Conference report
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Moving forward together: “we are making progress”
The 6th European Bioanalysis Forum Open Meeting
William D van Dongen* & Luuk C Renfurm
20–22 November 2013, Hesperia Tower Hotel, Barcelona, Spain
TNO Triskelion BV, Utrechtseweg 48, 3704 AV Zeist, The Netherlands *Author for correspondence: [email protected]
At the 6th European Bioanalysis Forum Open Meeting, held from 20–22 November 2013 in Hesperia Tower Hotel, Barcelona, Spain, bioanalytical experts from pharmaceutical industry, academia, contract laboratories and regulatory bodies discussed current topics of interest in bioanalysis. 450 delegates from more than 170 institutes and companies participated in 75 open and stimulating presentations regarding the new US FDA Guidance for industry, technology updates, including liquid chromatography– mass spectrometry of proteins and antibody–drug conjugates, dried blood spots, sampling and extraction and regulatory aspects of, for example, flow cytometry, parallelism, and interferences in ligand-binding assays. This article aims to provide an overview of the highlights discussed at the meeting.
6th European Bioanalysis Forum Open Meeting The 6th European Bioanalysis Forum Open Meeting was held from 20–22 November 2013 in Hesperia Tower Hotel, Barcelona, Spain. The EBF organization, moving forward under the enthusiastic and energetic leadership of Peter van Amsterdam and Philip Timmerman, assured that all the relevant developments and issues in the comprehensive field of bioanalysis are brought to the footlight. At least 450 bioanalytical experts, lab managers of pharmaceutical companies and contract research organizations shared scientific developments, procedures, opinions, regulatory aspects and global harmonization in the field of bioanalysis. Besides organizing open meetings, EBF also defines the most urgent and relevant bioanalytical themes and assigns them to topic teams, involving bioanalytical experts from pharmaceutical and CRO companies. The topic teams use surveys, its expertise, interviews, literature to provide clarity, produce recommendations and white papers [1,2]
10.4155/BIO.14.53 © 2014 Future Science Ltd
often resulting in improved guidance and regulatory documentation of the EMA. Several topic teams reported their progress on the open meeting, for example, ‘IS variability’, ‘the importance of parallelism during validation of ligand binding assays’ and ‘flow cytometry in regulated bioanalysis’. As always, the EBF meeting in beautiful Barcelona was an excellent opportunity for networking and informal meetings. The exhibition floor was filled with 45 booths and the attendees had intensive interactions with CROs en vendors of equipment. The highlights of the different sessions are discussed below. Scientific & technological developments in bioanalysis Peptide & protein analysis with LC–MS
Due to the large number of requests for presenting the subject of protein LC–MS bioanalysis, the organization took the opportunity to organize a last-minute session in this emerging field. Significant progress was reported in comparison to data reported at the
Bioanalysis (2014) 6(9), 1159–1165
part of
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Conference report EBF 2011 Focus Meeting ‘Large meets Small’ in Brussels, Belgium [3] . The session was kick-started by Nico van de Merbel (University of Groningen, The Netherlands). He postulated existential bioanalytical questions, such as what is ‘the concentration’ of a protein? Because of its multidimensional complex structure, a one dimensional concentration of a protein cannot be determined, as this depends on the sought information and the applied tools. Ligand-binding assay (LBA), the current standard for therapeutic protein bioanalysis, measures the unbound intact 3D domain of the therapeutic protein binding to the assay’s capture reagent, can be considered as a ‘biological concentration’. In contrast, van de Merbel explained that LC–MS will give a ‘chemical concentration’ by monitoring a unique primary structure piece of the protein. The advantages that LC–MS has over LBA is that no highly selective biological reagents have to be developed, which can take up to six months. He clearly explained that the biological and chemical protein level determined in the same sample with, respectively, LBA and LC–MS may not necessarily be similar. Rand Jenkins (PPD, Richmond, VA, USA) and William van Dongen (TNO Triskelion, Zeist, The Netherlands) continued this session by demonstrating the potential of immunoprecipitation, using protein A capture and LC–MS to support monoclonal antibody (mAb) development. Jenkins reviewed the progress of the development of a nano-LC–MS assay to simultaneously quantify trastuzumab and its soluble sHER2 ECD receptor, associated with HER2-overexpressing cancers. William van Dongen showed the feasibility of nano LC–MS to determine therapeutic mAbs at sub ng/ml levels. For their nano-LC experiments, Jenkins applied the Waters TRIZAIC™ bioanalytical nano-LC tile and Van Dongen, the Thermo EasySpray™ technology. The presented data illustrated that nano techniques have potential for bioanalytical quantitation of therapeutic proteins. Continued developments in DBS
EMA does not yet accept DBS technology to support drug-development studies and the US FDA requires that DBS delivers the same concentration data as conventional techniques. Since, DBS is a tool that has potential for regulated bioanalysis, EBF monitors innovations that overcome current essential limitations of DBS [4,5] . Variation in the blood hematocrit (Ht) level is one of the major issues as it influences spot size and extraction recovery and consequently may cause poor precision when only part of the spot is extracted [6] . Bert Ooms (Spark Holland, Emmen, The Netherlands) gave a potential solution to this issue using the
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fully automated DBS system of Spark Holland. This system automatically punches the whole bloodspot of DBS card followed by temperature-enhanced flowthrough desorption, on-line solid-phase extraction and mass spectrometry. Ooms demonstrated that hematocrit-variations can be handled by this system as near 100% recovery was obtained independent of HT values ranging from 0.3–0.7. Another major concern is the PK/TK measuring in blood, rather than plasma, can affect quantitation accuracy [7] . As a result of that plasma microsampling strategies have been reported in the literature. [8] . Jack Henion (Quintiles Bioanalytical and ADME laboratories, NY, USA) presented a device utilizing a dual sampling card where the upper layer filters the red blood cells (RBC) from applied low volumes of whole blood, so plasma micro samples are collected on the lower layer generating dried plasma spots. This device was inspired on a commercially available device, originally designed by Yorktest for at-home (pointof-care) measurement of homocysteine development. Ynze Mengerink (Chemelot, Geleen, The Netherlands) presented promising data about the use of new card material available that generates blood spot sizes independent of the Ht value. Innovative applications in sampling & extraction
Kathryn Chapman (NC3Rs, London, UK) introduced this session. She explained that the largest driver for the number of rodents in regulatory toxicology studies is the collection of samples for TK analysis [9] . The use of capillary microsampling techniques allows the reduction of the use of satellite animals for TK analysis. Kathryn summarized the conclusions of the NC3Rs workshop held in May 2013 all 80 delegates showed an interest in the use of microsampling for GLP regulatory toxicology studies at their companies. Nevertheless, there was still some hesitation to apply microsampling for main study animals, as its impact on the clinical pathology, pathology and other readouts as CNS effects is not known. During the workshop it was agreed that an evidence-base is needed to demonstrate that microsampling from main study samples does not compromise study end-points. In general, the conclusion of this session was that capillary microsampling is becoming more and more important, especially when samples have to be collected from children or small animals. A number of microsampling capillaries are commercially available and several methods using capillary microsampling have been validated in accordance with the FDA Guidance.
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Analytical challenges of antibody–drug conjugates
Antibody–drug conjugates (ADCs) are complex drugs consisting of cytotoxic drugs covalently attached to mAbs via a chemical linker, to achieve targeted delivery. The degree of complexity of ADCs is increased as it represents a heterogeneous mixture with different drug antibody ratios (DARs). It is important to assure the stability of the ADC, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk due to its high potency. Bernard Beckermann (Bayer Pharma, Berlin, Germany) explained that the above-mentioned complexity, heterogeneity and required stability is reflected in the desired bioanalytical methods for ADC development programs, for example, total amount of cytotoxic drug containing antibodies, drug-free mAbs, the released cytotoxic drug, the drug to antibody ratio (DAR) and the potentially formed antibodies against the ADC (ADA). Depending on the specific molecular design and the endpoints the bioanalytical strategy needs to be defined and usually includes ELISA formats for the antibody part, LC–MS for the released cytotoxic drug, high-resolution accurate mass for characterization of DAR during circulation and cell based assays for ADA testing. Steffen Gross (Paul-Ehrlich-Institut, Langen, Germany) discussed the challenges of the analytical characterization of ADC drug substance, where he also pointed out that special emphasis should be taken on the stability of the ADC. Jasja Wolthoorn (TNO Triskelion, Zeist, The Netherlands) gave a nice overview of the bioanalytical strategies that are recommended during the different stages of development of the ADC development program. Depending on the tools available and the (pre-) clinical phase, initial analysis might focus on the analysis of the antibody part (DAR≥0) of the ADC, the toxin, anti-drug antibodies and the average DAR. Later on it is recommended to use techniques that enables to get information on DAR distribution of the ADC.
Conference report
(TQMS) (Sciex APl4000) and a HRMS (Waters Synapt G2), under identical UPLC conditions. She demonstrated that for bioanalysis of a small molecule in bile, plasma and urine, the quantitative performance was comparable for HRMS and TQMS systems. The qualitative output of the HRMS was valuable, major excretion pathways have been identified as well as a circulating metabolite. However, as the UPLC conditions were optimized for quantitation, the metabolites did largely co-elute. Therefore, optimization with respect to the separation of parent and metabolites is in most cases required. ln practice, Lieve recommended to apply Quan/Qual bioanalysis only for selected cases, as it is a compromise between Quan efficiency and Qual data quality, and workflows are not as simple as promised. Mohammed Abrar (Unilabs York Bioanalytical Solutions, York, UK) compared TQMS (Waters TQS) and HRMS (Thermo QExactive) for bioanalysis of a 5 kDa therapeutic oligonucleotide in human plasma. Mohammed explained that LC–MS bioanalysis of oligonucleotides is challenging due to the presence of the multiple negatively charged phosphate groups in the analyte. For LC separation, the necessary ion pair chromatography is challenging to optimize and electrospray generates multiple charged states that are further complicated by the exchange of H+ for Na+, K+ at the phosphate groups. As a result bioanalytical LC–MS methods for oligonucleotides are relatively insensitive and subject to background ion interferences. Mohammed demonstrated that the very high resolving power of the Q-Exactive was able to give clean chromatograms of the 5 kDa therapeutic oligonucleotide at 0.4 ng/ml, whereas the TQ-MS gave significant background signal at this level. The overall outcome of this session was: apply HR-MS only for certain studies where metabolite lD is needed and in cases where the resolving power is required to obtain clean SRM chromatograms. Bioanalytical regulation & guidance updates
High-resolution MS
New FDA Guidance for Industry
The availability of well-priced accurate high-resolution mass spectrometers (HRMS), featuring enhanced sensitivity, resolving power, mass accuracy, acquisition speed and dynamic range makes this technique affordable for the bioanalytical laboratories. HRMS is capable of, besides target quantification of drugs and their anticipated metabolites, qualification of still undisclosed metabolites, endogenous compounds responsible for matrix effects, and/or potential biomarkers accessible in post-dose preclinical samples. Lieve Dillen (Janssen, Beerse, Belgium) made a bioanalytical comparison between a triple quadrupole
The FDA draft Guideline on bioanalytical method validation is available [10] , and as part of the feedback process, AAPS/FDA organized a meeting scheduled for December 2013 referred to as Crystal City–V (Baltimore). The EBF program of the 6th Open Symposium (20–22 November 2013, Barcelona, Spain) included a ‘Preparation session’. In this session feedback was received and collected from the EBF member companies. There were many comments. For example, for the ligand-binding assay part, many criteria have been copied directly from the chromatographic assays and modifications of the text are highly recommended. It was
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Conference report agreed that all feedback will be summarized and the EBF steering committee will represent all EBF companies at the Crystal City meeting in December 2013. Challenges for flow cytometry in regulated bioanalysis
• Discuss the project as early as possible with the CRO and assure that technical, operational, cultural differences between the laboratories are identified/understood;
Flow cytometry is increasingly used for safety/ exploratory purposes in (pre-) clinical trials, for example for immunogenicity, PK, potency testing, receptor occupancy testing. There are numerous advantages of flow cytometry such as quick quantitative analysis since multiple markers can be analyzed simultaneously. The main issue discussed in this session, organized by Topic Team 32, was the lack of guidance on how to perform flow analysis under regulated regime. Representatives from the FDA, AAPS, ICCS and ICSH request for clear guidelines. Topic Team 32 will soon publish a White Paper with the EBF’s view.
• Ensure noncommercial reagent early supply and continuity;
Assay transfer-expectations and practicalities
• Blame the CRO, ensure that solved issues go through a closing process.
