scholarly journals Anti-drug Antibody Validation Testing and Reporting Harmonization

2021 ◽  
Vol 24 (1) ◽  
Author(s):  
Heather Myler ◽  
João Pedras-Vasconcelos ◽  
Kelli Phillips ◽  
Charles Scott Hottenstein ◽  
Paul Chamberlain ◽  
...  
Keyword(s):  
Positive Control ◽  
Pharmaceutical Sciences ◽  
Performance Expectations ◽  
Assay Sensitivity ◽  
Cut Points ◽  
Health Authorities ◽  
Control Criteria ◽  
Antibody Validation ◽  
Immunogenicity Assay ◽  
Validation Testing

Abstract 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. Graphical Abstract

scholarly journals The Power of Phase I Studies to Detect Clinical Relevant QTc Prolongation: A Resampling Simulation Study

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Georg Ferber ◽  
Ulrike Lorch ◽  
Jörg Täubel
Keyword(s):  
Phase I ◽  
Healthy Subjects ◽  
Sparse Sampling ◽  
Positive Control ◽  
Qtc Prolongation ◽  
False Negatives ◽  
Assay Sensitivity ◽  
Phase I Studies ◽  
Sufficient Power

Concentration-effect (CE) models applied to early clinical QT data from healthy subjects are described in the latest E14 Q&A document as promising analysis to characterise QTc prolongation. The challenges faced if one attempts to replace a TQT study by thorough ECG assessments in Phase I based on CE models are the assurance to obtain sufficient power and the establishment of a substitute for the positive control to show assay sensitivity providing protection against false negatives. To demonstrate that CE models in small studies can reliably predict the absence of an effect on QTc, we investigated the role of some key design features in the power of the analysis. Specifically, the form of the CE model, inclusion of subjects on placebo, and sparse sampling on the performance and power of this analysis were investigated. In this study, the simulations conducted by subsampling subjects from 3 different TQT studies showed that CE model with a treatment effect can be used to exclude small QTc effects. The number of placebo subjects was also shown to increase the power to detect an inactive drug preventing false positives while an effect can be underestimated if time points aroundtmaxare missed.

scholarly journals Multiplexed Evaluation of a Cell-Based Assay for the Detection of Antidrug Neutralizing Antibodies to Panitumumab in Human Serum Using Automated Fluorescent Microscopy

2010 ◽  
Vol 15 (6) ◽  
pp. 644-652 ◽  
Author(s):  
Jason Pennucci ◽  
Steve Swanson ◽  
Arunan Kaliyaperumal ◽  
Shalini Gupta
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Human Serum ◽  
Neutralizing Antibodies ◽  
Egf Receptor ◽  
Fluorescent Microscopy ◽  
Positive Control ◽  
A431 Cells ◽  
Assay Sensitivity ◽  
Epidermal Growth

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay’s sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 µg/mL for the positive control antibody.

scholarly journals Suitability of Two Rapid Lateral Flow Immunochromatographic Assays for Predicting SARS-CoV-2 Neutralizing Activity of Sera

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Clara Frances-Gomez ◽  
Josep Ferrer ◽  
...  
Keyword(s):  
Predictive Value ◽  
Fluorescent Protein ◽  
Neutralizing Antibody ◽  
Antibody Test ◽  
Neutralization Assay ◽  
Positive Control ◽  
Lateral Flow ◽  
Control Line ◽  
Assay Sensitivity ◽  
Neutralizing Activity

Purpose: Assessment of commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially-available lateral flow immunochromatographic assays (LFIC) (Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for COVID-19 diagnosis in hospitalized patients, and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Patients and Methods: Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). Results: Overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%,93.9% and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers (≥1/160). Conclusions: Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.

A bridging immunogenicity assay for anti-cabiralizumab antibodies: overcoming the low assay cut point and drug tolerance challenges

Bioanalysis ◽  
2021 ◽  
Author(s):  
Long Yuan ◽  
Carol R Gleason ◽  
Dennis Stocker ◽  
Li Li ◽  
Jim X Shen ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Drug Tolerance ◽  
Normal Serum ◽  
Acid Dissociation ◽  
Reagent Concentration ◽  
Acid Pretreatment ◽  
Cut Points ◽  
Cut Point ◽  
Immunogenicity Assay ◽  
Assay Conditions

Background: To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Results: Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 μg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. Conclusion: A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.

scholarly journals Comparison of the ELISA and ECL Assay for Vedolizumab Anti-drug Antibodies: Assessing the Impact on Pharmacokinetics and Safety Outcomes of the Phase 3 GEMINI Trials

2020 ◽  
Vol 23 (1) ◽  
Author(s):  
Timothy Wyant ◽  
Lili Yang ◽  
Maria Rosario
Keyword(s):  
Neutralizing Antibodies ◽  
Credible Interval ◽  
Positive Control ◽  
Descriptive Statistics ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phase 3 ◽  
Drug Interference ◽  
Assay Sensitivity ◽  
Population Pk ◽  
The Impact

