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. A drug-tolerant, sensitive, generic ADA immunoassay has been developed and validated for measuring ADA in cynomolgus monkey serum samples, allowing for immediate qualification of future monoclonal antibody biotherapeutics. This approach allows us to differentiate complexed and free ADA in a rapidly deployable manner when needed. The testing of antidrug antibodies (ADA) in animal studies offers low value because the presence of animal ADA does not translate to human studies. However, the impact of ADA can be seen with reduced drug levels and/or safety findings in animal studies. Generic ADA methods offer a way to measure ADA leading to time and cost savings. This article details the testing of a generic plug-and-play method to measure ADA in monkey serum and how to qualify future drugs. To date, 16 drugs have been qualified using this method, which has also been applied to mouse, rat and rabbit serum.

During biotherapeutic drug development, immunogenicity is evaluated by measuring anti-drug antibodies (ADAs). The presence and magnitude of ADA responses is assessed using a multi-tier workflow where samples are screened, confirmed, and titered. Recent reports suggest that the assay signal to noise ratio (S/N) obtained during the screening tier correlates well with titer. To determine whether S/N could more broadly replace titer, anonymized ADA data from a consortium of sponsors was collected and analyzed. Datasets from clinical programs with therapeutics of varying immunogenicity risk levels (low to high), common ADA assay platforms (ELISA and MSD) and formats (bridging, direct, solid-phase extraction with acid dissociation), and titration approaches (endpoint and interpolated) were included in the analysis. A statistically significant correlation between S/N and titer was observed in all datasets, with a strong correlation (Spearman’s r > 0.8) in 11 out of 15 assays (73%). For assays with available data, conclusions regarding ADA impact on pharmacokinetics and pharmacodynamics were similar using S/N or titer. Subject ADA kinetic profiles were also comparable using the two measurements. Determination of antibody boosting in patients with pre-existing responses could be accomplished using similar approaches for titer and S/N. Investigation of factors that impacted the accuracy of ADA magnitude measurements revealed advantages and disadvantages to both approaches. In general, S/N had superior precision and ability to detect potentially low affinity/avidity responses compared to titer. This analysis indicates that S/N could serve as an equivalent and in some cases preferable alternative to titer for assessing ADA magnitude and evaluation of impact on clinical responses.

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. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.

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. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

BioanalysisVol. 12, No. 19 MethodologyNative high-resolution mass spectrometry analysis of noncovalent protein complexes up to 450 kDaZhuo Chen, John F Kellie, Charles S Hottenstein & Matthew E SzapacsZhuo Chen*Author for correspondence: Tel.: +1 610 917 2846; Fax: +1 610 270 5604; E-mail Address: zhuo.2.chen@gsk.comBioanalysis, Immunogenicity & Biomarkers, In-Vitro/In-Vivo Translation, R&D Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Rd, Collegeville, PA 19426, USA, John F KellieBioanalysis, Immunogenicity & Biomarkers, In-Vitro/In-Vivo Translation, R&D Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Rd, Collegeville, PA 19426, USA, Charles S HottensteinBioanalysis, Immunogenicity & Biomarkers, In-Vitro/In-Vivo Translation, R&D Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Rd, Collegeville, PA 19426, USA & Matthew E SzapacsBioanalysis, Immunogenicity & Biomarkers, In-Vitro/In-Vivo Translation, R&D Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Rd, Collegeville, PA 19426, USAPublished Online:24 Aug 2020https://doi.org/10.4155/bio-2020-0145AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: native mass spectrometrynon-covalent protein complextarget engagementPapers of special note have been highlighted as: • of interest; •• of considerable interestReferences1. Walsh G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 36(12), 1136–1145 (2018).Crossref, Medline, CAS, Google Scholar2. Terral G, Beck A, Cianferani S. Insights from native mass spectrometry and ion mobility-mass spectrometry for antibody and antibody-based product characterization. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1032, 79–90 (2016).Crossref, Medline, CAS, Google Scholar3. Gupta RK, Morton DL. Monoclonal antibody-based ELISA to detect glycoprotein tumor-associated-antigen-specific immune complexes in cancer patients. J. Clin. Lab. Anal. 6(5), 329–336 (1992).Crossref, Medline, CAS, Google Scholar4. Goldberg ME, Djavadi-Ohaniance L. Methods for measurement of antibody/antigen affinity based on ELISA and RIA. Curr. Opin. Immunol. 5(2), 278–281 (1993).Crossref, Medline, CAS, Google Scholar5. Vaisocherova H, Faca VM, Taylor AD, Hanash S, Jiang S. Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera. 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J. 81(6), 3503–3509 (2001).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDetermination of label efficiency and label degree of critical reagents by LC-MS and native MSAnalytical Biochemistry, Vol. 664Approaches to Heterogeneity in Native Mass Spectrometry2 September 2021 | Chemical Reviews, Vol. 122, No. 8Thermodynamic Evaluation of the Interactions between Anticancer Pt(II) Complexes and Model Proteins19 April 2021 | Molecules, Vol. 26, No. 8 Vol. 12, No. 19 Follow us on social media for the latest updates Metrics Downloaded 231 times History Received 27 May 2020 Accepted 27 July 2020 Published online 24 August 2020 Published in print October 2020 Information© 2020 Newlands PressKeywordsnative mass spectrometrynon-covalent protein complextarget engagementAcknowledgmentsThe authors gratefully appreciate the edits and suggestions on manuscript from T Sikorski. The authors also acknowledge C Evans and E Yang for supporting this research.Financial & competing interests disclosureThis research was supported by GlaxoSmithKline Research and Development. All authors are employees of GlaxoSmithKline, whom may be eligible for stock options or have stock ownership. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Ethical conduct of researchThe authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.PDF download

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. This review presents case studies and a regulatory perspective.

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.

BioanalysisVol. 9, No. 14 EditorialFree AccessPlatforms and techniques used for biomarker assays: where are we now?Charles Hottenstein, Matthew Szapacs, Kerensa Fuller & Christopher EvansCharles Hottenstein*Author for correspondence: E-mail Address: charles.s.hottenstein@gsk.com GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USA, Matthew Szapacs GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USA, Kerensa Fuller GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity and Biomarkers; Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK & Christopher Evans GlaxoSmithKline, PTS – In Vivo/In Vitro Translation; Bioanalysis, Immunogenicity & Biomarkers; 709 Swedeland Road, King of Prussia, PA 19406 USAPublished Online:24 Jul 2017https://doi.org/10.4155/bio-2017-0107AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: biomarkerligand binding assayLC–MSparallelismplatformsurrogate analytesurrogate matrixFirst draft submitted: 28 April 2017; Accepted for publication: 15 May 2017; Published online: 24 July 2017Currently, the term biomarker has many connotations, as such, it is important to properly define this term for the context of this editorial. In the broadest definition, biomarkers are "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention" [1]. However, in the context of biomarkers evaluated during the course of clinical drug trials, we can categorically divide the term into three categories which include biomarkers for determining risk (predisposition), biomarkers used for diagnosis (screening, prognosis and diagnosis) and biomarkers specific to pharmacology (predictive, target engagement, mechanism engagement, clinical engagement, surrogate and clinical end point).As the number of drugs targeting a specific biological mechanism increases, the pharmacodynamic (PD) biomarkers involved in that mechanism can provide a valuable understanding of target engagement, efficacy and/or safety to assist in guiding the drug development process for that mechanism. When measuring PD biomarkers in samples from preclinical and/or clinical studies, it is important that the robustness and limitations of the analytical methods as well as stability of biomarker in the matrix of interest is well understood allowing for minimal analytical variation and lack of biomarker degradation during sample collection.A variety of bioanalytical platforms are routinely applied to measurement of biomarker analytes, including: ligand-binding assays (LBA), LC–MS, flow cytometry and immunohistochemistry. Although each of these platforms can be used to understand the impact of a drug on PD end points, LBA and LC–MS are the two platforms most commonly utilized and will be the focus of this editorial. The most appropriate platform to monitor a given biomarker depends on the matrix, analyte and specific biological question to be answered. However, in some instances either platform could be used to support an analytical measurement, therefore understanding the biological question and/or problem and the benefits of a given platform should guide this selection. This editorial aims to address the challenges and benefits of either of these platforms in relation to the unique issues associated with specific biomarker analyte quantitation.Sensitive versus selective biomarker platformsAs a number of biomarkers of interest are present in matrices at very low concentrations (i.e., cytokines), development of a highly sensitive assay is a critical component to platform selection. However, as the need to develop ever more sensitive assays increases, problems with developing selective assays becomes more challenging. Historically, photometric LBAs were the most commonly used platform for both commercial and bespoke assays, however, the electrochemiluminescent format from Meso Scale Discovery (MSD) has over the past decade become the industry standard, with a large number of commercially available biomarker kits that can be used as is or optimized and validated for regulated use. Although the MSD platform offers improved selectivity and dynamic range over more traditional photometric methods, it represents a single vendor platform with proprietary instrumentation and microtiter plate labware. Where bespoke assays are developed, consistency in microtiter plate lot-to-lot variability of the background and overall signal range have caused issues when changing microtiter plate lots for established methods. Therefore, the benefits and risks should be closely considered when choosing single vendor platforms for biomarker quantitation.In addition, various vendors have been developing proprietary ultrasensitive instrumentation to allow quantitation of very low abundance biomarkers, including: MSD platform (S-Plex), Singulex and Quanterix Simoa with the various platforms providing different sensitivities depending on the analyte of interest [2,3]. Although