Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry, along with practical procedures for four different types of validation. The first, Basic Protocol 1 (the limited validation protocol), is recommended for research and non-regulated laboratories. Next, Basic Protocol 2) presents a reasonable, fit-for-purpose validation approach appropriate for biopharma and research settings. Basic Protocol 3 addresses the type of validation performed in clinical laboratories for moderate-risk tests developed in house. Finally, Basic Protocol 4 describes the process that should be applied whenever a method is being transferred from one facility to another. All four validation plans follow the fit-for-purpose validation approach, in which the validation parameters are selected based on the intended use of the assay. These validation protocols represent the minimal requirement and may not be applicable for every intended use such as high-risk clinical assays or data to be used as a primary endpoint in a clinical trial. The recommendations presented here are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (CLSI, 2021). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Limited validation Basic Protocol 2: Fit-for-purpose validation for biopharma and research settings Basic Protocol 3: Validation for moderate clinical risk laboratory developed tests Basic Protocol 4: Transfer validation.

The Evolution of Single-Cell Analysis and Utility in Drug Development.

This review provides a brief history of the advances of cellular analysis tools focusing on instrumentation, detection probes, and data analysis tools. The interplay of technological advancement and a deeper understanding of cellular biology are emphasized. The relevance of this topic to drug development is that the evaluation of cellular biomarkers has become a critical component of the development strategy for novel immune therapies, cell therapies, gene therapies, antiviral therapies, and vaccines. Moreover, recent technological advances in single-cell analysis are providing more robust cellular measurements and thus accelerating the advancement of novel therapies.Graphical abstract.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

High-sensitivity flow cytometric assays: Considerations for design control and analytical validation for identification of Rare events.

The current consensus recommendation papers dealing with the unique requirements for the analytical validation of assays performed by flow cytometry address the validation of sensitivity (both analytical and functional) only in general terms. In this paper, a detailed approach for designing and validating the sensitivity of rare event methods is described. The impact of panel design and optimization on the lower limit of quantification (LLOQ) and suggestions for reporting data near, or below, the LLOQ are addressed. This paper serves to provide best practices for the development, optimization, and analytical validation of flow cytometric assays designed to assess rare events. Note that this paper does not discuss clinical sensitivity validation, which addresses the positive and negative predictive value of the test result.

A Cytometrist's Guide to Coordinating and Performing Effective COVID-19 Research.

Cytometry is playing a crucial role in addressing the COVID-19 pandemic. In this commentary-written by a variety of stakeholders in the cytometry, immunology, and infectious disease communities-we review cytometry's role in the COVID-19 response and discuss workflow issues critical to planning and executing effective research in this emerging field. We discuss sample procurement and processing, biosafety, technology options, data sharing, and the translation of research findings into clinical environments. © 2020 International Society for Advancement of Cytometry.

Best practices for optimization and validation of flow cytometry-based receptor occupancy assays.

In the development of therapeutic compounds that bind cell surface molecules, it is critical to demonstrate the extent to which the drug engages its target. For cell-associated targets, flow cytometry is well-suited to monitor drug-to-target engagement through receptor occupancy assays (ROA). The technology allows for the identification of specific cell subsets within heterogeneous populations and the detection of nonabundant cellular antigens. There are numerous challenges in the design, development, and implementation of robust ROA. Among the most difficult challenges are situations where there is receptor modulation or when the target-antigen is expressed at low levels. When the therapeutic molecules are bi-specific and bind multiple targets, these challenges are increased. This manuscript discusses the challenges and proposes best practices for designing, optimizing, and validating ROA.

Flow cytometric method transfer: Recommendations for best practice.

As with many aspects of the validation and monitoring of flow cytometric methods, the method transfer processes and acceptance criteria described for other technologies are not fully applicable. This is due to the complexity of the highly configurable instrumentation, the complexity of cellular measurands, the lack of qualified reference materials for most assays, and limited specimen stability. There are multiple reasons for initiating a method transfer, multiple regulatory settings, and multiple context of use. All of these factors influence the specific requirements for the method transfer. This recommendation paper describes the considerations and best practices for the transfer of flow cytometric methods and provides individual case studies as examples. In addition, the manuscript emphasizes the importance of appropriately conducting a method transfer on data reliability.

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

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.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley & Sons, Inc.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

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.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society.

Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

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.

Saturation monitoring of VX15/2503, a novel semaphorin 4D-specific antibody, in clinical trials.

BACKGROUND: Receptor occupancy, or saturation, assays are often utilized in preclinical and clinical development programs to evaluate the binding of a biologic to a cellular target. These assays provide critical information regarding the dose of drug required to "saturate" the target as well as important pharmacodymamic (PD) data. A flow cytometric method was developed to measure the degree of Semaphorin 4D (SEMA4D; CD100) saturation by VX15/2303, an investigational monoclonal antibody specific for SEMA4D. METHODS: The assay detects VX15/2503, a human IgG4 specific for SEMA4D, with an IgG4 -specific monoclonal antibody. RESULTS: Data generated allowed assessment of two related SEMA4D-specific pharmacodynamic (PD) markers: (1) The measurement of cellular SEMA4D (cSEMA4D) saturation by VX15/2503, and (2) the cell membrane expression levels of cSEMA4D. CONCLUSIONS: This assay specifically and reproducibly measured cSEMA4D saturation and expression levels. Evaluation of the SEMA4D-specific PD markers were critical in determining the clinical saturation threshold of cSEMA4D by VX15/2503.