Biomarker context-of-use: how organizational design can impact the implementation of the appropriate biomarker assay strategy.

Since 2011, the European Bioanalysis Forum has been discussing the topic of context-of-use for biomarker assays, in support of a cross-industry implementation of its principles. The discussions have led to the acknowledgement of the challenges that we face as an industry in implementing these principles. In addition to scientific recommendations, the European Bioanalysis Forum has addressed these challenges by providing recommendations on organizational design, and what works in both sponsor and contract research organizations, to support and enable context-of-use across biomarker strategies. Here, we highlight the key considerations for organizational design to help ensure that biomarker assays are characterized and validated according to the right context-of-use, to ensure that the right decisions based on the biomarker data can be made during drug development.

Insulin detemir is characterized by a more reproducible pharmacokinetic profile than insulin glargine in children and adolescents with type 1 diabetes: results from a randomized, double-blind, controlled trial.

Insulin detemir (detemir) has previously been shown to be associated with lower within-subject variability compared with other basal insulin preparations in adults with type 1 diabetes mellitus (T1DM). This randomized, double-blind, crossover trial compared the within-subject variability of detemir and insulin glargine (glargine) in pharmacokinetic properties in children and adolescents with T1DM. The trial enrolled 32 children and adolescents (19 girls and 13 boys; mean +/- SD: age 13 +/- 2.5 yr and T1DM duration 6.3 +/- 3.0 yr) with a hemoglobin A1c (HbA1c) of 7.9 +/- 1.0%. Participants were randomized to a specific treatment sequence in which a dose of 0.4 U/kg of detemir and glargine was injected subcutaneously 24 h apart at each of two dosing visits. Insulin concentrations were measured at frequent intervals for a period of 16-h post-dosing. Detemir showed statistically significantly less within-subject variability compared with glargine with a 3.1-fold and 2.9-fold lower coefficient of variation (CV, %) for the area under the concentration-time curve [AUC((0-16) (h))] and the maximum concentration (C(max)), respectively. Separate analyses demonstrated a 2.5-fold and 2.9-fold lower CV (%) with detemir in children (8-12 yr) and a 4-fold and 3.8-fold lower CV (%) with detemir in adolescents (13-17 yr). No safety concerns were raised during the trial. In conclusion, within-subject variability in pharmacokinetic properties was significantly lower for detemir than for glargine in children and adolescents with T1DM. This indicates a less variable absorption with detemir, which is expected to be associated with a more predictable therapeutic effect also in this population.

EBF recommendation on practical management of critical reagents for PK ligand-binding assays.

Critical reagents play a crucial role in ligand-binding assays; the robustness and reliability of an assay is defined by the quality and long-term availability of these reagents. However, neither regulatory guidelines nor relevant scientific papers provide clear directions for set-up, life cycle management and, more importantly, the acceptance criteria required for the testing of the critical reagents for pharmacokinetic, biomarker and immunogenicity assays. The ambiguity from current guidelines can be a challenge for the bioanalytical community. Members of the European Bioanalysis Forum community undertook a more pragmatic approach on how to assess the impact of critical reagents. In this paper, a review and corresponding gap analysis of the current guidelines and relevant papers will be provided as well as decision trees proposed for lot-to-lot changes of critical reagents for pharmacokinetic assays.

Feedback from the European Bioanalysis Forum: focus workshop on current analysis of immunogenicity: best practices and regulatory hurdles.

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.

Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

Addressing the challenges of biomarker calibration standards in ligand-binding assays: a European Bioanalysis Forum perspective.

The analysis of biomarkers by ligand-binding assays offers significant challenges compared with the bioanalysis of small and large molecule drugs. The presence of endogenous analyte is a commonly cited issue. Also the sourcing and application of appropriate calibration or reference standards can present many issues. One of the main challenges is ensuring the continuity and validity of biomarker data when the source or lot number of calibration standard changes within or between studies. Several strategies exist in attempting to deal with this and standardize the biomarker data through the assay life or looking for ways to compare and normalize biomarker data. In this manuscript, the European Bioanalysis Forum view on dealing with calibration standards in biomarker assays is described.

C5a and C5aR are elevated in joints of rheumatoid and psoriatic arthritis patients, and C5aR blockade attenuates leukocyte migration to synovial fluid.

Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study was to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis-both with respect to expression and function. Synovial fluid, blood and synovial samples were obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue were analysed by immunohistochemistry and flow cytometry. SF and blood were analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration was determined using a Boyden chamber. Appropriate statistical tests were applied for comparisons. C5aR+ cells were detected in most rheumatoid arthritis, in all psoriatic arthritis, but not in non-inflammatory control synovia. C5aR+ cells were primarily neutrophils and macrophages. C5aR+ macrophages were mainly found in lymphoid aggregates in close contact with T cells. C5a levels were increased in both rheumatoid arthritis and psoriatic arthritis synovial fluid compared to osteoarthritis, and in blood from rheumatoid arthritis compared to healthy subjects. Neutrophil and monocyte migration to rheumatoid arthritis synovial fluid was significantly inhibited by anti-C5aR. The data support that the C5a-C5aR axis may be driving the infiltration of inflammatory cells into the synovial fluid and synovium in both rheumatoid and psoriatic arthritis, and suggest that C5a or C5aR may be a promising treatment target in both diseases.

Tiered approach into practice: scientific validation for chromatography-based assays in early development - a recommendation from the European Bioanalysis Forum.

