Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

Lack of analytical interference of dydrogesterone in progesterone immunoassays.

OBJECTIVES: Progesterone, a sex steroid, is measured in serum by immunoassay in a variety of clinical contexts. One potential limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Dydrogesterone (DYD), an orally active stereoisomer of progesterone, is used for various indications in women's health. Herein, we report a systematic in vitro investigation of potential interference of DYD and its active metabolite 20α-dihydrodydrogesterone (DHD) in seven widely used, commercially available progesterone assays. METHODS: Routine human plasma samples were anonymized and pooled to create three graded concentration levels of progesterone (P4 high, P4 medium, P4 low). Each pooled P4 plasma sample (6-7 mL) was spiked at high, medium, and "none" concentration with DYD/DHD and was divided into 0.5 mL aliquots. The blinded aliquots were analyzed by seven different laboratories with their routine progesterone assay (six different immunoassays and one liquid chromatography-tandem mass spectrometry assay, respectively) within the Dutch working group on endocrine laboratory diagnostics of the Dutch Foundation for Quality Assessments in Medical Laboratories. RESULTS: The sample recovery rate (P4 result obtained for sample spiked with DYD/DHD, divided by the result obtained for the corresponding sample with no DYD/DHD × 100) was within a ±10% window for the medium and high P4 concentrations, but more variable for the low P4 samples. The latter is, however, attributable to high inter- and intra-method variability at low P4 concentrations. CONCLUSIONS: This study does not indicate any relevant interference of DYD/DHD within routinely used progesterone assays.

Dydrogesterone: pharmacological profile and mechanism of action as luteal phase support in assisted reproduction.

The pharmacological and physiological profiles of progestogens used for luteal phase support during assisted reproductive technology are likely to be important in guiding clinical choice towards the most appropriate treatment option. Various micronized progesterone formulations with differing pharmacological profiles have been investigated for several purposes. Dydrogesterone, a stereoisomer of progesterone, is available in an oral form with high oral bioavailability; it has been used to treat a variety of conditions related to progesterone deficiency since the 1960s and has recently been approved for luteal phase support as part of an assisted reproductive technology treatment. The primary objective of this review is to critically analyse the clinical implications of the pharmacological and physiological properties of dydrogesterone for its uses in luteal phase support and in early pregnancy.

Stabilization of clinical samples collected for quantitative bioanalysis--a reflection from the European Bioanalysis Forum.

In bioanalysis of small molecules, the analyte concentration in the measured samples should reflect the concentration during sample collection. Precautions may be needed to prevent over- or under-estimation of the obtained result. This might require the addition of stabilizers to prevent degradation or nonspecific binding. For unstable drugs, it is essential to know how analytes can be stabilized before the start of the clinical study. Although the stabilization methods are well documented, the impact of the stabilization on the clinical workflow is not properly addressed. Already during method development, the clinical implications in terms of personnel safety, ease of use, training possibilities and staff capacity should be taken into account, and validation of the bioanalytical method should reflect collection procedures.

European Bioanalysis Forum: recommendation for dealing with internal standard variability.

Adequate monitoring of internal standard (IS) response across an analytical run and identification of anomalies is now a common expectation. However, the means to conduct this assessment in an appropriate manner is unclear and differs widely between laboratories. A European Bioanalysis Forum (EBF) topic team was formed to survey current practices within European Bioanalysis Forum member companies and to recommend a best practice approach for dealing with IS response variability.

Pharmacokinetic interaction between simvastatin and fenofibrate with staggered and simultaneous dosing: Does it matter?

Simvastatin and fenofibrate are frequently co-prescribed at staggered intervals for the treatment of dyslipidemia. Since a drug-drug interaction has been reported when the two drugs are given simultaneously, it is of clinical interest to know whether the interaction differs between simultaneous and staggered combinations. A study, assessing the impact of both combinations on the interaction, was conducted with 7-day treatment regimens using simvastatin 40 mg and fenofibrate 145 mg: (A) simvastatin only (evening), (B) simvastatin and fenofibrate (both in evening), and (C) simvastatin (evening) and fenofibrate (morning). Eighty-five healthy subjects received the respective treatments in a randomized, 3-way cross-over study. The pharmacokinetics of simvastatin and the active metabolite simvastatin acid were determined. There was a limited reduction in the AUC0-24h of simvastatin acid of 21 and 29% for simultaneous and staggered combination, respectively. The geometric mean AUC0-24h ratio of simvastatin acid for the two combined dosing regimens (B/C) and 90% confidence interval were 111% (102-121). The interaction apparently had no impact on lipid markers. The findings imply that the observed pharmacokinetic interaction is unlikely clinically relevant, and support the combined use of simvastatin and fenofibrate not only given at staggered interval but also given simultaneously.

