2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines ( - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17 Workshop on Recent Issues in Bioanalysis (17 WRIB) took place in Orlando, FL, USA on 19-23 June 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 17 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.

Development of a Prototype Immunohistochemistry Assay to Measure Programmed Death Ligand-1 Expression in Tumor Tissue.

Context: - With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation.

Prognostic Significance of PD-L1 in Patients with Non-Small Cell Lung Cancer: A Large Cohort Study of Surgically Resected Cases.

Introduction: The aim of our analysis was to evaluate the prognostic effect of programmed cell death ligand-1 (PD-L1) expression in patients with non-small cell lung cancer (NSCLC).

Methods: PD-L1 expression among 1070 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Data were analyzed using Cox proportional hazard models adjusting for age, sex, smoking status, histologic type, stage, and performance status.

Influence of Pre-Analytical Factors on Thymus- and Activation-Regulated Chemokine Quantitation in Plasma.

Thymus- and activation-regulated chemokine (TARC) in serum/plasma associates with the disease activity of atopic dermatitis (AD), and is a promising tool for assessing the response to the treatment of the disease. TARC also exists within platelets, with elevated levels detectable in AD patients. We examined the effects of pre-analytical factors on the quantitation of TARC in human EDTA plasma.

A clinical biomarker assay to quantitate thymic stromal lymphopoietin in human plasma at sub-pg/ml level.

Background: Thymic stromal lymphopoietin (TSLP) is an attractive therapeutic target for the treatment of allergic diseases, and plasma TSLP is a potential patient selection marker in the development of therapeutic agents.

Results: We developed and validated an ultrasensitive electrochemiluminescence assay for measurement of TSLP in plasma with a lower limit of quantitation of 0.12 pg/ml, which allowed the quantitation of TSLP in approximately 90% of human plasma samples tested.

Workshop report: Crystal City V--quantitative bioanalytical method validation and implementation: the 2013 revised FDA guidance.

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion.

Development and fit-for-purpose validation of a LC-MS/MS assay for fibrinogen peptide A quantitation in human plasma.

Background: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma.

A candidate plasma protein classifier to identify Alzheimer's disease.

Biomarkers currently used in the aid for the diagnosis of Alzheimer's disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing.

Navigating the rapids: the development of regulated next-generation sequencing-based clinical trial assays and companion diagnostics.

Over the past decade, next-generation sequencing (NGS) technology has experienced meteoric growth in the aspects of platform, technology, and supporting bioinformatics development allowing its widespread and rapid uptake in research settings. More recently, NGS-based genomic data have been exploited to better understand disease development and patient characteristics that influence response to a given therapeutic intervention. Cancer, as a disease characterized by and driven by the tumor genetic landscape, is particularly amenable to NGS-based diagnostic (Dx) approaches.

An ultra-sensitive clinical biomarker assay: quantitation of thymus and activation-regulated chemokine in human plasma.

Background: Thymus and activation-regulated chemokine (TARC) is a Th2 type, pro-allergic secreted chemokine. TARC in plasma/serum has been proposed as a marker for disease activity of atopic dermatitis (AD) and as a pharmacodynamic readout in the clinical development of novel agents for the treatment of AD.

Results: An ultra-sensitive electrochemiluminescence assay for TARC in human plasma was developed and analytically validated.

Practical guidance for implementing predictive biomarkers into early phase clinical studies.

The recent U.S. Food and Drug Administration (FDA) coapprovals of several therapeutic compounds and their companion diagnostic devices (FDA News Release, 2011, 2013) to identify patients who would benefit from treatment have led to considerable interest in incorporating predictive biomarkers in clinical studies.

Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation.

Development and validation of an ultra-sensitive method for the measurement of plasma renin activity in human plasma via LC-MS/MS.

Background: Renin catalyzes the conversion of angiotensinogen to angiotensin I (Ang I), the first and rate-limiting step in the renin-angiotensin-aldosterone system. Plasma renin activity (PRA) is an important target engagement biomarker in the clinical development of renin inhibitors. We have developed and validated an improved PRA assay that incorporates an Ang I trapping antibody followed by extraction and quantification of Ang I using a highly sensitive and specific LC-MS/MS method.