these platforms can provide high sensitivity assays, they also suffer from additional challenges associated with selectivity from individual samples due to the low levels of analyte that are being detected and often lead to a trade-off between assay sensitivity and overall selectivity. It should be noted that the Singulex and Quanterix Simoa are also single-vendor platforms and the same considerations listed above for the MSD platform should apply when selecting these higher sensitivity instrument for use.One platform that has emerged to address inherent selectivity challenges identified in LBA platforms is LC–MS/MS. Through the use of immunocapture reagents followed by enzymatic digestion and selection of a surrogate peptide which is coupled to LC–MS, we and others [4–6] have shown that this platform can be used to develop highly selective and sensitive assays utilizing immunocapture reagents that may not be suitable for use in a typical LBAs. In addition to mitigating the observed selectivity issues in LBAs, there are also occasions when the LC–MS platform may provide the more sensitive analytical option as the selectivity and overall S/N of the assay is improved; therefore platform selection needs to be made on a case-by-case basis.As well as sensitivity, LBA formats often require less expensive specialized equipment and are often a more economical option than LC–MS platforms. In our laboratories, we employ all of the above described approaches for biomarker quantitation depending on the availability of reagents, while balancing the benefits of sensitivity and selectively when choosing one platform over another allowing one to accurately answer to biological question at hand.Measuring biomarkers in the presence of endogenous analyteAnother common challenge encountered during quantitation of biomarkers is determination of the absolute concentration of a biomarker. Unlike pharmacokinetic methods, where the calibration matrix is typically analyte free, allowing for the detection of absolute drug concentrations; biomarker matrices often contain some level of endogenous analyte and the biomarker bioanalyst often relies on either a depleted matrix approach to provide analyte free matrices for calibration purposes or the use of low- and high-concentration megapools with subsequent admixing of these pools to create a uniform control and potentially use for calibration purposes [7].However, both approaches are not without issue as depleting the matrix may remove other components and the megapools often are not available for novel or less well-studied biomarkers. With this in mind, the use of surrogate matrix or surrogate analyte strategies to generate a standard calibration curve without interference from endogenous biomarkers continues to be standard practice. Surrogate matrix strategies are the most common approaches and use buffer with added proteins like bovine serum albumin, commercially available matrices (SeraSub, UriSub) or other in-house mixtures of proteins in an aqueous buffer all with spiked analyte. The use of surrogate matrix requires that a parallelism assessment is completed by spiking analyte in both surrogate matrix and authentic matrix across the assay range. If the surrogate matrix curve is 'parallel' to the authentic matrix curve then the surrogate matrix is representative of the authentic matrix [8].As LC–MS continues to grow as a platform for biomarker bioanalytical support, the use of a surrogate analyte approach has become more commonplace. Surrogate analyte methodologies utilize an isotopically labeled biomarker that can be used to create a standard calibration curve in the authentic matrix as it has a unique mass that can be differentiated from the authentic, endogenous analyte using LC–MS [9]. This technique has enormous promise for development of biomarker assays as the authentic matrix that is identical to that of the samples can be used for study support and allow for absolute quantitation. In addition, the use of longer gradients or high-resolution LC–MS provides increased confidence and confirmation of the endogenous analyte being measured and demonstrates that it is not a cross-reactive species or a molecule with a similar mass transition in the MS.Measuring the various forms of biomarker targetsOne of the most common challenges for biotherapeutics is the development of a free, total and/or complex target engagement assays. Traditionally, LBA-based platforms have been used for the quantitation of free, total or complex target engagement and have been reviewed previously [10]. However, the ex vivo manipulation of samples required before analysis (i.e., sample dilution, extraction, etc.) can cause an equilibrium bias between free, total and complex forms in solution. Therefore, the development of assays that provide 'true' free, total and/or complex measurements is quite challenging and the assays often do not demonstrate agreement when used in combination.In our laboratories, we prefer the combination of total and complex assay measurements to understand biomarker