Strategy and validation of a nonclinical generic plug-and-play antidrug antibody method for human monoclonal antibody biotherapeutics.

The measurement of antidrug antibodies (ADA) in nonclinical studies provides limited value because the formation and incidence of nonclinical ADA does not translate to clinical experience. The formation and presence of ADA in nonclinical species can, however, correlate to reduced drug exposure and safety observations including vasculitis and immune complex disease. Generic ADA methods for humanized monoclonal antibody biotherapeutics mitigate the need to develop bespoke ADA methods during nonclinical drug development.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities.

Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity.

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway.

Bioanalytical challenges and unique considerations to support pharmacokinetic characterization of bispecific biotherapeutics.

Compared with conventional (monospecific) therapeutics, bispecific protein therapeutics present unique challenges for pharmacokinetic (PK) characterization - namely, the characterization of multiple functional domains as well as the consideration of biotransformation or interference by the formation of antitherapeutic antibodies against each functional domain. PK characterization is essential to the success of the overall drug development plan and for molecules with multiple binding domains; multiple bioanalytical methods may be needed to answer critical questions for each phase of drug development. The number of bispecific protein therapeutics entering drug development continues to increase, and therefore, a bioanalytical strategy for the PK characterization of bispecific molecules and study of their in vivo structure-function relationship is needed.

A whole-molecule immunocapture LC-MS approach for the in vivo quantitation of biotherapeutics.

Aim: Large-molecule biotherapeutic quantitation in vivo by LC-MS has traditionally relied on enzymatic digestion followed by quantitation of a 'surrogate peptide' to infer whole-molecule concentration. MS methods presented here measure the whole molecule and provide a platform to better understand the various circulating drug forms by allowing for variant quantitation.

Results: An immunocapture LC-MS method for quantitation of a biotherapeutic monoclonal antibody from human plasma is presented.

Assessment of the effect of ofatumumab on cardiac repolarization.

Ofatumumab is a human monoclonal antibody that binds to a unique CD20 epitope on the surface of B lymphocytes, resulting in efficient lysis of CD20-expressing cells via complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. The potential effect of ofatumumab on cardiac repolarization and the relationship between ofatumumab concentration and change in corrected QT interval (ΔQTcF) were evaluated in data from three clinical trials in 82 patients with chronic lymphocytic leukemia receiving ofatumumab alone (n = 14), ofatumumab with chemotherapy (n = 33), and chemotherapy alone (n = 35). Because of ofatumumab accumulation, baseline QTcF interval was recorded prior to the first infusion for each patient.

The Gyrolab™ immunoassay system: a platform for automated bioanalysis and rapid sample turnaround.

Background: The Gyrolab™ workstation benefits from fully automated transfer of reagents and samples originating from a storage microplate onto a compact disc containing solid-phase microstructures composed of a 15 nl streptavidin-derivitized bead bed.

Results: This paper describes the development, full validation and use of the method in a regulated environment to measure a humanized bispecific monoclonal antibody-domain antibody (GSK-A) molecule using the Gyrolab immunoassay system in cynomolgus nonhuman primate plasma ranging from 5 to 250 µg/ml. The method was subsequently used in support of the TK portion of a regulated preclinical study in monkeys.

Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass spectrometry and immunoassay.

Background: Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb.

Results: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody.