Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction.

Aim: Quantitative LC-MS analysis of oligonucleotides (OGNs) in biological matrices is needed to support candidate selection of new therapeutic OGNs. Methodology & results: A set of 20 single stranded antisense oligonucleotides (ASO) and five siRNAs were extracted from plasma and tissue homogenates. Anion Exchange (AEX) SPE was selected as generic extraction approach, resulting in recoveries from plasma >70%. Extraction from tissue homogenates showed often more variation and lower recoveries. A proof of concept of a novel tailored hybridization extraction is demonstrated for two 16-mer reference OGNs. Conclusion: Two methods for extraction of OGNs were investigated and applied for quantitative analysis. The AEX-SPE is considered a more generic approach preferred when multiple compounds are evaluated. Hybridization extraction has great potential but critical reagents per analyte are needed.

In vitro evaluation of poloxamer in situ forming gels for bedaquiline fumarate salt and pharmacokinetics following intramuscular injection in rats.

The objective of this study was to evaluate in vitro and in vivo drug release from in situ forming gels prepared with poloxamer 338 (P338) and/or 407 (P407) in N-methyl-2-pyrrolidone (NMP)/water mixtures for the model compound bedaquiline fumarate salt. The impact of total poloxamer concentration (20%-25% (w/w)), P338/P407 ratio (100/0%-0/100% (w/w)) and NMP/water ratio (0/100%-25/75% (v/v)) on gel point temperature (GPT) was investigated via a design of experiments (DoE), showing that GPT decreased mainly with increasing poloxamer concentration and decreasing P338/P407 ratio, while the relation with NMP/water ratio was more complex resulting in a flexion. Based on the DoE, four formulations with 10 mg/g bedaquiline fumarate salt, a fixed NMP/water ratio of 25/75% (v/v) and varying total poloxamer concentration and P338/P407 ratio were selected for evaluation of gel erosion in vitro. The fastest eroding formulation had the lowest total poloxamer concentration (20% (w/w)) and the lowest P338/P407 ratio (20.4/79.6% (w/w)), while the formulation with the highest total poloxamer concentration (23.5% (w/w)) and highest P338/P407 ratio (100/0% (w/w)) showed the lowest gel erosion rate. These fast and slow eroding formulations showed a similar trend for in vitro drug release and in vivo pharmacokinetics after intramuscular (IM) injection in rats. In vivo tmax of the IM administered poloxamer in situ forming gels was about 6 h and a short-term sustained drug release was observed in vivo in rats up to 24 h after dosing, similar to a solution of bedaquiline fumarate salt in polyethylene glycol (PEG400)/water. In conclusion, IM administration of the evaluated poloxamer in situ forming gels may be useful for drugs that require a short-term sustained release, but is not able to extend drug release rates up to weeks or months.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 1--small molecules by LCMS).

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 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 3 - LBA and immunogenicity).

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.

European Bioanalysis Forum: recommendation on dealing with hemolyzed and hyperlipidemic matrices.

Recent guidelines on bioanalytical method validation have recommended to investigate matrix effects in special matrices such as hemolytic and hyperlipidemic plasma. However, these guidelines were not clear on how to implement these recommendations. The European Bioanalysis Forum has discussed this topic in depth and has asked for feedback from member companies. Those discussions have resulted in more specific guidance on how to define hemolytic and hyperlipidemic plasma, how to validate bioanalytical methods for these matrices and how to deal with hemolytic and hyperlipidemic study samples. These recommendations are presented in this manuscript.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 2 - hybrid LBA/LCMS, ELN & regulatory agencies' input).

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).

Managing scientific, technical and regulatory innovation in regulated bioanalysis: a discussion paper from the European Bioanalysis Forum.

On 12-13 June 2012, the European Bioanalysis Forum hosted its third Focus Meeting in Brussels (Belgium). At the meeting, a panel discussion was held on the hurdles that the bioanalytical community encounters when adopting new technologies or managing regulated bioanalysis expectations around emerging technologies. Over the last few years, the industry has seen many new technologies maturing. As they became available, the bioanalytical scientist has observed that implementing these technologies in the regulated environment has become increasingly challenging. For one, scientific developments and regulatory expectations may not go hand in hand. At the same time, the pharmaceutical industry has become increasingly risk averse in their response to these real or perceived higher expectations in regulated bioanalysis. As a downstream consequence, the potential result of overinterpretation of guidance or occasional widespread and premature implementation of responses to health authority inspections, industry may be contributing significantly to raising the bar on some processes related to day-to-day practices in the bioanalytical laboratory. Last but not least, with the community being satisfied with the performance of the current tools, potential complacency can be observed in the regulated bioanalytical community because existing technologies, such as LC-MS/MS and ligand-binding assays, have served and still are serving them extremely well. Hence, the question 'what's next after LC-MS/MS or ELISA?' is not resonating with many scientists as pertinently compared with 'What's next after RIA, GC or LC-UV?', which was the key question in the 1990s, certainly in the context of an increasing effort needed to validate these new tools. With this article, the European Bioanalysis Forum aims to stimulate an open dialogue between all stakeholders in regulated bioanalysis to positively influence how we balance science, process and regulations in day-to-day work. This discussion should facilitate the evaluation and the subsequent implementation of innovative techniques for the benefit of the patient, while stimulating our community to raise the bar on added-value science, but at the same time removing the bar on processes with limited or no added value.

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.

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index.

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

Direct injection of whole blood for liquid chromatography/tandem mass spectrometry analysis to support single-rodent pharmacokinetic studies.

Mass spectrometric developments in the last decade enable (sub)nanomolar detection of drug compounds in biological matrices in a few microliters of blood. However, the sampling and especially the handling of these small blood volumes is not straightforward. We studied the feasibility of a recently developed 'sorbent sampling technique' to handle these small blood volumes and the application to support pharmacokinetic (PK) screening programs. This technique applies 5-10 microL of blood on a fibrous material packed into a cartridge. Blood samples absorbed on these cartridges are eluted directly, on-line onto a solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE-LC/MS/MS) system. It is shown that the sorbent sampling technique can be applied for a range of drug compounds. In spite of issues with recovery and sample clean-up that need further improvement, the sorbent sampling technique provided similar data as compared to conventional analytics. The technique was successfully applied to derive kinetic data from individual mice, thereby decreasing the number of required mice for a PK study from 21 to 3.

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H(2)O(2).

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H(2)O(2) at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.