RESUMO
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 3 Main Workshops and 7 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 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. 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 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
Assuntos
Receptores de Antígenos Quiméricos , Vacinas , Biomarcadores/análise , Sistemas CRISPR-Cas , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Imunoterapia Ativa , Reação em Cadeia da PolimeraseRESUMO
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.
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Anticorpos , Bioensaio , Europa (Continente) , Estados UnidosRESUMO
The first author's name was published incorrectly as "Gorovits Boris". The correct name is "Boris Gorovits".
RESUMO
Neublastin (NBN) is a neurotrophic growth factor that promotes the survival and regenerative properties of nociceptive neurons and has been tested in clinical trials as a treatment for neuropathic pain in individuals with sciatica and painful lumbosacral radiculopathy. Like many low molecular weight heparin binding proteins, NBN is rapidly cleared from the blood following systemic administration. To explore ADME properties of NBN in rats, we used metabolically 35S-labeled NBN following IV and SC administration quantifying counts and intact protein in kidney, liver, brain, serum, and urine at 5â¯min, 8â¯h, 24â¯h and 48â¯h, and biodistribution in whole body carcasses by QWBA at 2, 8, 48, 96, and 168â¯h post dose. NBN is rapidly taken up by tissues mainly by liver and kidney and then degraded. Products of degradation are excreted in urine or recycled and utilized for resynthesis. The data we generated for NBN provides a first look at the complex clearance mechanisms for this protein and should aid in the design of ADME studies for other heparin binding proteins.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Isótopos de Enxofre/metabolismo , Distribuição Tecidual/fisiologia , Animais , Proteínas de Transporte/metabolismo , Heparina/metabolismo , Masculino , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Ratos , Ratos Long-EvansRESUMO
Viral vector-based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bioensaio , Dependovirus/imunologia , Terapia Genética , Vetores Genéticos/imunologia , Testes Imunológicos , Linhagem Celular , Dependovirus/genética , Genes Reporter , Vetores Genéticos/genética , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives 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, immunogenicity and gene therapy. 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 LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.
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Bioensaio/métodos , Biomarcadores/metabolismo , Citometria de Fluxo/métodos , Terapia Genética/métodos , United States Food and Drug Administration/normas , História do Século XXI , Humanos , Estados UnidosRESUMO
Aim: Replicate sample testing has long been regarded as a necessity for bioanalytical laboratory testing, especially in the realm of ligand-binding assays (LBAs). In an era in which results were derived from crude test tube-based assays, the replication of results was warranted. Those assays were often imprecise and required multiple replicates to arrive at results that approached accuracy. However, given technological advancements and excellent accuracy and precision of many modern LBAs, the practice of replicate testing should be re-evaluated. Although most regulatory guidelines allow for singlet testing when sufficient robustness and precision are demonstrated during validation, duplicate testing is still common practice. Recently however, several articles have been published that support singlet analysis for LBAs performed on a platform with automated liquid handling. Results: Data from five pharmacokinetic assay validations and five clinical and preclinical studies originally run in duplicate were re-evaluated in singlet and found to be nearly identical to the original duplicate results. Conclusion: We confirm that well-developed LBAs produce comparable data whether evaluated in singlet or duplicate. Additionally, automation is not requisite for singlet testing.
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Bioensaio/métodos , Bibliotecas de Moléculas Pequenas/metabolismo , Animais , Ligantes , Macaca fascicularis , Participação nas Decisões , Bibliotecas de Moléculas Pequenas/farmacocinética , Distribuição TecidualRESUMO
The 2018 12th Workshop on Recent Issues in Bioanalysis 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 LCMS, hybrid LBA/LCMS 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 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.
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Antígenos/análise , Bioensaio/normas , Citometria de Fluxo/normas , Terapia Genética/normas , Farmacocinética , Antígenos/imunologia , Bioensaio/métodos , Biomarcadores/análise , Biotecnologia , Citometria de Fluxo/métodos , Órgãos Governamentais , Humanos , Valores de ReferênciaRESUMO
This manuscript aims to provide insights and updates on emerging technologies from a throughput and multiplexing perspective and to update readers on changes in previously reported technologies. The technologies discussed range from nascent (ultrasensitive Cira, Intellicyt®, Dynaxi and Captsure™) to the more established (Ella and SQIDlite™). For the nascent technologies, there was an emphasis on user interviews and reviews, where available, to help provide an unbiased view to our readers. For the Ella, a review of published user data as well as author and other user experiences are summarized. Due to their emergent nature, all the technologies described are applicable in the early drug development stage, may require an upfront investment of capital and may not perform as expected.
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Citocinas/sangue , Drogas em Investigação/análise , Imunoensaio , Técnicas Analíticas Microfluídicas , Anticorpos Monoclonais/química , Automação Laboratorial , Humanos , Limite de DetecçãoRESUMO
Anti-drug antibodies (ADA) pose a potential risk to patient safety and efficacy and are routinely monitored during clinical trials. Pre-existing drug-reactive antibodies are present in patients without prior drug exposure and are defined by their ability to bind to a component of the drug. These pre-existing drug-reactive antibodies are frequently observed and could represent an adaptive immune response of an individual who has been previously exposed to antigens with structural similarities to the biotherapeutic. Clinical consequences of these antibodies can vary from no impact to adverse effects on patient safety, exposure, and efficacy, and are highly dependent on biotherapeutic modality, disease indications, and patient demographics. This paper describes how the immunogenicity risk assessment of a biotherapeutic integrates the existence of pre-existing drug-reactive antibodies, and provides recommendations for risk-based strategies to evaluate treatment-emergent ADA responses.