The process of assay transfer requires intensive communication such as clear definition of final goal of the project, a mutually understood and accepted plan, and regular communication between sending and receiving lab. Especially assays for biopharmaceutical, due to their size and complexity, and the inability to characterize biopharmaceuticals fully, their instability, makes assay transfer comparably more complex than for small molecules. Cheryl McAlpine (Merck Millipore, Oxford, UK) discussed the highly challenging transfer of neutralizing anti-drug antibodies (nAb) assays. For a receiving lab to be successful it is critical to establish at the set-up stage of any assay transfer, the assay status at the transferring lab. This can vary from merely conceptual to the transfer of a fully validated protocol. In either case, it is essential that the transferring partner fully understands the mechanism of action, and that the most relevant of multiple signaling pathways of the in vivo effect is used. Also one should aim that, after implementation at the receiving laboratory, target expression levels are adequate, stable and specific, the assay’s dynamic range is known and cell growth conditions/seeding density are optimized. Matrix interference effects from human plasma/serum of different populations must also be assessed and the final assay must meet the requirements of clinical sample analysis. Fabienne Deckert-Salva (Novartis Pharma, Mulhouse Area, France) presented a useful do/do not list with respect to outsourcing of biological assay transfer: Do: • Outsourcing is a win–win collaboration, both parties have the same goals;
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• Build mutual trust;
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• Ask for regular feedback on methods, coordination and monitoring processes and so on. Do not: • Underestimate CRO expertise (CROs are exposed to other external expertise and opinions); • Hide any weakness/complexity of the transferred method;
From biomarker to diagnostics or personalized medicine
John Mills (AstraZeneca, Stockport, UK) introduced this session by stating “more than 90% of drugs only work in 30–50% of the people.” Personalized healthcare (PHC) can significantly improve this number and has the potential to benefit prescribers, payers, regulators, and obviously patients by aiming to deliver the right medicine, to the right patient at the right dose and time. The potential to use biomarkers for classifying patients into groups that are less or more likely to benefit from a certain therapy, is anticipated to have a major impact on the integrated development of new drugs and their companion diagnostic method. John presented some illustrative examples in which therapies (e.g., Vemurafenib, Crizotinib) are matched with specific patient population characteristics using companion diagnostics, and discussed the challenges and implications of this approach to a drug development program and the involved bioanalysis and regulatory requirements. John made it clear that companion diagnostics are considered to be high risk and therefore likely to need to meet the highest regulatory hurdles in the US, for example, Class III, Pre Market Approval (PMA) required [11] . Suntje Sander-Struckmeier (Abbott, Weesp, The Netherlands) and Peter van Amsterdam (Abbott, Weesp, The Netherlands) presented an example of optimization treatment efficacy diagnosis of pancreatic exocrine insufficiency (PEI). They discussed the use of faster, better and less laborious tools for determine the efficacy of
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pancreatic enzyme replacement therapy (PERT). Instead of complete stool fat excretion measurement during 72-h collection, measurement in only sparse stool samples was evaluated. Potentially more efficient alternatives under investigation are 13C-mixed triglyceride breath testing and the malabsorption blood test. Interpretation & implementation of guidance on haemolyzed/hyperlipidemic plasma, & co-administered drugs
Benno Ingelse (MSD, Oss, The Netherlands) presented the progress of EBF’s topic team 15 sorting out “how to deal with haemolysed and hyperlipidemic samples for LC–MS bioanalysis”. The EMA guidelines [12] mention that hemolyzed and hyperlipidemic matrices must be included as part of the selectivity assessment. Hemolyzed plasma contains hemoglobin from damaged red blood cells. Hyperlipidemic plasma, originating from humans having a higher risk for heart disease, has higher triglyceride and cholesterol levels. Plasma samples with >240 mg/dL cholesterol, corresponding to high heart disease risk are considered to be hyperlipidemic. Hemolyzed and hyperlipidemic matrix samples can result in ion-suppression or enhancement during LC–MS analysis. The stable isotope-labeled (SIL) IS do not always compensate sufficiently for these effects especially close to LLOQ. Benno explained how to prepare suitable hemolyzed and hyperlipidemic matrices for matrix effect testing. Haemolysed plasma can be self-prepared by spiking plasma with 2% (V/V) hemolyzed blood to plasma. Hemolyzed blood is typically prepared by subjecting control whole blood to three freeze-thaw cycles. There is still discussion how hyperlipidemic samples should be obtained; one can either use plasma with a predefined minimum (>240 mg/dL) cholesterol level or triglyceride level, or selfprepare samples by spiking plasma with Intralipid. Based on the outcome of a recently held EBF survey an EBF recommendation on haemolysed/hyperlipidemic samples will be published soon. Progress was also reported in the issue of co-administered drugs. The EBF survey held on co-medication testing will soon result in a risk-based tiered approach recommendation, where also scheduled or nonscheduled co-medication will be taken into account. Interpretation & implementation of Guidance expectations IS variability
As also discussed in the previous section, SIL IS do not always compensate sufficiently during LC–MS bioanalysis. Based on an EBF survey, an EBF recommendation will be issued soon. The recommendation will address both individual irregularities as well as systematic variability.
