2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Immunogenicity of Cemiplimab: Low Incidence of Antidrug Antibodies and Cut-Point Suitability Across Tumor Types.

The immunogenicity of cemiplimab, a fully human immunoglobulin G4 monoclonal antibody directed against programmed cell death 1, was assessed in patients across multiple tumor types. The development of antidrug antibodies (ADAs) against cemiplimab was monitored using a validated bridging immunoassay. To identify ADA-positive samples in the assay, statistically determined cut points were established by analyzing baseline clinical study samples from a mixed population of different tumor types, and this validation cut point was used to assess immunogenicity in all subsequent studies. Regulatory guidance requires that ADA assay cut points be verified for appropriateness in different patient populations. Thus, for the cemiplimab ADA assay, we evaluated whether each new oncology population was comparable with the validation population used to set the cut point. Assay responses from 2393 individual serum samples from 8 different tumor types were compared with the validation population, using established statistical methods for cut-point determination and comparison, with no significant differences observed. Across tumor types, the immunogenicity of cemiplimab was low, with an overall treatment-emergent ADA incidence rate of 1.9% and 2.5% at intravenous dose regimens of 3 mg/kg every 2 weeks and 350 mg every 3 weeks, respectively. Moreover, no neutralizing antibodies to cemiplimab were detected in patients with ADA-positive samples, and there was no observed impact of cemiplimab ADAs on pharmacokinetics. Study-specific cut points may be required in some diseases, such as immune and inflammatory diseases; however, based on this analysis, in-study cut points are not required for each new oncology disease indication for cemiplimab.

Subcutaneous Administration of Monoclonal Antibodies: Pharmacology, Delivery, Immunogenicity, and Learnings From Applications to Clinical Development.

Subcutaneous (s.c.) administration of monoclonal antibodies (mAbs) can reduce treatment burden for patients and healthcare systems compared with intravenous (i.v.) infusion through shorter administration times, made possible by convenient, patient-centric devices. A deeper understanding of clinical pharmacology principles related to efficacy and safety of s.c.-administered mAbs over the past decade has streamlined s.c. product development. This review presents learnings from key constituents of the s.c. mAb development pathway, including pharmacology, administration variables, immunogenicity, and delivery devices. Restricted mAb transportation through the hypodermis explains their incomplete absorption at a relatively slow rate (pharmacokinetic (PK)) and may impact mAb-cellular interactions and/or onset and magnitude of physiological responses (pharmacodynamic). Injection volumes, formulation, rate and site of injection, and needle attributes may affect PKs and the occurrence/severity of adverse events like injection-site reactions or pain, with important consequences for treatment adherence. A review of immunogenicity data for numerous compounds reveals that incidence of anti-drug antibodies (ADAs) is generally comparable across i.v. and s.c. routes, and complementary factors including response magnitude (ADA titer), persistence over time, and neutralizing antibody presence are needed to assess clinical impact. Finally, four case studies showcase how s.c. biologics have been clinically developed: (i) by implementation of i.v./s.c. bridging strategies to streamline PD-1/PD-L1 inhibitor development, (ii) through co-development with i.v. presentations for anti-severe acute respiratory syndrome-coronavirus 2 antibodies to support rapid deployment of both formulations, (iii) as the lead route for bispecific T cell engagers (BTCEs) to mitigate BTCE-mediated cytokine release syndrome, and (iv) for pediatric patients in the case of dupilumab.

Statistical Approaches for Establishing Appropriate Immunogenicity Assay Cut Points: Impact of Sample Distribution, Sample Size, and Outlier Removal.

