Following up on our most recent discussion paper focusing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what?) and another on the practical considerations (i.e., the how). In this paper, we discuss 'the what'. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

Following up on our most recent discussion paper focussing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated Bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what) and another on the practical considerations (i.e., the how). In this paper, we discuss 'the how'. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

Within the bioanalytical community, the use of blank matrix from preclinical animals for bioanalytical method validation and sample analysis is common practice and required in the context of guidelines for bioanalytical method validation. At the same time, its use has been challenged by the scientific community for decades, since there is ample scientific evidence to allow the use surrogate matrices for this purpose. Nevertheless, legacy and current regulatory thinking continues to be reluctant to allow the use of surrogate matrices in bioanalytical testing except for so-called rare matrices. As part of ongoing discussions in relation to the ICH M10 Guideline, the European Bioanalysis Forum re-challenges the unnecessary use of blank matrices from preclinical animals and believes that, as part of community responsibility and ethical standards and when supported by data, the use of surrogate matrices should become widely accepted. It is in this context that targeted experiments were conducted within the European Bioanalysis Forum to gather additional data and re-open the discussions with all involved and that it should become acceptable to use surrogate matrices wherever possible.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

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 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The 14 th edition of the Workshop on Recent Issues in Bioanalysis (14 th 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 14 th 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, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) 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 covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.