Generation, Characterization, and Quantitative Bioanalysis of Drug/Anti-drug Antibody Immune Complexes to Facilitate Dedicated In Vivo Studies

Abstract

Purpose Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. Methods Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. Results Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. Conclusions The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation ( Part 3 –Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays)

“…SEC and asymmetric flow field flow fractionation (A4F) can detect CICs but provide no actual size information if not coupled to specific detectors such as Multi Angle Light Scattering (MALS). Furthermore, application of these technologies to analyze biological samples is challenging with regard to sensitivity and specificity [87,88]. There is a need to develop methods to better characterize CIC, particularly in biological samples, in terms of their size and to better understand their time of onset, structure, and their impact on PK.…”

The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance

“…The dosing solutions were prepared as described by Hoffman et al ., where also different conditions and concentrations of the components were tested. 11 The dosing solutions for the control groups contained monomeric IgG 1 (drug, germline sequence, no target-binding specificity) with a WT-Fc domain (drug WT ) or a PGLALA mutation in the Fc domain (drug PGLALA ). 17 The dosing solutions for the IC groups were generated by mixing one of the two drug variants with one of three ADA surrogates (monoclonal or polyclonal IgG against the CDR of the drug and a monoclonal IgG against the Fc domain of the drug) in a ratio of 1:1.5.…”

“…The detailed interaction between ADA and drug, that is, the formation of drug/ADA ICs, is well understood. 11 , 12 Although it is well known from literature that large ICs are cleared faster than smaller ICs, quantitative information about the exact size or structure of the ICs, as well as their actual impact on drug PK is missing. 7 , 13–16 …”

“…To study the effects of IC formation on the PK of two therapeutic mAbs, in vivo studies in rats with pre-formed and defined IC preparations were performed. 11 Animals were dosed with monomeric/uncomplexed monoclonal IgG 1 (hereinafter referred to as “drug”) or IgG 1 that was fully complexed with different ADA surrogates targeting either the Fc or CDR of the drug (anti-Fc ADA → <Fc>, anti-CDR ADA → <CDR>).…”

“…The applied bioanalytical assay panel was previously described and allowed the quantification of total drug (free and fully complexed) and size-specific IC PK evaluation in the collected matrices. 11 …”