The statistical assessments needed to establish anti-drug antibody (ADA) assay cut points (CPs) can be challenging for bioanalytical scientists. Poorly established CPs that are too high could potentially miss treatment emergent ADA or, when set too low, result in detection of responses that may have no clinical relevance. We evaluated 16 validation CP datasets generated with ADA assays at Regeneron's bioanalytical laboratory and compared results obtained from different CP calculation tools. We systematically evaluated the impact of various factors on CP determination including biological and analytical variability, number of samples for capturing biological variability, outlier removal methods, and the use of parametric vs. non-parametric CP determination. In every study, biological factors were the major component of assay response variability, far outweighing the contribution from analytical variability. Non-parametric CP estimations resulted in screening positivity in drug-naïve samples closer to the targeted rate (5%) and were less impacted by skewness. Outlier removal using the boxplot method with an interquartile range (IQR) factor of 3.0 resulted in screening positivity close to the 5% targeted rate when applied to entire drug-naïve dataset. In silico analysis of CPs calculated using different sample sizes showed that using larger numbers of individuals resulted in CP estimates closer to the CP of the entire population, indicating a larger sample size (~ 150) for CP determination better represents the diversity of the study population. Finally, simpler CP calculations, such as the boxplot method performed in Excel, resulted in CPs similar to those determined using complex methods, such as random-effects ANOVA.

Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay.

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.

Comparison of Titer and Signal to Noise (S/N) for Determination of Anti-drug Antibody Magnitude Using Clinical Data from an Industry Consortium.

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.

Interference in a Neutralizing Antibody Assay for Odronextamab, a CD20xCD3 Bispecific mAb, from Prior Rituximab Therapy and Possible Mitigation Strategy.

A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy.

2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Assay pH and critical reagent optimization in measuring concentrations of a monoclonal antibody and its target.

Aim: To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Results: Mild acidic assay conditions and capture and detection antibodies with different affinities and t1/2 under different assay pHs were used to mitigate interference in the total drug and total target assays. A free target assay was also developed using a lower-affinity capture antibody with a much slower association and dissociation rate. The impact of sample incubation, dilution and storage on the accurate detection of the free target was also evaluated. Conclusion: The total drug, total and free target assays can accurately quantitate drug and target concentrations when tested with a subset of clinical study samples.

Quantitation of reduced IL-33 levels in human serum: mitigating interference from endogenous binding partners.

Aim: IL-33 is a potential therapeutic target but commercially available assays for the quantitation of systemic IL-33 have poor reliability. Results: In commercial IL-33 kits, interference from endogenous binding partners (e.g., soluble ST2) causes under-quantitation. Mitigating this required acid dissociation and addition of the detection reagent simultaneously with the capture step. This enabled detection of total, reduced (active) levels of IL-33 in human serum (LLOQ 6.25 pg/ml). Conclusion: Acid treatment of serum samples dissociates IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml. The modified method was specific for reduced endogenous IL-33. Analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no difference in serum IL-33.

REGEN-COV® antibody cocktail bioanalytical strategy: comparison of LC-MRM-MS and immunoassay methods for drug quantification.

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Therapeutic Antibody Cocktail REGEN-COV Concentrations in COVID-19 Patient Serum.

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.

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).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Isotyping and Semi-Quantitation of Monkey Anti-Drug Antibodies by Immunocapture Liquid Chromatography-Mass Spectrometry.

There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.

Assessment of clinically relevant immunogenicity for mAbs; are we over reporting ADA?

Immunogenicity is recognized as a possible clinical risk due to the development of anti drug antibodies (ADAs) that can adversely impact drug safety and efficacy. Although robust assays are currently used to assess the ADA, there is a debate on how best to generate the most appropriate immunogenicity data. There are several factors that can trigger ADA formation including the immunity status of the target population and the severity of the disease indication. Immunogenicity testing has defaulted to the most conservative approach regardless of the inherent risk of the molecule or the patient population. For low-risk biotherapeutics such as human monoclonal antibodies, ADA data that provide clinically relevant information should be prioritized when establishing immunogenicity monitoring plans.

Drug Removal Strategies in Competitive Ligand Binding Neutralizing Antibody (NAb) Assays: Highly Drug-Tolerant Methods and Interpreting Immunogenicity Data.

Neutralizing anti-drug antibody (NAb) assays often have lower drug tolerance (DT) than trough drug concentrations, potentially under-estimating NAb incidence. To improve DT, drug-specific proteins were coupled to magnetic beads to deplete drug in the sample. To avoid interference from carryover, drug-specific proteins that did not interfere in the NAb assay, such as target or non-blocking anti-drug antibodies, were selected. With the drug depletion step, DT improved by > 10-fold in two competitive ligand binding NAb assays. Analysis of anti-drug antibody positive clinical samples with elevated drug levels demonstrated that NAb incidence was under-estimated without the drug depletion step. However, these NAb-positive samples had low titer and no impact on drug concentrations.

