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.
Background: Accurate quantitation of immune markers is critical for ensuring reliable assessment of vaccines efficacy against infectious diseases. This report presents the standardization and harmonization of test results of SARS-CoV-2 assays amongst the initial seven laboratories (North America, Europe and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN) used to support evaluation of COVID-19 vaccine candidates.Methods: Three ELISA (Spike, Receptor Binding Domain and Nucleocapsid), a Microneutralization (MNA), a pseudotyped virus-based neutralization (PNA), and an Interferon gamma (IFN-γ) T-cell ELISpot assays were developed, validated/qualified, and transferred to participating laboratories in accordance with rigorous Quality Control Systems to ensure reliable performance across all laboratories. Findings: High reproducibility of results across all labs was demonstrated with interlaboratory precision of 4.1 to 7.7 % CV for all three ELISAs, 3.8 to 19.5% for PNA and 17.1 to 24.1% for MNA, over a linear range of 11 to 30,760 ELU/mL (ELISAs), 14 to 7,876 NT50/mL (PNA) and 21 to 25,587 ND50/mL (MNA). The MNA was also adapted for detection of neutralizing antibodies against the major SARS-CoV-2 Variants of Concern. The results of the PNA and MNA (R2=0.819) and ELISA and PNA (R2=0.876) were highly correlated. The IFN γ ELISpot interlaboratory variability was 15.9 to 49.9 % CV. Sensitivity and specificity were near 100% for all assays. Interpretation: Statistical evidence suggests these assays are well standardized for providing accurate quantitation of immune responses and are suitable to support Vaccine Clinical trials.Funding: This work was funded by CEPI.Declaration of Interest: All work performed in this study was funded by CEPI under contractual agreements with the respective laboratories. The agreement provides for salaries, materials, equipment and travel as directly required for the conduct of this study. The authors do not receive any royalties, licenses, stocks, stock options, or other financial benefit directly related to this work.Ethical Approval: All human samples used in this study were obtained from either commercial sources of human blood samples or as part of Phase I-III vaccine clinical trials. All samples were collected under Informed Consent in accordance with Regulatory Requirements for Protection of Human Subjects and were tested in a blinded manner, with neither CEPI nor its partner laboratories having any links to individual subject IDs.
The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th 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.Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agencies experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2024 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 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 3) covers in the Part 3A the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 4 and 5 (2025), respectively.
The regulatory control of pertussis vaccines, as for other biological products, requires that they conform to specified standards of safety and efficacy. The current potency test for whole cell vaccines, the intracerebral mouse protection test (AMPT) is still the only such assay that has shown a correlation with protection in children. An alternative in vivo assay based on non-lethal aerosol challenge of mice has been assessed as a replacement for the current AMPT. An in vitro assay based on determination of reactive nitrogen/oxygen intermediates produced as a result of macrophage activation has also been investigated as a potential replacement for the in vivo challenge test. On the other hand, for safety testing, an enzymatic-HPLC coupled assay using a fluorescein-labelled G alpha(i3)C20 peptide to measure the enzymatic ribosylation activity of active pertussis toxin was evaluated for its suitability as a replacement for the current histamine sensitisation test (HIST). An assay for adenylate cyclase toxin (ACT)-related toxicity, based on measuring the ACT-induced oxidative burst in macrophage-like cell cultures has also been investigated. Although some questions still need to be answered in relation to the development of suitable replacements for in vivo tests of pertussis vaccines, the prospects for further improvements are promising.
Brucellosis is a highly contagious bacterial zoonosis that affects millions of people worldwide. The infection induces debilitating and incapacitating severe diseases, with mild to serious complications. The intracellular localization of Brucella spp. makes treatment difficult, since most antibiotics, although effective in vitro, do not actively pass through cellular membranes. Therefore, complete eradication of the microorganisms is difficult to achieve, and the incidence of relapses is rather high. Hence, drug delivery systems such as liposomes and biodegradable microspheres should be most useful for targeting these intracellular sites where the organisms are located. Many examples and promising results of the use of drug delivery systems for the treatment of intracellular infectious diseases already contribute to the literature and demonstrate the great potential of these technologies. In the present manuscript, we review our work on the development of new approaches to an effective pharmaceutical dosage form for the treatment of brucellosis. For this purpose, we prepared and evaluated a number of liposomal and microparticulate formulations for the efficient encapsulation of gentamicin and a competent therapeutic activity towards infected models in vitro and in vivo. Liposomes loaded with gentamicin sulphate eliminated completely intracellular Brucella in infected cultured monocytes-macrophages. In vivo, gentamicin-loaded cationic liposomes produced a protective effect in mice challenged with a lethal dose of B. abortus. Further, gentamicin sulphate microencapsulated into poly(lactide) (PLA) and its copolymers with glycolide (PLGA) provided controlled release of the drug, typically with biphasic profiles, what may represent an advantage over the use of liposomes. In vitro, this biodegradable particulate delivery system was efficiently phagocytosed, induced cell activation and reduced intracellular Brucella infection in cultured monocyte-macrophages. In conclusion, liposomes and biodegradable microspheres loaded with antibiotics appear useful for targeting monocytes and reducing intracellular Brucella infection; these delivery systems should represent a promising alternative approach for the treatment of brucellosis. Keywords: drug delivery systems, gentamicin sulphate, liposome stability
The intrinsic complexity and heterogeneity of therapeutic monoclonal antibodies is built into the biosimilarity paradigm where critical quality attributes are controlled in exhaustive comparability studies with the reference medicinal product. The long-term success of biosimilars will depend on reassuring healthcare professionals and patients of consistent product quality, safety and efficacy. With this aim, the World Health Organization has endorsed the need for public bioactivity standards for therapeutic monoclonal antibodies in support of current controls. We have developed a candidate international potency standard for rituximab that was evaluated in a multi-center collaborative study using participants' own qualified Fc-effector function and cell-based binding bioassays. Dose-response curve model parameters were shown to reflect similar behavior amongst rituximab preparations, albeit with some differences in potency. In the absence of a common reference standard, potency estimates were in poor agreement amongst laboratories, but the use of the candidate preparation significantly reduced this variability. Our results suggest that the candidate rituximab standard can support bioassay performance and improve data harmonization, which when implemented will promote consistency of rituximab products over their life-cycles. This data provides the first scientific evidence that a classical standardization exercise allowing traceability of bioassay data to an international standard is also applicable to rituximab. However, we submit that this new type of international standard needs to be used appropriately and its role not to be mistaken with that of the reference medicinal product.
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