevant to measure.1 Due to the bivalency intrinsic to mAbs, multiple forms of the drug can exist simultaneously GSK1070916 in vivo in the presence of soluble target.2 These include non-complexed free drug and partially or completely complexed forms where one or both binding sites are occupied. Multiple complexed forms may also exist if the soluble target has more than one binding site.3 The presence of ligand or shed receptor in sera often depends on the mechanism of action of the drug and the biology of the disease. The total and free drug species are often equivalent in the absence or at low levels of circulating target.2 High concentrations of circulating target, however, could potentially have a significant effect on PK1 and may result in nonlinear and linear mixed profiles. It is often difficult to predict the concentrations of a circulating target/shed receptor, especially after drug treatment because target levels may become elevated. In addition, the level of target being shed from GSK1070916 cell membranes may be variable depending on the disease biology. Therefore, estimation of the potential effect on PK results often comes from experimental data rather than theoretical predictions. The actual drug species being measured often depends on the tools/reagents available for assay development. The target binding enzyme-linked immunosorbent assay (direct ELISA) is the method of choice for quantifying free drug levels, but this format frequently measures a mixture of different drug species because of the bivalency of mAb therapeutics, choice of detection reagents and the dilution scheme used for samples. Other formats for PK measurements include use of polyclonal [polyclonal anti-therapeutic complementarity-determining region (CDR) assay, or PAC assay], or monoclonal antibodies (monoclonal anti-therapeutic CDR assay, or MAC assay). PAC assays are generally used to detect total drug by utilizing multiple binding epitopes of polyclonal antibodies against the analyte; however, it is difficult to predict which drug species (total or free) are detected by mAbs. As reagents, mAbs are often selected to detect GSK1070916 the CDR region of antibody therapeutics and therefore are believed to block drug-target interactions, but this is not always the case. In this study, we compared the effect of different assay formats and reagents on the PK profiles of three anti-CD20 molecules, chimeric rituximab and two humanized Abs, ocrelizumab and v114 (PRO131921)4,5, which share similar antigen-binding characteristics as rituximab. The MAC assay format for ocrelizumab in rheumatoid arthritis (RA) patients resulted in significant differences in the PK profiles compared with that of rituximab PK profiles in the same patient population; however, a PAC assay similar to that used in the rituximab studies generated comparable PK profiles for ocrelizumab and rituximab. For v114, the MAC and PAC assays generated equivalent clinical PK data in non-Hodgkin lymphoma (NHL) patients. To illustrate that our observations with ocrelizumab and rituximab PK assays were not simply due to the biology of the disease (RA vs. NHL), the MAC and the PAC PK profiles for ocrelizumab in both RA and NHL populations were evaluated. Reagent characterizations provided potential explanations highlighting the importance of assay format and choice of reagents on PK parameters. Results Comparison of PK Data for Rituximab, Ocrelizumab and v114 The serum concentrations of ocrelizumab in RA patients treated during the Phase1/2 ACTION trial (ClinicalTrials.gov identifier: NCT00077870)6_ENREF_4 were initially quantified using the MAC assay format, the serum concentration-time profiles are presented in Figures?1B and ?and1C.1C. The data generated from this Egf trial had unexpected PK differences, compared with historical data for rituximab.7 Concentration-time profiles of rituximab and ocrelizumab following intravenous (IV).