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The fibronectin (FN) isoform including the extradomain B (EDB) segment (EDB + FN) is a promising tumor target and is highly expressed in some tumor types, such as breast, head, and neck cancer. To date, mostly immunohistochemistry (IHC) and Western blot have been used for the analysis of EDB + FN. However, complete quantitative measurements of EDB + FN expression in a tumor and circulation are important for the development of anti-EDB therapeutics. To this end, a method using protein enrichment followed by online antipeptide antibody enrichment coupled with a nanoflow LC-MS/MS was developed to quantify EDB + FN in human and cynomolgus plasma, patient-derived xenograft (PDX) tumors, and PDX formalin-fixed paraffin-embedded (FFPE) samples. Mouse plasma EDB + FN was analyzed using a protein immunoaffinity method followed by nanoflow LC-MS/MS. EDB + FN concentrations were 63.1 pmol/g in PDX breast cancer tumor and 49.6 pmol/g in PDX head and neck tumor. Mean plasma concentration was 1.1 nM (pmol/mL, 47.4 ng/mL) in normal healthy humans and 0.35 nM (15.1 ng/mL) in naive cynomolgus. The assay sensitivity was 0.018 nM based on calibration with recombinant human EDB + FN (rhEDB + FN).

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Purpose A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. Procedures NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. Results The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. Conclusion Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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, GeneCell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay ComparabilityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

The 14

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

Catch a Glimpse of Me is an ongoing project that uses video to help staff deliver more person-centered care for people with dementia living in long-term care. Focus groups consisting of residents, family and staff members were conducted to develop a template for the development of the videos. The five themes they identified as being important to include are: family; interests and hobbies; memories and moments; life space and getting personal. The article describes the process of developing the videos and discusses the ongoing potential of the Catch a Glimpse of Me project.

A mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model was used for preclinical to clinical translation of inotuzumab ozogamicin, a CD22-targeting antibody-drug conjugate (ADC) for B cell malignancies including non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL). Preclinical data was integrated in a PK/PD model which included (1) a plasma PK model characterizing disposition and clearance of inotuzumab ozogamicin and its released payload N-Ac-γ-calicheamicin DMH, (2) a tumor disposition model describing ADC diffusion into the tumor extracellular environment, (3) a cellular model describing inotuzumab ozogamicin binding to CD22, internalization, intracellular N-Ac-γ-calicheamicin DMH release, binding to DNA, or efflux from the tumor cell, and (4) tumor growth and inhibition in mouse xenograft models. The preclinical model was translated to the clinic by incorporating human PK for inotuzumab ozogamicin and clinically relevant tumor volumes, tumor growth rates, and values for CD22 expression in the relevant patient populations. The resulting stochastic models predicted progression-free survival (PFS) rates for inotuzumab ozogamicin in patients comparable to the observed clinical results. The model suggested that a fractionated dosing regimen is superior to a conventional dosing regimen for ALL but not for NHL. Simulations indicated that tumor growth is a highly sensitive parameter and predictive of successful outcome. Inotuzumab ozogamicin PK and N-Ac-γ-calicheamicin DMH efflux are also sensitive parameters and would be considered more useful predictors of outcome than CD22 receptor expression. In summary, a multiscale, mechanism-based model has been developed for inotuzumab ozogamicin, which can integrate preclinical biomeasures and PK/PD data to predict clinical response.

Seema Kumar is a Principal Scientist at Pfizer. She leads a group that provides regulated bioanalytical support including assay development, validation and sample analysis for the PK and immunogenicity assessment for preclinical and clinical development of Pfizer's biotherapeutics portfolio. She is also responsible for scientific oversight of regulated studies outsourced at CROs. Prior to Pfizer, Dr Kumar held a similar role as Director of CLIA certified Clinical Bioanalytical Laboratory at XBiotech USA, Inc. She holds a PhD in Biophysical Chemistry from Johns Hopkins University, and has published several publications in peer-reviewed journals, and contributed to book chapters. The objective of antibody–drug conjugate (ADC) bioanalysis at different stages of drug development may vary and so are the associated bioanalytical challenges. While at early drug discovery stage involving candidate selection, optimization and preliminary nonclinical assessments, the goal of ADC bioanalysis is to provide PK, toxicity and efficacy data that assists in the design and selection of potential drug candidates, the late nonclinical and clinical drug development stage typically involves regulated ADC bioanalysis that delivers TK data to define and understand pharmacological and toxicological properties of the lead ADC candidate. Bioanalytical strategies and considerations involved in developing successful ligand binding assays for ADC characterization from early discovery to late nonclinical stages of drug development are presented here.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.