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Anticorpos/imunologia , Produtos Biológicos/imunologia , Terapia Biológica , Medição de Risco , Humanos , Segurança do PacienteRESUMO
AIM: ImmunoPCR technology combines the advantages of specificity and robustness of a ligand binding assay with the amplification potential of PCR. We describe through three case studies a plug-and-play immuno polymerase chain reaction (iPCR) technique to measure biomolecules. RESULTS: Case Study 1 demonstrated feasibility of measurement of IgG1 in cerebrospinal fluid at the desired level of sensitivity with minimal cost and timelines of clinical assay implementation. Case Study 2 translated the iPCR protocol to measure multiple IgG1 analytes in cerebrospinal fluid. Case Study 3 demonstrated broad applicability of the technique to yet another analyte IL-6. CONCLUSION: The advantages of our iPCR approach were: lack of reliance on a single vendor for technology platform/software, minimal reliance on proprietary reagents and reduced method development times and cost.
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Imunoensaio/métodos , Reação em Cadeia da Polimerase/métodos , Estudos de Viabilidade , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Interleucina-6/sangueAssuntos
Produtos Biológicos/imunologia , Produtos Biológicos/uso terapêutico , Proteínas/imunologia , Proteínas/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Produtos Biológicos/efeitos adversos , Terapia Biológica/efeitos adversos , Terapia Biológica/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Proteínas/efeitos adversos , Resultado do TratamentoRESUMO
A comprehensive cross-platform and cross-assay evaluation using nine technology platforms and four cytokine immunoassays (IL-6, TNFα, IL-17a, IL-2) was performed by comparing assay precision, sensitivity, parallelism and data correlation between platforms. The precision was acceptable for most evaluated assays. In addition to comparing the analytical assay sensitivity using a spiked recombinant analyte in buffer, forty serum samples from both normal controls and multiple sclerosis patients were used to measure the frequency of endogenous analyte detection (FEAD) as a parameter of each assay's ability to detect the endogenous analyte. The highest FEAD measurements were observed on the Simoa™, Erenna®, Milliplex® and Imperacer® platforms. However, only Simoa and Erenna results showed a high correlation across all evaluated cytokine assays, followed by a more moderate correlation of results across platforms for the V-plex™, high sensitivity ELISA and the Ella™ IL-6 and TNFα assays. In contrast, results from the evaluated cytokine assays on the Milliplex, AMMP™ ViBE® and Imperacer platforms did not correlate to each other nor to other evaluated assays. Acceptable parallelism was observed for the Simoa, Erenna, V-plex and Ella assays but not for the Milliplex, AMMP ViBE and Imperacer assays. In conclusion, the Simoa, Erenna,V-plex and Ella platforms performed well in one or more evaluated cytokine assays. Among those, the Simoa and Erenna assays had the highest sensitivity for detection of cytokines present at sub-pg/mL levels in human serum. In addition, the cross-platform and cross-assay comparisons demonstrated that different immunoassays may yield different results, which underscores the importance of performing such comparative evaluations, especially in the absence of reliable reference standards for the quantitative assessments of biomarkers in immunoassays.
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Citocinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Soro/química , Humanos , Variações Dependentes do Observador , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.
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Anticorpos Monoclonais/imunologia , Anticorpos/análise , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Anticorpos Monoclonais/uso terapêutico , Terapia Biológica , Tolerância a Medicamentos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Imunoensaio/normas , Reação em Cadeia da Polimerase/métodosRESUMO
Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed.
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Anticorpos Monoclonais/sangue , Produtos Biológicos/sangue , Terapia Biológica/métodos , Animais , Anticorpos Monoclonais/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Produtos Biológicos/imunologia , HumanosRESUMO
The purpose of this manuscript is to provide a summary of the evaluation done by the Throughput and Multiplexing subteam on five emerging technologies: Single molecule array (Simoa™), Optimiser™, CyTOF® (Mass cytometry), SQIDLite™, and iLite™. Most of the information is presented with a minimum amount of published data and much is based on discussions with users and the vendor, to help provide the reader with an unbiased assessment of where the subteam sees each technology fitting best in the bioanalysis of large molecules. The evaluation focuses on technologies with advantages in throughput and multiplexing, but is wide enough to capture their strengths in other areas. While all platforms may be suited to support bioanalysis in the discovery space, because of their emergent nature, only Optimiser and SQIDLite are currently ready to be used in the regulated space. With the exception of Optimiser, each instrument/technology requires an up-front investment from the bioanalytical lab that will need justification during capital budget discussions. Ultimately, the platform choice should be driven by the quality of data, project needs, and the intended use of the data generated. In a time- and resource-constrained environment, it is not possible to evaluate all emergent technologies available in the market; we hope that this review gives the reader some of the information needed to decide which technology he/she may want to consider evaluating to support their drug development program in comparison to the options they already have in their hands.
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Ensaios de Triagem em Larga Escala/métodos , Imunoensaio/métodos , Descoberta de Drogas/métodos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/tendências , Imunoensaio/instrumentação , Imunoensaio/tendências , Ligantes , Microfluídica/métodos , Ligação Proteica , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The goal of this article is to discuss the fundamental key questions around parallelism assessments: why to do it, when to do it and how to do it, with consideration for different molecule types and the scientific rationale that drives different approaches. Current practices for both PK and biomarker assays regarding which samples to pick, whether to pool or not pool samples, as well as generally accepted acceptance criteria are discussed, while also highlighting the many outstanding questions that remain. In order to reach the long-term goal of understanding and developing best practices for implementation of parallelism testing for both PK and biomarker assays, industry and regulators will need to keep the conversations going, and commit to generating and reviewing data for the purposes of our own education. Means to easily share data in open forums to facilitate and build common understandings should continue and will be necessary to expedite resolution of many of these questions.