Use of polarity switching for the simultaneous bioanalysis of analytes with three orders of magnitude difference in concentration by LC-MS/MS.

BACKGROUND: The challenge of quantifying two compounds in a single assay with drastic dynamic ranges is to obtain linearity without source or detector saturation at the mass spectrometer. RESULTS: In positive-ionization mode, the nonlinear relationships for Desmethyl Mebeverine Acid (DMAC) were demonstrated using three common strategies to overcome this issue: using offset voltage parameters, less-sensitive product ion or 13C mass SRM transitions. On the contrary, nonlinear relationships for DMAC were overcome if negative-ionization mode was used. Due to Mebeverine analytical LLOQ, dilution was not suitable for a single assay of Mebeverine and DMAC. However, polarity switching in negative mode for DMAC was successfully found to compensate for the nonlinearity at the mass spectrometer while preserving Mebeverine linear regression model in positive mode. CONCLUSION: The polarity switching strategy has demonstrated the advantage of improving linearity for analytes having different ionization polarities and three orders of magnitude difference in concentration.

The European Bioanalysis Forum community's evaluation, interpretation and implementation of the European Medicines Agency guideline on Bioanalytical Method Validation.

The European Medicines Agency's (EMA) 2011 guideline on bioanalytical method validation (BMV) was evaluated and subsequently intensely discussed by the European Bioanalysis Forum (EBF) during a 2-day workshop (EBF Workshop on the implementation of the EMA guideline on BMV, Château de Limelette, Limelette, Belgium, 15-16 March 2012). The goal of the evaluation and discussions was to come to a uniform interpretation of the guideline and thus to help facilitate a smooth implementation at our laboratories. Up front preparations for the workshop by dedicated teams concentrated on challenges on implementation: ambiguities, technical or operational challenges and issues in general. In addition, common understandings were identified as well as main differences to the 2011 US FDA guideline. The guideline was perceived as being well written with a clear structure, separating method validation from sample analysis and treating all relevant aspects one-by-one in a logical order. It is the first BMV guideline clearly addressing the specifics for ligand binding assays and it shows a good match with current scientific thinking. The EBF community considers the EMA BMV guideline an excellent basis for countries that are in the process of developing or updating their own BMV guideline.

Conference Report: the 3rd EBF Focus Meeting: 'Hatching'--emerging technologies approaching the regulated bioanalysis laboratory. Brussels, Belgium, 12-13 June 2012.

One hundred and eighty scientists from industry and academia discussed the progress in emerging technologies approaching regulated bioanalysis, with a focus on what potential hurdles prevent them becoming broadly accepted by industry and regulators. The conference delegates agreed that moving innovative technologies forward can only be achieved by providing solid data to support the application. In addition, also establishing an open dialogue with health authorities is key for success. By successfully integrating new technologies in the bioanalytical laboratory we contribute to bringing more safe and efficacious therapies faster to patients.

'Large Meets Small': connecting the bioanalytical community around peptide and protein bioanalysis with LC-MS(/MS).

This conference report provides an overview of the discussions at the 2nd European Bioanalysis Forum (EBF) Focus Meeting 'Large Meets Small' held on 20 and 21 June 2011 in Brussels. The meeting discussed scientific progress in the bioanalysis of peptides and proteins with MS-based techniques. Bioanalytical experts in ligand-binding assays (LBAs) and MS from industry and academia presented at the meeting or joined the discussion. The conference hosted sessions on technology developments, validation requirements, cutting edge (bio)analytical approaches for both proteins and peptides and discussions on the analytical challenge presented by the metabolism of peptides or proteins. The engagement of the scientists as well as the bioanalytical challenges identified were real: by shifting peptide or protein analysis from the LBAs laboratory into the LC-MS laboratory, the bioanalytical scientist is moving into partially uncharted territory. The conference delegates strongly shared the feeling that success in overcoming the challenges of peptide and protein bioanalysis will require further integration of the expertise of LBAs and LC-MS/MS experts.