Bio-layer interferometry (BLI) is a label-free technology that can be used for kinetic characterization of proteins. Although other label-free platforms have been used for quantitation purposes (most notably surface plasmon resonance), little work has been done using BLI. Here we present rationale and strategies for the development and analytical qualification of a BLI assay for the quantitation of a humanized antibody therapeutic in cynomolgus monkey plasma. Results of the qualification were compared to those of a validated ELISA used to quantitate the same therapeutic. Selectivity, matrix effect, and precision and accuracy were similar between the two methods. Target interference was more pronounced in the BLI assay compared to the ELISA. The main difference between the two assays was in the dynamic range (0.1–10 μg/mL for ELISA vs. 0.4–50 μg/mL for BLI). The monkey plasma BLI assay was applied to rat plasma for the comparison of study samples generated in the same matrix by ELISA. A direct quantitation comparison of sample results for the two methods shows a high degree of agreement (r2 = 0.979, slope = 1.017). However, an evaluation of low concentration samples showed a bias of over-recovery in the BLI compared to the ELISA. In addition to utilizing the quantitative capabilities of the platform, we evaluated the utility of using the kinetic properties of the quantitative assay to detect anti-drug antibodies (ADA) and illustrated the potential for ADA to cause either over recovery (non-neutralizing ADA) or under recovery (neutralizing ADA) of a biotherapeutic using the BLI assay.

We propose convergent plasma lenses, possibly from current sheets, as a generic solution to strong interstellar scattering. These lenses resolve the overpressure problem by geometric alignment as noted by Goldreich and Shridhar (2006). They further quantitatively explain properties of extreme scattering events, and pulsar parabolic arcs. This model makes quantitative predictions testable by VLBI on scattering events. It differs conceptually from previous models by occurring through rare, localized underdense sheets. Such sheets are thermally and kinematically stable, and could be consequences of reconnection. The apparent diffractive effects are a result of coherent interference of refractive images. We propose that these lenses can be used for precision distance determination to pulsars, enabling accurate gravity source localization.

Most therapeutic monoclonal antibodies are designed to bind a specific antigen to elicit pharmacological effects. Accurate quantification of a therapeutic monoclonal antibody in biological matrices is essential for assessing its pharmacokinetics and selecting an effective dosing regimen. Therapeutic antibodies may exist in free, partially bound and fully bound forms in the bloodstream. The choice of which form(s) to measure and how to measure them is gaining much attention with the increase in the number of soluble therapeutic targets. This article will review the bioanalytical methods used in supporting the clinical development of the US FDA-approved therapeutic monoclonal antibodies and also discuss how different factors, such as assay format, target and antibody concentrations, and sample dilutions, can have an impact on the measurement of each form of antibody. Appreciation of which form of drug is being measured and what factors may impact measurement under different conditions are important for interpretation of the pharmacokinetics of therapeutic antibodies.

Although weight concerns and smoking are related, anorexic and bulimic women, both of whom have elevated weight concerns, have significant differences in smoking status. Fewer anorexic women smoke, compared with bulimic women, suggesting that weight concerns do not fully explain smoking status. This study investigated the contribution of one factor, cognitive style, to differences in smoking status among college women with different levels of weight concerns: Anorexic tendencies (n = 47), bulimic tendencies (n = 62), weight concerns (n = 56), and no weight concerns (n = 76). Nearly 47% of women with bulimic tendencies, 37.5% of women with weight concerns, and 27.7% of women with anorexic tendencies were current smokers. A total of 22% of women without weight concerns were current smokers. A logistic regression model revealed that race, age, the Personal Standards subscale of the Frost Multidimensional Perfectionism Scale, the Self-Control and Self-Esteem subscale of the Brief Mizes Anorectic Cognitions Questionnaire, and membership in the bulimic tendencies category were significant independent predictors of smoking status. Both weight concerns and cognitive-style variables, including perfectionism and self-control, carried "weight" in the prediction of smoking among college women. In light of these findings, treatment research should explore both the behavioral and cognitive factors associated with weight-concerned college smokers.

Objective Ankylosing spondylitis (AS) is a progressive disease in which chronic inflammation can lead to extensive new bone formation throughout the spine. At present, few measures of the activity or extent of the disease are available. In this study, we sought to determine whether markers of cartilage synthesis and degradation could provide such quantitative measures. Methods Serum samples from 23 patients receiving infliximab treatment for AS were obtained at baseline and at weeks 2, 6, 14, and 22. Patients were stratified with respect to joint involvement and baseline levels of inflammatory markers, and responders were defined according to the Assessments in Ankylosing Spondylitis 20% criteria. Serial measurements of interferon-γ, tumor necrosis factor α, transforming growth factor β (TGFβ), interleukin-10 (IL-10), and IL-1 were done at each time point. The following biomarkers were measured by enzyme-linked immunosorbent assay: the proteoglycan aggrecan 846 epitope, a marker of cartilage turnover; C-propeptide of type II collagen (CPII), a biosynthesis marker; and the Col2-3/4long mono (C2C) and Col2-3/4short (C1–2C) neoepitopes, reflecting collagen cleavage of type II collagen and type I/type II collagen, respectively. Results At baseline, patients with AS demonstrated significant elevations in serum levels of CPII, the 846 epitope, and the CPII-to-C2C (CPII:C2C) ratio (but not C2C or C1–2C) compared with normal controls. Of the biomarkers examined, only CPII:C2C showed a correlation with the C-reactive protein (CRP) level. Among the biomarker–cytokine relationships, TGFβ demonstrated a trend toward a positive correlation with the 846 epitope. Conclusion In AS, elevated serum levels of CPII and the 846 epitope may be related to biosynthetic turnover of hyaline cartilage and the intervertebral discs but may also reflect progressive bone formation as a result of endochondral ossification. The correlation of the CPII:C2C ratio with CRP suggests that the CPII:C2C ratio might prove to be a useful marker of disease activity in AS.

