A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor–mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey–adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05–3.2 μg/mL as well as be tolerant to more than 400 μg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.

[1]  J. Panse,et al.  The complement C5 inhibitor crovalimab in paroxysmal nocturnal hemoglobinuria. , 2020, Blood.

[2]  J. Nezu,et al.  Antibody engineering to generate SKY59, a long-acting anti-C5 recycling antibody , 2018, PloS one.

[3]  Mark Dysinger,et al.  A Gyrolab Assay for the Quantitation of Free Complement Protein C5a in Human Plasma , 2018, The AAPS Journal.

[4]  Julia Heinrich,et al.  Evaluation of the potential use of hybrid LC-MS/MS for active drug quantification applying the 'free analyte QC concept'. , 2017, Bioanalysis.

[5]  P. Gatault,et al.  Development and validation of an enzyme-linked immunosorbent assay to measure free eculizumab concentration in serum. , 2017, Bioanalysis.

[6]  J. Nezu,et al.  Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases , 2017, Scientific Reports.

[7]  T. Igawa,et al.  Fc Engineering to Improve the Function of Therapeutic Antibodies. , 2016, Current pharmaceutical biotechnology.

[8]  L. King.  Parallelism experiments in biomarker ligand-binding assays to assess immunological similarity. , 2016, Bioanalysis.

[9]  A. Papadimitriou,et al.  Validation of a ligand-binding assay for active protein drug quantification following the 'free analyte QC concept'. , 2016, Bioanalysis.

[10]  C. Hottenstein,et al.  Ligand-Binding Assay Development: What Do You Want to Measure Versus What You Are Measuring? , 2015, The AAPS Journal.

[11]  K. Stubenrauch,et al.  Proposal for a harmonized descriptive analyte nomenclature for quantitative large-molecule bioanalysis. , 2015, Bioanalysis.

[12]  Melody Y. Pai,et al.  Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform , 2015, The AAPS Journal.

[13]  M. Sank,et al.  Development and characterization of a free therapeutic ligand binding assay with assistance from kinetics modeling. , 2015, Journal of immunological methods.

[14]  R. Porcher,et al.  Assessing complement blockade in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab. , 2015, Blood.

[15]  Roland F Staack,et al.  Free analyte QC concept: a novel approach to prove correct quantification of free therapeutic protein drug/biomarker concentrations. , 2014, Bioanalysis.

[16]  Shobha Purushothama,et al.  Parallelism: considerations for the development, validation and implementation of PK and biomarker ligand-binding assays. , 2014, Bioanalysis.

[17]  A. Rafique,et al.  Minimizing target interference in PK immunoassays: new approaches for low-pH-sample treatment. , 2013, Bioanalysis.

[18]  R. Kelley,et al.  Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG , 2013, mAbs.

[19]  V. Wroblewski,et al.  Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments , 2013, mAbs.

[20]  Mark Ma,et al.  Implementation of a universal analytical method in early-stage development of human antibody therapeutics: application to pharmacokinetic assessment for candidate selection. , 2012, Bioanalysis.

[21]  G. Nakamura,et al.  The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters , 2012, mAbs.

[22]  Heather Myler,et al.  Bioanalytical Approaches to Quantify “Total” and “Free” Therapeutic Antibodies and Their Targets: Technical Challenges and PK/PD Applications Over the Course of Drug Development , 2011, The AAPS Journal.

[23]  K. Ozawa,et al.  Safety and efficacy of the terminal complement inhibitor eculizumab in Japanese patients with paroxysmal nocturnal hemoglobinuria: the AEGIS Clinical Trial , 2011, International journal of hematology.

[24]  A. Avrameas,et al.  A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion. , 2010, Journal of pharmaceutical and biomedical analysis.

[25]  Bing Kuang,et al.  Therapeutic monoclonal antibody concentration monitoring: free or total? , 2010, Bioanalysis.

[26]  Jean W Lee,et al.  Method validation and application of protein biomarkers: basic similarities and differences from biotherapeutics. , 2009, Bioanalysis.

[27]  N. Nishimoto,et al.  Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. , 2008, Blood.

[28]  Viswanath Devanarayan,et al.  Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement , 2006, Pharmaceutical Research.

[29]  R. Hamilton,et al.  Immunological methods for quantifying free and total serum IgE levels in allergy patients receiving omalizumab (Xolair) therapy. , 2005, Journal of immunological methods.

[30]  L. Matis,et al.  Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv. , 1996, Molecular immunology.

[31]  R. Ogata,et al.  Primary structure of the fifth component of murine complement. , 1987, Biochemistry.

[32]  D. Peters,et al.  Metabolism of the fifth component of complement, and its relation to metabolism of the third component, in patients with complement activation. , 1977, The Journal of clinical investigation.

[33]  H. Müller-Eberhard,et al.  ISOLATION OF β1F-GLOBULIN FROM HUMAN SERUM AND ITS CHARACTERIZATION AS THE FIFTH COMPONENT OF COMPLEMENT , 1965, The Journal of experimental medicine.