The principles of tiered approach have been part of the bioanalytical toolbox for some years. Nevertheless, an in spite of many valuable discussions in industry, they remain difficult to apply in a harmonized way for a broad array of studies in early drug development where these alternative approaches to regulated validation would make sense. The European Bioanalysis Forum has identified the need to proposes some practical workflows for five categories of studies for chromatography based assays where scientific validation will allow additional freedom while safeguarding scientific rigor and robust documentation: quantification of metabolites in plasma in relation to ICH M3(R2), urine analysis, tissue homogenate analysis, and preclinical and clinical studies in early stages of drug development. The recommendation would introduce a common language and harmonized best practice for these study categories and can help to refocus towards optimized scientific and resource investments for bioanalysis in early drug development.

Clinical pharmacokinetics of the anti-interleukin-20 monoclonal antibody NNC0109-0012 in healthy volunteers and patients with psoriasis or rheumatoid arthritis.

INTRODUCTION: NNC0109-0012, a novel human monoclonal antibody that binds to and neutralizes the activity of interleukin-20, was investigated as a potential treatment for inflammatory diseases. Pharmacokinetic (PK) modeling was performed using data from four completed clinical phase 1/2 trials to better understand the clinical PK of NNC0109-0012. METHODS: The populations included were patients with rheumatoid arthritis (RA), chronic plaque psoriasis, and healthy volunteers. NNC0109-0012 was administered subcutaneously at various dose levels (0.01-3 mg/kg) as single dose, once weekly, or multiple doses every second week for up to 12 doses. Noncompartmental methods were used to describe the PK parameters. Population PK was analyzed using nonlinear mixed-effects modeling, with body weight as the main covariate and gender, age, and population as additional covariates. RESULTS: Across studies (N = 116), mean age and body weight ranged from 38 to 58 years and 72 to 96 kg, respectively. NNC0109-0012 displays linear PK. Time to maximum plasma concentration occurred at approximately 1 week, and the terminal half-life was approximately 3 weeks. Clearance and volume of distribution increased proportionally to body weight. No difference in clearance or volume of distribution was observed between gender or different age groups; however, clearance was slightly lower in healthy volunteers than in patients with RA. CONCLUSION: The PK profile of NNC0109-0012 is similar to other monoclonal antibodies directed against soluble targets.

Emerging technologies to increase ligand binding assay sensitivity.

Ligand binding assays (LBAs) have been the method of choice for protein analyte measurements for more than four decades. Over the years, LBA methods have improved in sensitivity and achieved larger dynamic ranges by using alternative detection systems and new technologies. As a consequence, the landscape and application of immunoassay platforms has changed dramatically. The introduction of bead-based methods, coupled with single molecule detection standardization and the ability to amplify assay signals, has improved the sensitivity of many immunoassays, in some cases by several logs of magnitude. Three promising immunoassay platforms are described in this article: Single Molecule Counting (SMC™) from Singulex Inc, Single Molecule Arrays (Simoa™) from Quanterix Corporation, and Immuno-PCR (Imperacer®) from Chimera Biotec GmbH. These platforms have the potential to significantly improve immunoassay sensitivity and thereby address the bioanalytical needs and challenges faced during biopharmaceutical drug development.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3--LBA, biomarkers and immunogenicity).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 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 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Synthesis and structure-activity relationship of aminobenzophenones. A novel class of p38 MAP kinase inhibitors with high antiinflammatory activity.

We wish to report the synthesis and structure-activity relationship (SAR) of a series of 4-aminobenzophenones, as a novel compound class with high antiinflammatory activity. Our initial lead, (4-[(2-aminophenyl)amino]phenyl)(phenyl)methanone (3), was systematically optimized and resulted in compounds that potently inhibited the release of the proinflammatory cytokines IL-1beta and TNF-alpha in human peripheral blood mononuclear cells stimulated by LPS. One of the most potent compounds, among others, was (4-[(2-aminophenyl)amino]-2-chlorophenyl)(2-methylphenyl)methanone (45) with IC(50) values of 14 and 6 nM for the inhibition of IL-1beta and TNF-alpha, respectively. Furthermore, we found these types of compounds to be potent and selective p38 MAP kinase inhibitors, e.g. 45 had an IC(50) value of 10 nM. Molecular modeling was used to rationalize our SAR data and to propose a model for the interaction of compound 45 with the p38 MAP kinase. The model involved a favorable hydrogen bond between the carbonyl group of the benzophenone and the NH of Met-109, positioning ring A in the hydrophobic pocket I of the enzyme. Good antiinflammatory effects were demonstrated in two murine models of dermatitis after topical application (oxazolone and TPA model).

How the bioanalytical scientist plays a key role in interdisciplinary project teams in the development of biotherapeutics - a reflection of the European Bioanalysis Forum.

The bioanalytical scientist plays a key role in the project team for the drug development of biotherapeutics from the discovery to the marketing phase. Information from the project team members is required for assay development and sample analysis during the discovery, preclinical and clinical phases of the project and input is needed from the bioanalytical scientist to help data interpretation. The European Bioanalysis Forum target team 20 discussed many of the gaps in information and communication between the bioanalytical scientist and project team members as a base for providing a perspective on the bioanalytical scientist's role and interactions within the project team.

Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2.

Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-alpha protein expression, but similar TNF-alpha mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-alpha expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1beta caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-alpha protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1beta. This increase in TNF-alpha protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-alpha, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-alpha protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.