2012 white paper on recent issues in bioanalysis and alignment of multiple guidelines.

Over 400 professionals representing pharmaceutical companies, CROs, and multiple regulatory agencies participated in the 6th Workshop on Recent Issues in Bioanalysis (WRIB). Like the previous sessions, this event was in the format of a practical, focused, highly interactive and informative workshop aiming for high-quality, improved regulatory compliance and scientific excellence. Numerous 'hot' topics in bioanalysis of both small and large molecules were shared and discussed, leading to consensus and recommendations among panelists and attendees representing the bioanalytical community.

Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies.

High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10-3,000 ng/mL.

2011 White paper on recent issues in bioanalysis and regulatory findings from audits and inspections.

The 5th Workshop on Recent Issues in Bioanalysis (WRIB) was organized by the Calibration and Validation Group as a 2-day full immersion workshop for pharmaceutical companies, CROs and regulatory agencies to discuss, review, share perspectives, provide potential solutions and agree upon a consistent approach to recent issues in the bioanalysis of both small and large molecules. High quality, better compliance to regulations and scientific excellence are the foundation of this workshop. As in the previous editions of this significant event, recommendations were made and a consensus was reached among panelists and attendees, including industry leaders and regulatory experts representing the global bioanalytical community, on many 'hot' topics in bioanalysis.

Translational biomarkers: from preclinical to clinical a report of 2009 AAPS/ACCP Biomarker Workshop.

There have been some successes in qualifying biomarkers and applying them to drug development and clinical treatment of various diseases. A recent success is illustrated by a collaborative effort among the US Food and Drug Administration, the European Medicines Agency, and the pharmaceutical industry to provide a set of seven preclinical kidney toxicity biomarkers for drug development. Other successes include, but are not limited to, clinical biomarkers for cancer treatment and clinical management of heart transplant patients.

Characterization and development of a Luminex(®)-based assay for the detection of human IL-23.

Background: IL-23 is a cytokine produced by dendritic cells, T-cells and macrophages that plays a critical regulatory role in the inflammatory and autoimmune responses. We describe the development and preclinical validation of a highly sensitive Luminex(®) assay specific to IL-23 that is suitable for its measurement in support of early-phase clinical trials.

Results: Intra-assay precision for the BioSource™ ELISA was under 12.

Electrochemiluminescence in bioanalysis.

The discovery of electrochemiluminescence (ECL) and its development as a means of detection is truly a success story. Although studies describing ECL were published in the early 1960s, most studies using ECL as a means of detection were not widely published until the mid 1990s. Incorporating ECL into assays provides increased sensitivity, several logs of dynamic range and the ability to electronically control the reaction.

Immunoassay-based measurement of clinical biomarkers for monitoring changes in nasal cavity.

Background: Many drugs for treatment of allergies, migraine headaches, inflammation, and other indications are administered into the nasal cavity providing access to the immune and central nervous systems. One of the concerns for using this route of administration is potential damage to the nasal epithelium and mucosal regions. We assembled a panel of clinical biomarkers that can be used to monitor changes in the nasal epithelium, mucosa, and olfactory regions in preparation for clinical trials involving drugs administered via intranasal route.

Workshop report and follow-up--AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples--implications of Crystal City recommendations.

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR.

Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions.

Fit-for-purpose method development and validation for successful biomarker measurement.

Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate.

Bioanalytical method validation for macromolecules in support of pharmacokinetic studies.

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.

Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules.

Purpose: With this publication a subcommittee of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) makes recommendations for the development, validation, and implementation of ligand binding assays (LBAs) that are intended to support pharmacokinetic and toxicokinetic assessments of macromolecules.

Methods: This subcommittee was comprised of 10 members representing Pharmaceutical, Biotechnology, and the contract research organization industries from the United States, Canada, and Europe. Each section of this consensus document addresses a specific analytical performance characteristic or aspect for validation of a LBA.