AbstractVedolizumab immunogenicity has been assessed using an enzyme-linked immunosorbent assay (ELISA) with a ~ 0.5 μg/mL drug interference, which may underestimate on-drug immunogenicity. We aimed to compare immunogenicity results between ELISA and the new drug-tolerant electrochemiluminescence (ECL) assay (and the two versions of neutralizing assays, drug-sensitive versus drug-tolerant). The ECL assay drug tolerance is ~ 100 times higher than that of the ELISA (≥ 50 μg/mL vs. 0.5 μg/mL with a 500 ng/mL positive control), and assay sensitivity is < 5 ng/mL for both assays. Vedolizumab immunogenicity was assessed in 2000 GEMINI 1 and 2 patients originally tested by ELISA and retested by ECL assay. Anti-drug antibody (ADA) impact on infusion-related reactions and pharmacokinetics (PK) was examined using descriptive statistics and population PK analyses. By ECL assay, 6% (86/1427) of patients treated with vedolizumab as induction and maintenance therapy tested ADA-positive. Of these, 20 patients were persistently positive and 56 had neutralizing antibodies. By ELISA, 4% (56/1434) of these patients were ADA-positive, 9 were persistently positive, and 33 had neutralizing antibodies. Among 61 patients with infusion-related reactions, 6 (10%) were ADA-positive (2 persistently positive) by ECL assay. By ELISA, 3 (5%) patients were both ADA-positive and persistently positive. Most results (96%) were similar with both assays. In the updated population PK model, ADA-positive status was estimated to increase vedolizumab linear clearance by a factor of 1.10 (95% credible interval 1.03–1.17), which is consistent with previous reports. The impact of ADA on safety and PK modeling remained generally consistent using either ELISA or ECL assay. ClinicalTrials.gov: NCT00783718 and NCT00783692

Enhancing efficiency and quality of statistical estimation of immunogenicity assay cut points through standardization and automation

2015 ◽  
Vol 425 ◽  
pp. 88-96 ◽  
Author(s):  
Cheng Su ◽  
Lei Zhou ◽  
Zheng Hu ◽  
Winnie Weng ◽  
Jayanthi Subramani ◽  
...  
Keyword(s):  
Statistical Estimation ◽  
Cut Points ◽  
Immunogenicity Assay

scholarly journals Evaluation of the Roche Elecsys Anti-SARS-CoV-2 Assay

2020 ◽  
Author(s):  
CS Lau ◽  
SP Hoo ◽  
SF Yew ◽  
SK Ong ◽  
LT Lum ◽  
...  
Keyword(s):  
Positive Control ◽  
Negative Control ◽  
Cross Reactivity ◽  
Assay Sensitivity ◽  
Automated Assay ◽  
Extensive Evaluation ◽  
Positive Antibody ◽  
Assay Time ◽  
Polymerase Chain ◽  
Assay Precision

ABSTRACTBackgroundLittle is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on the Cobas e801/e602 immunoassay analysers.MethodsWe assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 714 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples (syphilis, RF, ANA, ds-DNA, influenza, dengue, HBV, HCV) and the anti-SARS-CoV-2 kinetics.ResultsThe assay cut-off index (COI) was linear up to 90.7. The inter-assay precision was 2.9% for a negative control (COI=0.1) and 5.1% for a positive control (COI=3.0). Assay time is 18min and results are available 1 minute later; throughput for 300 samples was 76 minutes. No cross-reactivity was observed with other antibody positive samples; specificity was 100%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 subjects in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to “sero-conversion”) on average is 3 days (range 1-6 days) and 4 more days (range 1-7) for the anti-SARS-CoV-2 to plateau.ConclusionThe Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and sero-conversion appears quite early.

scholarly journals Evaluation of an Electrochemiluminescent SARS-CoV-2 Antibody Assay

2020 ◽  
Vol 5 (6) ◽  
pp. 1313-1323
Author(s):  
C S Lau ◽  
S P Hoo ◽  
S F Yew ◽  
S K Ong ◽  
L T Lum ◽  
...  
Keyword(s):  
Positive Control ◽  
Negative Control ◽  
Cross Reactivity ◽  
Assay Sensitivity ◽  
Automated Assay ◽  
Double Stranded Dna ◽  
Extensive Evaluation ◽  
Positive Antibody ◽  
Assay Time ◽  
Polymerase Chain

Abstract Background Little is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on Cobas e801/e602 immunoassay analyzers. Methods We assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 715 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples [syphilis, rheumatoid factor (RF), antinuclear antibody (ANA), double-stranded DNA (ds-DNA), influenza, dengue, hepatitis B (HBV), hepatitis C (HCV)] and the anti-SARS-CoV-2 kinetics. Results The assay cut-off index (COI) was linear up to 90.8. The interassay precision was 2.9% for a negative control (COI = 0.1) and 5.1% for a positive control (COI = 3.0). Assay time is 18 min and results are available 1 min later; throughput for 300 samples was 76 min. Only 1 case positive for HBsAg tested falsely positive; specificity was 99.9%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 patients in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to “seroconversion”) on average is 3 days (range 1–6 days) and 4 more days (range 1–7) for the anti-SARS-CoV-2 to plateau. Conclusion The Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and seroconversion appears quite early.