PDs in relation to the administered drug and feel that free assays are the most challenging and often generate less robust data for a number of reasons including equilibrium shifts from native sample matrix upon dilution, the density of the solid phase capture mechanism concentration compared with analyte concentration in solution and the length of primary incubation times. Typically, we avoid free assays unless the methods are highly characterized and the quality of the data can be assured. Overall, drug target complex and total target assays are less susceptible to these issues and have been shown to demonstrate the best measure of target engagement and target concentration [11].ConclusionThe best-suited quantitation platform for a given biomarker measurement depends on a number of factors including the required sensitivity, selectivity with the ability to use the actual matrix and actual biomarker analyte or will a surrogate or combination approach need to be used. The ability to select from more than one analytical option in many situations is beneficial for the biomarker bioanalyst and allows for the ability to proactively address analytical issues allowing for high-quality reliable data for internal or pivotal decision-making purposes regarding the safety or efficacy of a drug candidate over the course of the drug development process. With the recent bioanalytical advancements and ability to couple platforms (hybrid LBA/LC–MS), the biomarker bioanalyst now has the tools that are critical to address and resolve the unique biological questions and challenges that exist over the course of the drug development process.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Atkinson AJ, Colburn WA, Degruttola VG, Demets DL. Surrogate endpoints: preferred definitions and conceptual framework* biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin. Pharmacol. Ther. 69(3), 89–95 (2001).Crossref, Medline, Google Scholar2 Oberoi P, Suschak J, Stenglin M et al. True sensitivity of immunoassays: are concentrations of low abundance analytes real or artifacts? J. Immunol. 196(Suppl. 1), 138–139 (2016).Google Scholar3 Fischer SK, Joyce A, Spengler M et al. Emerging technologies to increase ligand binding assay sensitivity. AAPS J. 17(1), 93–101 (2015).Crossref, Medline, CAS, Google Scholar4 Bronsema KJ, Bischoff R, Pijnappel WP, van der Ploeg AT, van de Merbel NC. Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC–MS/MS. Anal. Chem. 87(8), 4394–4401 (2015).Crossref, Medline, CAS, Google Scholar5 Palandra J, Finelli A, Zhu M, Masferrer J, Neubert H. Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC–MS/MS. Anal. Chem. 85(11), 5522–5529 (2013).Crossref, Medline, CAS, Google Scholar6 Ackermann BL, Berna MJ. Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers. Expert Rev. Proteomics 4(2), 175–186 (2007).Crossref, Medline, CAS, Google Scholar7 Arnold ME, Booth B, King L, Ray C. Workshop Report: Crystal City VI – Bioanalytical Method Validation for Biomarkers. AAPS J. 18(6), 1366–1372 (2016).Crossref, Medline, CAS, Google Scholar8 Jones BR, Schultz GA, Eckstein JA, Ackermann BL. Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules. Bioanalysis 4(19), 2343–2356 (2012).Link, CAS, Google Scholar9 Jenkins RG. Accuracy: a potential quandary in regulated bioanalysis of 'endogenous' analytes. Bioanalysis 8(23), 2393–2397 (2016).Link, CAS, Google Scholar10 Talbot JJ, Calamba D, Pai M, Ma M, Thway TM. Measurement of free versus total therapeutic monoclonal antibody in pharmacokinetic assessment is modulated by affinity, incubation time, and bioanalytical platform. AAPS J. 17(6), 1446–1454 (2015).Crossref, Medline, CAS, Google Scholar11 Lee JW, Kelley M, King LE 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. AAPS J. 13(1), 99–110 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByProgress with Metabolomic Blood Tests for Gastrointestinal Cancer Diagnosis—An Assessment of Biomarker Translation10 October 2022 | Cancer Epidemiology, Biomarkers & Prevention, Vol. 31, No. 12An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatricesJournal of Chromatography A, Vol. 1676One-dollar microfluidic paper-based analytical devices: Do-It-Yourself approachesMicrochemical Journal, Vol. 165Development of a Meso Scale Discovery ligand-binding assay for measurement of free (drug-unbound) target in nonhuman primate serumYun Liu, Ronald Robinson, Thao Ung, Chrysanthe Spais, Justin Schreiber, Jacquelyn Lyons, Jean Husten, Hussein Hallak & Thelma Angeles22 March 2021 | Bioanalysis, Vol. 13, No. 7High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths4 February 2019 | Analytical Chemistry, Vol. 91, No. 5A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?Cytokine, Vol. 111 Vol. 9, No. 14 Follow us on social media for the latest updates Metrics History Published online 24 July 2017 Published in print July 2017 Information© 2017 Future Science LtdKeywordsbiomarkerligand binding assayLC–MSparallelismplatformsurrogate analytesurrogate matrixFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