Safety, pharmacokinetics, and immunogenicity of a co-formulated cocktail of three human monoclonal antibodies targeting Ebola virus glycoprotein in healthy adults: a randomised, first-in-human phase 1 study.

BACKGROUND: REGN3470-3471-3479 is a co-formulated cocktail of three human monoclonal antibodies targeting three non-overlapping epitopes on Ebola virus. We investigated safety, tolerability, pharmacokinetics, and anti-drug antibodies in healthy adults. METHODS: This randomised, double-blind, placebo-controlled, dose-escalation study was done at a phase 1 unit in the USA. Healthy adults, aged 18-60 years, with a body-mass index of 18·0-30·0 kg/m2 were randomly assigned (3:1) to receive a single intravenous dose of REGN3470-3471-3479 or placebo on day 1 (baseline) in one of the four sequential ascending intravenous dose cohorts (3 mg/kg, 15 mg/kg, 60 mg/kg, and 150 mg/kg). Site investigators and participants were masked to the treatment assignment, whereas designated personnel at the site who prepared and generated the study medication were aware of the randomisation treatment assignments. The primary outcome was safety and the secondary outcomes were the pharmacokinetic profiles and immunogenicity. Study assessments were done the day before study drug administration, on the day of drug administration, on day 2 (before discharge), on days 3, 4, 8, 15, 29, 57, 85, 113, and 141, and at the end of study on day 169. The safety analysis included all randomised participants who received study drug. This trial is registered with ClinicalTrials.gov, number NCT002777151. FINDINGS: Between May 18, 2016, and October 27, 2016, 70 adults were screened and 24 participants were enrolled in the study. 18 participants were assigned to and received REGN3470-3471-3479, and six participants were assigned to and received placebo as a single intravenous infusion. 19 treatment-emergent adverse events occurred in the combined REGN3470-3471-3479 treatment groups, and four treatment-emergent adverse events occurred in combined placebo groups. Adverse events were transient and mild-to-moderate in severity. The most common treatment-emergent adverse event was headache (six [33%] of 18 participants in the combined REGN3470-3471-3479 group vs none of six participants in the placebo group. Headaches were mild-to-moderate in severity, with onset between 2 h and 27 days after start of study drug infusion. There were no deaths, serious adverse events, or adverse events that led to study discontinuation. The pharmacokinetics of each antibody was linear, with mean half-lives of 27·3 days for REGN3471, 21·7 days for REGN3470, and 23·3 days for REGN3479. No participants tested positive for anti-REGN3470, anti-REGN3471, or anti-REGN3479 antibodies. INTERPRETATION: REGN3470-3471-3479 was well tolerated, displayed linear pharmacokinetics, and did not lead to detectable immunogenicity. These data support further clinical development of REGN3470-3471-3479 as a single-dose therapeutic drug for acute Ebola virus infection. FUNDING: The Department of Health and Human Services, the Office of the Assistant Secretary for Preparedness and Response, and the Biomedical Advanced Research and Development Authority.

2017 White Paper on recent issues in bioanalysis: a global perspective on immunogenicity guidelines & biomarker assay performance (Part 3 - LBA: immunogenicity, biomarkers and PK assays).

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.

Bridging immunogenicity assays for IgG4 therapeutics: mitigating interference from Fc-Fc interactions.

AIM: A bridging immunogenicity assay for a human IgG4 mAb therapeutic was transferred to an automation system to increase throughput. However, background signal increased five- to six-fold during the 6- to 8-h run. RESULTS: Noncovalent Fc contacts formed between labeled IgG4 drugs in reagent solutions stored during the automation run. This generated substantial background signal, reducing assay sensitivity by approximately sixfold. Fc interactions also significantly impacted the confirmation assay. Fc contacts formed between labeled and unlabeled drug, significantly increasing signal inhibition (∼7-70%) in the 6-h run. CONCLUSION: Storing labeled antibody solutions separately and combining them immediately before adding to samples reduced interference from Fc interactions. Preincubation time for reagent solutions should be strictly controlled for anti-drug antibody assays with IgG4 drugs to avoid false-positive results.