scholarly journals Assay concept for detecting anti-drug IgM in human serum samples by using a novel recombinant human IgM positive control

Bioanalysis ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 253-263
Author(s):  
Christian Künzel ◽  
Afsaneh Abdolzade-Bavil ◽  
Alfred M Engel ◽  
Mirko Pleitner ◽  
Eginhard Schick ◽  
...  
Keyword(s):  
Immune Response ◽  
Phase I Trial ◽  
Positive Control ◽  
Assay Development ◽  
Clinical Samples ◽  
Serum Samples ◽  
Assay Sensitivity ◽  
Clinical Phase ◽  
Regulatory Guidelines

Aim: Development and qualification of an easy-to-use ELISA for detection of IgM anti-drug antibodies (ADA) and its use in a clinical Phase I trial. Results & methodology: During the assay development two positive control (PC) approaches, the preparation of a chemically conjugated and a recombinant PC, were pursued. With both PCs, the assay was developed and successfully qualified considering the regulatory guidelines. For a case study, the IgM ADA isotyping assay with the recombinant PC was selected. Different courses and intensities of immune response regarding IgM signals were demonstrated. Conclusion: The easy-to-use ELISA allowed IgM-ADA detection in clinical samples. Conjugated and recombinant IgM PCs were comparable regarding assay sensitivity, precision and suitability.

scholarly journals 114 An Assessment of QTc Effects With SPN-812 (Extended-Release Viloxazine) in Healthy Adults

CNS Spectrums ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 274-274
Author(s):  
Azmi Nasser ◽  
Shamia L. Faison ◽  
Tesfaye Liranso ◽  
Toyin Adewole ◽  
Maurizio Fava ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Qt Interval ◽  
Phase 1 ◽  
Plasma Concentrations ◽  
Positive Control ◽  
Negative Slope ◽  
Assay Sensitivity ◽  
Supratherapeutic Dose ◽  
The Mean ◽  
The Relationship

Abstract:Study Objective:SPN-812 (extended-release viloxazine) is a structurally distinct, bicyclic, Serotonin Norepinephrine Modulating Agent (SNMA) under investigation as a treatment for attention-deficit/hyperactivity disorder (ADHD). One concern for any new drug is prolongation of the QT interval, which is associated with increased risk for potentially very harmful ventricular cardiac arrhythmias such as torsades de pointes (TdP). The objective of this study was to assess the effects of SPN-812 at a supratherapeutic dose (1800 mg once daily [QD]) on cardiac repolarization (QTc) in healthy adults.Method:This study was a Phase 1, double-blind (except for the positive control moxifloxacin), randomized, 3-period, 6-sequence crossover design in healthy adult male and female subjects evaluating the electrocardiographic effects of SPN-812. Subjects were randomized to receive a sequence of all 3 treatments – placebo, 400 mg moxifloxacin (positive control), and 1800 mg SPN-812 (supratherapeutic dose). Treatment was given for 2 consecutive days (separated by a washout of at least 4 days). The primary endpoint was based on concentration-QTc effect modeling, evaluating the relationship between plasma concentrations of SPN-812 and its metabolite 5-hydroxyviloxazine glucuronide (5-HVLX-gluc) with the placebo-adjusted change from baseline in QTcI, ΔΔQTcI (QT interval corrected for HR based on the individual-specific QT interval correction method). Secondary endpoints included time point change from baseline in QTcI, QTcF, HR, PR, and QRS; evaluation of the relationship between the plasma concentration of viloxazine and 5-HVLX-gluc and the placebo-adjusted change from baseline in HR, PR, QRS, and QTcF; evaluation of the relationship between the plasma concentration of moxifloxacin and ΔΔQTcI to demonstrate assay sensitivity; and changes in ECG morphology. Safety endpoints included assessment of adverse events and other parameters.Results:The relationship between ΔΔQTcI and viloxazine plasma concentration demonstrated a negative slope (p=0.0012). Predicted mean ΔΔQTcI (2-sided 90% CI) for SPN-812 was -9.7 ms (-11.3, -8.1) at the mean Cmax of 12.4 μg/mL. The relationship of 5-HVLX-gluc and ΔΔQTcI similarly demonstrated a predicted negative slope (p=0.0007) with a predicted mean ΔΔQTcI (2-sided 90% CI) of -9.2 ms (-10.8, -7.8) at the mean Cmax of 10.0 μg/mL. Assay sensitivity was confirmed. Concentration-effect modeling demonstrated no relationship between plasma concentrations of viloxazine and 5-HVLX-gluc and other ECG parameters. The secondary time point analyses demonstrated no effect of SPN-812 on QTcI or other ECG intervals. SPN-812 produced no changes in ECG T wave or U wave morphology.Conclusions:Data from this Phase 1 thorough QT study demonstrate that a supratherapeutic dose of SPN-812, 1800 mg QD, has no effect on cardiac repolarization or other ECG parameters, and is thus not associated with a risk for cardiac arrhythmias such as TdP.Funding Acknowledgements:This research was funded by Supernus Pharmaceuticals, Inc., Rockville, MD.

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