This chapter covers the use of ligand binding assays (LBAs) in a regulated bioanalysis environment. The various platforms employed in the analysis of protein therapeutics are varied and selection of the appropriate platform for a particular application is highlighted. The suitability, procurement and implementation of critical reagents are of major importance for LBAs and considerations for their careful selection and use are provided. In addition, various strategies, including Design of Experiments (DoE) are discussed as a means of optimizing assay conditions to produce robust and rugged assays during method development. The key parameters for the validation of LBAs are discussed in depth as well as many considerations, including automation, that can be employed when analyzing study samples in a production environment.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Future Science Book SeriesCurrent Issues in Ligand Binding Assay Bioanalysis Considerations and rationale for the selection of LC-MS/MS or ligand binding assays for bioanalysis of therapeutic proteinsCharles Scott Hottenstein, Eric Dobrzynski, Joshua Albert, Jonathan Kehler & Matthew SzapacsCharles Scott Hottenstein, Eric Dobrzynski, Joshua Albert, Jonathan Kehler & Matthew SzapacsPublished Online:3 Mar 2016https://doi.org/10.4155/fseb2013.14.379AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail View chapterReferences1. Hoofnagle AN , Wener MH . The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry . J. Immunol. Methods 347 ( 1 ), 3 – 11 ( 2009 ). Crossref, Medline, CAS, Google Scholar2. Barnidge DR , Goodmanson MK , Klee GG et al. Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-MS/MS using protein cleavage and isotope dilution mass spectrometry . J. Proteome Res. 3 ( 3 ), 644 – 652 ( 2004 ). Crossref, Medline, CAS, Google Scholar3. Plumb RS , Fujimoto G , Mather J et al. Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS . Bioanalysis 4 ( 5 ), 605 – 615 ( 2012 ). Crossref, Medline, CAS, Google Scholar4. Boscato LM , Stuar MC . Heterophilic antibodies: a problem for all immunoassays . Clin. Chem. 34 ( 1 ), 27 – 33 ( 1988 ). Crossref, Medline, CAS, Google Scholar5. Butch AW . Dilution protocols for detection of hook effects/prozone phenomenon . Clin. Chem. 46 ( 10 ), 1719 – 1720 ( 2000 ). Crossref, Medline, CAS, Google Scholar6. Lippi G , Aloea R , Meschib T et al. Interference from heterophilic antibodies in troponin testing. Case report and systematic review of the literature . Clin. Chim. Acta 426 , 79 – 84 ( 2013 ). Crossref, Medline, CAS, Google Scholar7. Kragstrup TW , Vorup-Jensen T , Deleuran B et al. A simple set of validation steps identifies and removes false results in a sandwich enzymelinked immunosorbent assay caused by antianimal IgG antibodies in plasma from arthritis patients . Springerplus 2 ( 1 ), 1 – 20 ( 2013 ). Crossref, Medline, Google Scholar8. Jager WD , Rijkers GT . Solid-phase and bead-based cytokine immunoassay: a comparison . Methods 38 ( 4 ), 294 – 303 ( 2006 ). Crossref, Medline, Google Scholar9. Bumbaca D , Wong A , Drake E et al. Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4 . mAbs 3 ( 4 ), 376 – 386 ( 2011 ). Crossref, Medline, Google Scholar10. Montrose-Rafizadeh C , Yang H , Rodgers BD et al. High potency antagonists of the pancreatic glucagon-like peptide-1 receptor . J. Biol. Chem. 272 ( 34 ), 21201 – 21206 ( 1997 ). Crossref, Medline, CAS, Google Scholar11. Alcantara AI , Morales M , Delgado E et al. Exendin-4 agonist and exendin (9–39) amide antagonist of the GLP-1(7–36) amide effects in liver and muscle . Biochem. Biophys. 341 ( 1 ), 1 – 7 ( 1997 ). Crossref, Medline, CAS, Google Scholar12. Fraley KJ , Abberley L , Hottenstein CS et al. The GyrolabTM immunoassay system: a platform for automated bioanalysis and rapid sample turnaround . Bioanalysis 5 ( 14 ), 1765 – 1774 ( 2013 ). Crossref, Medline, CAS, Google Scholar13. Murphy RE , Kinhikar AG , Shields MJ et al. Combined use of immunoassay and two-dimensional liquid chromatography–mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples . J. Pharma. Biomed. Anal. 53 ( 3 ), 221 – 227 ( 2010 ). Crossref, Medline, CAS, Google Scholar14. Kay RG , Roberts A . Bioanalysis of biotherapeutic proteins and peptides: immunological or MS approach . Bioanalysis 4 ( 8 ), 857 – 860 ( 2012 ). Crossref, Medline, CAS, Google Scholar15. Damen CWN , Rosing H , Schellens JM et al. Quantitative aspects of the analysis of the monoclonal antibody trastuzumab using high-performance liquid chromatography coupled with electrospray mass spectrometry . J. Pharma. Biomed. Anal. 46 ( 3 ), 449 – 455 ( 2008 ). Crossref, Medline, CAS, Google Scholar16. Damen CWN , de Groot ER , Heij M et al. Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma . Anal. Biochem. 391 ( 2 ), 114 – 120 ( 2009 ). Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Current Issues in Ligand Binding Assay BioanalysisMetrics Downloaded 14 times History Published online 3 March 2016 Published in print March 2016 Information© Future Science Ltd© Future Science LtdPDF download

