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 (PART 2 - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17th Workshop on Recent Issues in Bioanalysis (17th 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 17th 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 "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.

2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 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 17th 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 "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. 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 16th 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 ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority 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 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. 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 16th 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 ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority 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 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

Mouse models and human islet transplantation sites for intravital imaging.

Human islet transplantations into rodent models are an essential tool to aid in the development and testing of islet and cellular-based therapies for diabetes prevention and treatment. Through the ability to evaluate human islets in an in vivo setting, these studies allow for experimental approaches to answer questions surrounding normal and disease pathophysiology that cannot be answered using other in vitro and in vivo techniques alone. Intravital microscopy enables imaging of tissues in living organisms with dynamic temporal resolution and can be employed to measure biological processes in transplanted human islets revealing how experimental variables can influence engraftment, and transplant survival and function. A key consideration in experimental design for transplant imaging is the surgical placement site, which is guided by the presence of vasculature to aid in functional engraftment of the islets and promote their survival. Here, we review transplantation sites and mouse models used to study beta cell biology in vivo using intravital microscopy and we highlight fundamental observations made possible using this methodology.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

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.

Evaluation of the accuracy of an electronic point-of-care analyzer to quantify blood creatinine concentration in goats.

BACKGROUND: There are no commercially available handheld blood creatinine analyzers validated in goats. OBJECTIVES: The objective of the study was to validate the accuracy of a handheld point-of-care (POC) analyzer (Nova StatSensor) for quantifying blood creatinine concentration in goats. A secondary objective was to compare this POC against a chemistry analyzer to classify goats as normal or having mild or moderate azotemia. ANIMALS: Sixty-three goats admitted to a referral hospital. METHODS: Cross-sectional study. Venous blood was obtained, and creatinine concentration was measured by the POC in duplicate. Plasma was submitted for creatinine determination via the chemistry analyzer (gold standard). RESULTS: A total of 101 blood samples were collected from 63 goats. There was high repeatability for creatinine concentrations obtained by the POC (adjusted R2 = .97, P < .0001). Correlation of POC concentrations with those reported by the chemistry analyzer was moderate (adjusted R2 = .57, P < .0001). When correctly categorizing goats with mild azotemia, the POC demonstrated a sensitivity of 73.3% and a specificity of 88.3%. For moderate to severe azotemia, the POC had a sensitivity of 75.0% and specificity of 97.5%. CONCLUSION AND CLINICAL IMPORTANCE: The Nova StatSensor POC provided above average accuracy for measuring blood creatinine concentration in goats compared with the gold standard test.

Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction.

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105-1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

Maternal Vaccination With a Monocomponent Pertussis Toxoid Vaccine Is Sufficient to Protect Infants in a Baboon Model of Whooping Cough.

Background: Bordetella pertussis is a human pathogen responsible for serious respiratory illness. The disease is most severe in infants too young to be vaccinated with most hospitalizations and deaths occurring within this age group. The Advisory Committee on Immunization Practices recommended immunization of pregnant women to protect infants from birth until their first vaccination at 6-8 weeks of age. We previously demonstrated that maternal vaccination with licensed acellular pertussis vaccines protected newborn baboons from disease. We hypothesized that protection was due to toxin-neutralizing, maternal anti-pertussis toxin antibodies and predicted that maternal vaccination with a pertussis toxoid (PTx)-only vaccine would protect newborns from disease. Methods: Infant baboons born to unvaccinated mothers or mothers vaccinated with a PTx-only vaccine were challenged with B. pertussis at 5 weeks of age and followed for infection and signs of disease. Results: Although all challenged infants were heavily colonized, the infant baboons born to mothers vaccinated with PTx-only vaccine were free from clinical disease following exposure to B. pertussis. In contrast, disease was observed in infants born to unvaccinated mothers. Conclusions: Our results demonstrated that maternal vaccination with a PTx-only vaccine is sufficient to protect newborn baboons from disease following exposure to pertussis.

Towards replacement of the acellular pertussis vaccine safety test: Comparison of in vitro cytotoxic activity and in vivo activity in mice.

Because of the exquisite sensitivity of the murine histamine sensitization test (HIST) in detecting minute amounts of active pertussis toxin (PTx), this animal-based test has been used to assure the safety of acellular pertussis vaccines in the United States and other countries around the world. Prompted by humane considerations, efforts are underway to find a suitable in vitro replacement assay that has critical attributes comparable to that of the HIST. In this study, we compared the sensitivity of the in vivo HIST with an in vitro Chinese Hamster Ovary (CHO) cell-based assay. Using vaccine samples that had been spiked with PTx, we found that both assays were capable of detecting as little as 4-10 ng of active pertussis toxin per dose of vaccine; thus, the sensitivities of the two assays are comparable. Because the strength of adsorption of PTx to the vaccine adjuvant could change over time, we also used both assays to examine the bioavailability of PTx in spiked vaccine samples that had been stored at 25 °C for 9 weeks, mimicking long term vaccine storage conditions. We found that both assays detected similar amounts of active PTx in these samples, indicating that bioavailability of the toxin in stored samples was similar. Taken together, our results indicate that critical attributes of the HIST are met by the CHO cell assay used in this study and provide proof of concept that the CHO cell assay may be further considered as a replacement for the in vivo HIST.

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Role of the N-terminal amino acid of Bacillus anthracis lethal factor in lethal toxin cytotoxicity and its effect on the lethal toxin neutralization assay.

The cytotoxic activity of lethal factor (LF), a critical reagent used in the cell-based lethal toxin neutralization assay to assess anthrax vaccines, was shown to depend on the identity of its N-terminal amino acid, which plays a role in the targeting of LF to the proteasome for degradation. These results demonstrate that care must be taken to ensure that LF preparations used in standardized cell-based assays are not altered at their N-terminal ends.

Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants.

The only US-licensed anthrax vaccine for human use, as well as several experimental vaccines containing solely purified recombinant protective antigen (rPA), are formulated using aluminum hydroxide (Al(OH)3) as an adjuvant. It has been suggested that effective adjuvanticity of aluminum salts for protein antigens depends, at least partially, on the degree of adsorption of the antigen to the adjuvant. On the other hand, the ease of antigen desorption from the adjuvant in a quantitative fashion may facilitate the assessment of vaccine characteristics in the laboratory. In this regard, aluminum phosphate (AlPO4), although deemed a "weaker" adjuvant than Al(OH)3, appears superior to the latter. To investigate the possibility of formulating rPA vaccines with AlPO4, as well as the significance of the adsorption of this antigen to the aluminum salt for adjuvanticity, we studied the effect of AlPO4 and Al(OH)3 on the induction of anti-rPA antibodies in mice. In a first immunization experiment the adjuvanticity of AlPO4 combined with rPA was examined. Antibodies against rPA were measured using an ELISA. Results indicated that AlPO4 is able to significantly increase the antibody response to rPA, irrespective of its degree of adsorption to the adjuvant. Based on these results, in a second experiment mice were immunized twice, with different formulations of rPA containing either AlPO4 or Al(OH)3, and rPA-antibodies were measured using ELISA and an in vitro toxin neutralization assay. Comparable immune responses to rPA were obtained with both aluminum salts. Additionally, results with AlPO4 as adjuvant confirmed that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response induced.