'Less is More': defining modern bioanalysis.

The 4th Open Symposium of the European Bioanalytical Forum entitled 'Less is More' was held on 16-18 November 2011 at the Hesperia Tower Hotel, Barcelona, Spain. More than 50 interesting presentations were delivered covering areas with interest for the small- and large-molecule community - biomarker validation; regulations, including an update on new and emerging guidelines and on Global harmonization; technology updates; incurred sample stability; microdosing; dried blood spots and microsampling; challenges of 'free' and 'total' macromolecule quantification; stability issues in ligand binding assays or anomalous results. In excess of 450 delegates from more than 170 institutes and companies (industry, regulators and academia) from all global regions participated in the open and stimulating discussions. This manuscript provides an overview of the highlights discussed at the meeting.

Anticoagulant counter ion impact on bioanalytical LC-MS/MS assays: results from discussions and experiments within the European Bioanalysis Forum.

BACKGROUND: In regulated bioanalysis, the need for partial validation when changing the counter ion of the anticoagulant is currently being debated within the bioanalytical community. To date, industry and the health authorities have not yet reached a consensus on this issue. The aim of the present study was to evaluate the impact of a change in counter ion when using the same anticoagulant on LC-MS/MS assay performance for a broad array of new chemical entities, compiling data generated at companies within the European Bioanalysis Forum (EBF). RESULTS: In all, 15 EBF member companies provided experimental data on partial validation. In total, data from 42 LC-MS/MS assays were evaluated. The results show that a change in counter ion when using the same anticoagulant had no impact on assay performance. CONCLUSION: Based on these results and on conclusions from previous studies, the EBF recommends that in regulated bioanalysis, plasma samples containing different counter ions, but the same anticoagulant, should be regarded as equal matrices, thus removing any need for partial validation.

2011 White paper on recent issues in bioanalysis and regulatory findings from audits and inspections.

The 5th Workshop on Recent Issues in Bioanalysis (WRIB) was organized by the Calibration and Validation Group as a 2-day full immersion workshop for pharmaceutical companies, CROs and regulatory agencies to discuss, review, share perspectives, provide potential solutions and agree upon a consistent approach to recent issues in the bioanalysis of both small and large molecules. High quality, better compliance to regulations and scientific excellence are the foundation of this workshop. As in the previous editions of this significant event, recommendations were made and a consensus was reached among panelists and attendees, including industry leaders and regulatory experts representing the global bioanalytical community, on many 'hot' topics in bioanalysis. This 2011 White Paper is based on the conclusions from this workshop, and aims to provide a practical reference guide on those topics.

From challenges to solutions. European Bioanalysis Forum 3rd Annual Open Symposium, Hesperia Towers, Barcelona, Spain, 1-3 December 2010.

The European Bioanalysis Forum is a bioanalytical nonprofit organization comprised of European pharmaceutical companies (27 members to date) and currently expanding to include CROs as well. The European Bioanalysis Forum provides a broad European bioanalytical network for the discussion of scientific, technological and regulatory topics of bioanalytical interest. The 3rd Annual Open Symposium was again much anticipated after the two previous successful meetings. The symposium included sessions on thinking outside the 'commodity' box, bioanalytical challenges with blood, global harmonization, assay platforms, dried blood spots, immunogenicity, matrix effects, anomalous results, biomarkers and two plenary technology sessions hosted by the Platinum sponsors. Experts and key opinion leaders were invited as guest speakers. A total of 424 delegates registered from 113 companies representing a large percentage of the European bioanalytical community. In addition to 48 oral presentations, 88 posters were presented and there was a vendor exposition of 40 companies.

2010 white paper on recent issues in regulated bioanalysis & global harmonization of bioanalytical guidance.

The 4th Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis, a 2-day full immersion workshop, was organized by the Calibration and Validation Group. Contract research organizations, pharmaceutical companies and regulatory agencies came together to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges and to reach a consensus among panelists and attendees on many points regarding method validation of small and large molecules.

2009 White Paper on recent issues in regulated bioanalysis from the 3rd Calibration and Validation Group Workshop.

The 3rd Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis was organized by the Calibration and Validation Group as a 1.5-day full immersion workshop for contract research organizations, pharmaceutical companies and regulatory agencies to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges. A consensus was reached among panelists and attendees on many points regarding method validation of small molecules.