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Aim: Volumetric absorptive microsampling (VAM) is being increasingly applied in nonclinical pharmacokinetic studies. Although there are published results for VAM use in small molecule pharmacokinetics (PK) studies, there is limited data on the utility of VAM for protein therapeutics. Results: We describe the use of Mitra® microsampler for blood sampling, ELISA quantitation and PK analysis of two marketed therapeutic monoclonal antibodies administered to rat. Results generated for these monoclonal antibodies using Mitra® were compared with both serum and whole blood sampling methods in the same study. Conclusion: The low relative standard deviation among the three sets of PK data suggest that Mitra® microsampler could be useful in early nonclinical PK studies for protein therapeutics where reduction and refinement of animal use is desirable.

Aim: The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. Results: Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the “know-how” of therapeutic modality by design.

Prior to clinical studies, the pharmacokinetics (PK) of antibody-based therapeutics are characterized in preclinical species; however, those species can elicit immunogenic responses that can lead to an inaccurate estimation of PK parameters. Immunodeficient (SCID) transgenic hFcRn and C57BL/6 mice were used to characterize the PK of three antibodies that were previously shown to be immunogenic in mice and cynomolgus monkeys. Four mouse strains, Tg32 hFcRn SCID, Tg32 hFcRn, SCID and C57BL/6, were administered adalimumab (Humira®), mAbX and mAbX-YTE at 1 mg/kg, and in SCID strains there was no incidence of immunogenicity. In non-SCID strains, drug-clearing ADAs appeared after 4–7 days, which affected the ability to accurately calculate PK parameters. Single species allometric scaling of PK data for Humira® in SCID and hFcRn SCID mice resulted in improved human PK predictions compared to C57BL/6 mice. Thus, the SCID mouse model was demonstrated to be a useful tool for assessing the preclinical PK of immunogenic therapeutics.

Volumetric absorptive microsampling (VAM) is being increasingly applied in nonclinical pharmacokinetic studies. Although there are published results for VAM use in small molecule pharmacokinetics (PK) studies, there is limited data on the utility of VAM for protein therapeutics.We describe the use of MitraThe low relative standard deviation among the three sets of PK data suggest that Mitra

Background: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). Discussion & conclusion: This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

Targeting CD40-CD40L interactions has been an interesting drug concept for the treatment of autoimmune diseases given this pathway’s role in the development of both humoral and cell mediated immune responses. While preclinical blockade of CD40L is well documented in various autoimmune animal models of disease, limited data exists for CD40 blockade due to lack of a truly antagonistic anti-mouse CD40 tool antibody (Ab). Here we describe the in vitro and in vivo characterization of a fully antagonistic anti-mouse CD40 monoclonal Ab (BI CD40-1); a chimeric rat Fv anti-mouse CD40 mAb engineered with a mouse IgG2a Fc containing mutations to abrogate Fcγ receptor binding. BI CD40-1 blocks molecular CD40-CD40L interactions (IC50 = 0.25 nM) and exhibits potent binding to CD40 expressed on mouse B cells (EC50 = 0.42 nM ± 0.08). In vitro profiling of BI CD40-1 using a mouse splenocyte proliferation assay confirmed potent antagonistic activity (IC50 = 0.27 nM ± 0.09) as well as absence of any agonistic properties (stimulation index < 2 @ 67 nM). Administration of BI CD40-1 to mice prior to ovalbumin (OVA) immunization resulted in dose-dependent blockade of OVA-specific IgG responses (100, 100, 74, 0% inhibition at 10, 3, 1, and 0.3 MPK; day 13) correlating with similar dose dependent receptor occupancy and inhibition of B cell activation as measured by ex vivo CD54 upregulation. Prophylactic dosing in cGVHD model of scleroderma showed dose dependent protection in disease development as seen by decreased dermal thickness, myofibroblast counts and collagen deposition. BI CD40-1 represents a novel fully antagonistic anti-mouse CD40 mAb, an important tool for mechanistic understanding of the therapeutic value of targeting of CD40 in inflammatory diseases.