This paper uses results of two research projects investigating the pressures on volunteers in UK sport to illustrate the implications of partnerships between national governing bodies of sport and the central government. National governing bodies of sport receive funding from the central government via Sport England and UK Sport. This funding has conditions attached. The conditions will affect volunteers at the national level of the NGB, but will also cascade down to volunteers in the sports clubs. Thus, they add to the complexity and scale of tasks performed by club level volunteers, who require additional support to perform them. For most NGBs, this external funding is a very significant proportion of their income, so it may have a corresponding impact on the development of the sports and the work of volunteers. The conditions attached to support can be understood in the broader context of a professionalisation of sport in the voluntary sector. As such, the pressures from the central government ...

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

This article shows how the concept of a recruitment niche can be valuable in understanding the difficulties the Guide Association in the United Kingdom has in recruiting new volunteers. Understanding Guiding as career volunteering, within serious leisure, shows how the distinctive ethos of the existing volunteers contributes to the social construction of the recruitment niche. The defining boundaries of the niche restrict the ability to recruit new volunteers. Thus the article gives an example of how a recruitment niche for a voluntary organization can be defined using the socially constructed ethos of volunteers involved in career volunteering rather than by characteristics such as level of educational attainment. It also demonstrates the implications of this for voluntary organizations wishing to increase recruitment.

Author for correspondence: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Eastern Point Rd, Groton, CT 06340, USA Tel.: +1 860 715 0641 Fax: +1 860 715 9501 E-mail: tracey.clark@pfizer.com Xiaogang Han Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Lindsay King Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Frank Barletta Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer, NY, USA Antibody–drug conjugate (ADC) therapeutics utilize the specificity of monoclonal antibodies (mAbs) and potency of highly toxic small molecules. ADCs are typically composed of a mAb with a cytotoxin conjugated to it, resulting in a heterogeneous mixture of mAb with various numbers of toxins. Due to this heterogeneity, characterized by the therapeutic drug-to-antibody ratio (DAR), the selection of bioanalytical tools, biomeasure assays and analytes used to understand and develop ADCs can be challenging [1]. Since the therapeutic has both largeand small-molecule components, one can use bioanalytical tools in both spaces. Questions for ADC programs are typically: what should be measured, when, and by which method? Since all assays have limitations [1], a combination of bioanalytical tools is typically used to understand the ADC in vitro/in vivo, understand payload delivery to the site of action and to establish an exposure–response relationship.

Objective To investigate whether short-term changes in serum biomarkers of type II collagen degradation (C2C) and types I and II collagen degradation (C1,2C), as well as the biomarker for the synthesis of type II procollagen (CPII) can predict radiographic progression at 1 year following initiation of biologic therapy in patients with inflammatory arthritis. Methods Serum levels of biomarkers were measured at baseline and at 1, 3, 6, 9, and 12 months after initiation of biologic therapy. A composite score reflecting changes from baseline in all 3 biomarkers (ΔCOL) was calculated. Associations with clinical responses according to the 28-joint count Disease Activity Score and with radiographic progression according to the modified Sharp/van der Heijde score (SHS) were assessed. Results The 1-year increase in the SHS correlated with the 1-month change in C2C results (r = 0.311, P = 0.028) and the ΔCOL score (r = 0.342, P = 0.015). Radiographic progression was predicted by increases in serum C2C at 1 month (P = 0.031). The ΔCOL score was significantly associated with 1-year radiographic progression after 1 (P = 0.022), 3 (P = 0.015), 6 (P = 0.048), and 9 (P = 0.019) months of therapy. Clinical remission was predicted by 1-month decreases in serum levels of C2C (P = 0.008) and C1,2C (P = 0.036). By regression analysis, 1-month changes in C2C, C1,2C, and CPII levels were independently associated with, and correctly predicted radiographic outcome in, 88% of the patients. Conclusion Short-term changes in serum levels of collagen biomarkers following initiation of biologic therapy may better predict long-term clinical and radiographic outcomes. These collagen biomarkers may therefore be valuable new early indicators of short-term biologic treatment efficacy in clinical trials and in individual patients with inflammatory erosive arthritis.