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. Sensitivity, precision and accuracy for each molecular portion are presented along with an example of glycoform variant quantitation. Conclusion: The method is presented as a basic platform to be further developed for Good Practice (GxP) applications, critical quality attribute analysis or general understanding of molecular forms present as required for the wide range of drug development processes.

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. No patient had a post‐baseline QTcF interval >480 milliseconds or a ΔQTcF >60 milliseconds; five patients (four on ofatumumab) had a ΔQTcF between 30 and 60 milliseconds. At cycle 6 (week 21; 308 μg/mL), there was an increase in QTcF in patients on ofatumumab treatment, with an estimated between‐treatment difference (90% CI) of 12.5 (4.5, 20.5) milliseconds. However, at the visit with the highest median concentration (week 8; 1386 μg/mL), median ΔQTcF was 4.8 milliseconds. There was no significant relationship between ofatumumab plasma concentration and ΔQTcF. Ofatumumab did not have a clinically significant effect on cardiac repolarization.

In this study, we evaluated the safety and pharmacodynamic effects of the Fc-inactivated anti-β-amyloid (anti-Aβ) monoclonal antibody GSK933776 in patients with mild Alzheimer’s disease and mild cognitive impairment. Aβ and tau levels were investigated in cerebrospinal fluid (CSF), and the relationship between Aβ levels and Aβ modulation in plasma was explored. The feasibility of a continuous sampling method using a lumbar catheter was assessed.

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. CONCLUSION:The Gyrolab immunoassay system proved to be a viable alternative to traditional immunoassays and was used to support a regulated preclinical TK study. The speed of analysis that the Gyrolab provides was beneficial in meeting timelines to complete this project as multiple assays and repeat sample analysis could be completed in the same day.

Background: Domain antibodies (dAbs; similar to 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. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed. Conclusion: The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.