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M. of SEC and ELISA allowed unbiased reconstruction of IC oligomeric says in serum. Conclusions The developed IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination Tanshinone IIA sulfonic sodium between different oligomeric states of ICs and can be bench marking for future studies with ICs. Keywords: Anti-drug antibodies, bioanalysis, ELISA, immune complex, immunogenicity, therapeutic proteins Introduction The therapy of a variety of diseases was revolutionized by the development of biotherapeutics. Especially monoclonal antibodies (mAbs) are widely used for the treatment of diseases such as cancer and autoimmune deficiencies. Technological progress allows the generation of fully human mAbs. Although fully human biotherapeutics are much better tolerated by patients, immune responses against these drugs can occur (1). Immunogenicity against a mAb can be influenced by different extrinsic and intrinsic factors, like manufacturing, formulation, co-medication, immunological status or glycosylation pattern (2,3). Although fully human, immunogenicity can also be triggered by antibody-specific structural amino acid patterns, especially in the complementary determining region (CDR) of Abs (2,4). Consequently, anti-drug antibodies (ADAs) can be generated by the immune system, which bind to the drug and may lead to the formation of immune complexes (ICs) of drug and ADAs. Neutralizing ADAs for example can constrain the PRKM10 binding of the drug to its target. Therefore, formation of ICs can have influence on efficacy and pharmacodynamics of the drug. Additionally, pharmacokinetics (PK) of the drug can be altered by increasing or decreasing drug clearance (5). To enable dedicated studies investigating the influence of IC formation on drug PK, this paper describes an model for the generation and characterization of defined ICs, as well as the development of bioanalytical methods to quantify drug fully complexed with ADAs to allow IC size-specific PK assessment. In contrast to Tanshinone IIA sulfonic sodium previous studies with no information Tanshinone IIA sulfonic sodium about the exact size of formed or found ICs (6,7), our goal was to have a quantitative determination of generated ICs facilitating advanced studies. A human non-binding IgG1 (PGLALA) mAb was chosen as drug model (8C11). As ADA surrogate a polyclonal Ab (pAb) against the CDR (pAb ADA) of the drug was used. Additionally, a mAb Tanshinone IIA sulfonic sodium against the CDR and the Fc region of the drug were used as ADA surrogates for a more detailed understanding of ADA clonality and epitope on IC formation (see Fig.?1 and discussion). Open in a separate window Fig. 1 Schematic overview of used ADA surrogates, pAb ADA, mAb ADA, mAb??ADA and their binding sites to the hIgG1 drug. Materials and Methods Compounds As drug surrogate a human monoclonal IgG1 without target specificity was used. Three different Abs were used as ADA surrogates: a mouse-derived monoclonal IgG against the Fc portion of human IgG, a mouse-derived monoclonal IgG against the CDR of the drug, and a polyclonal rabbit-derived IgG against the CDR of the drug. All mentioned Abs, as part of the ICs or as capture and detection reagents were produced in house (Roche Diagnostics GmbH, Penzberg, Germany). Biotin (Bi)- and digoxigenin (Dig)-labeling of appropriate Abs was performed in house. Reagents Bovine serum albumin (BSA), Roche universal buffer for ELISA (RUB), phosphate-buffered saline (PBS), and Tween20 (10% (studies with preformed ICs, we decided to generate ICs with fully complexed drug. Furthermore, excess of ADA should be as low as possible. Different drug:ADA ratios were tested with increasing ADA concentrations. The effect on the overall IC pattern and the excess monomeric fraction was visualized by UV-SEC. The absence of monomeric drug was tested by two different ways: with fluorescently labeled drug (data not shown) and with an ELISA-based method (see below). Increasing ADA concentrations had little effect on the IC pattern always resulting in comparable SEC chromatograms (Fig.?6). A drug to ADA ratio of 1 1:1.5 fulfilled all set criteria (no monomeric drug, little ADA excess) and was.