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Conference report
Analysis of parallelism for biomarkers & therapeutic proteins
Parallelism is defined by the lack of an increase or decrease in concentration along the standard curve after correction of the dilution factor of a sample. During the assessment of parallelism a plot of the determined concentrations of the analyte in an incurred sample at multiple dilutions against the expected concentrations at each dilution should have a slope near 1.0. Incurred samples are recommended, since it is an intrinsic sample issue, and not related to the applied method. The incurred samples should obviously be individual and not pooled to avoid averaging out effects. Also preferably later time point samples should be tested for parallelism to determine the binding protein effects. Issues that were discussed during this session were: • What to do if samples do not dilute parallel? A potential solution might be to use a quasi-quantitative approach by comparing pre- with post-dose samples. Factors to consider in case of non-parallelism: drug aggregation, drug stability in vivo, antidrug antibody presence, endogenous binding partners, and different 3D structural forms of the drug; • Often no access to study samples during validation; • GCP issue: the use of incurred samples required for parallelism testing is often not covered by an informed consent; • Global view on parallelism by the GBC: parallelism testing should be science based and testing on a routine basis is not recommended. Some examples of parallelism were presented, for example, a bridging ELISA for PK analysis of a mAb, where the coating concentration was critical for good assay performance. Bruno Boulanger (Arlenda, Liège, Belgium) discussed the assessment of parallelism in bioassays from a statistical point of view. There are two approaches. The four-parameter logistic curve approach was based on measuring the ratio of two slopes and determination of the EC50 differences between the lines, which should be close to one. The other approach, the linear assay approach, takes the linear range of the S-curve into account. Boulanger recommended the “4-parameter logistic curve approach”, as it is easy to implement on the laboratory and there are less biases in the method. Interferences & surprises in ligand-binding assay method development
Critical reagents (CR) in ligand binding assays (LBAs) has become a topic of attention with the publication by the AAPS Ligand binding in the 21st century:
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Conference report recommendations for characterization and supply of critical reagents [13] . Critical reagents are the backbone of all LBAs, and appropriate quality management of CRs minimizes assay performance issues caused by declining reagent activity throughout all stages of the drug development process. The first speaker in this session, Helen Young (Quotient Bio Analytical Sciences, Teddington, Middlesex, UK), discussed the practical issues around monitoring of critical reagents in a clinical laboratory. She gave an extensive list of CRs: reference standards, antibodies, in-house conjugated reagents, cell lines, matrix (e.g. serum, especially for biomarker assays), precoated assay plates, commercial kits reagents, blocking reagents, assay buffers, substrate, etc. Young recommends to implement a procedure that defines what CRs are, how to monitor the performance of the CRs, what steps to take when the performance of the CRs change, and how to store, treat, and document CRs. Martin Nemansky (PRA Bioanalytical, Zuidlaren, The Netherlands) zoomed in on the practical interpretation of the guidelines or white papers for immunogenicity of anti-drug antibody (ADA) testing. He mentioned that “guideline interpretation is challenging when there is an assay issue” and fit-for-purpose solutions are required. He illustrated this by several examples, e.g. a low positive-control sample was screened positive for ADAs, but could not be confirmed in the confirmatory assay due to low noise. A practical solution was to increase the screening cut point more close to the confirmatory cut point. Bioanalysis Young Investigator Award Each year, Bioanalysis run the Young Investigator Award to identify and reward promising early-career researchers in bioanalysis. Peter van Amsterdam presented the 2013 Bioanalysis Young Investigator Award. This year 18 young scientists were nominated for the Award. This year’s winner of the Award was Anthony O’Donoghue (University of California, CA, USA) for his work on the development of a technique to simultaneously assay all proteases in a biological sample at exquisitely low concentrations. In continuation, Anthony presented his award winning research on the development of affinity microcolumns for high throughput bio interaction analysis. This global identification of protease specificity allowed him to determine the proteolytic signatures of cancer cells and
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parasitic organisms and to subsequently identify the major proteolytic enzymes involved. Conclusion & future agenda Again, the open meeting successfully connected the bioanalytical community and accomplished this year’s mission of ‘moving forward together’. Almost 75 interesting presentations were shared in 18 different sessions. Significant progress was reported in the new emerging field of bioanalytical LC–MS of therapeutic proteins. Developments in DBS, sampling and extraction, the possibilities and limitations of HRMS for bioanalysis were all very well covered in their respective sessions. In addition, the contours of feasible bioanalytical strategies for ADCs, currently the biggest challenge for the bioanalytical specialist, are becoming distinct. Regulation and guidance updates considering flow cytometry, hemolyzed/hyperlipidemic plasma, IS variability, assay transfer-expectations and practicalities, parallelism for biomarkers and therapeutic proteins, ligand-binding assay interferences and biomarkers and diagnostics for personalized medicine were all comprehensively covered. Again, the EBF organizers succeeded very well in their mission to share, discuss, optimize and seek alignment on a broad array of bioanalytical topics including science, technology and regulatory issues. For 2014, the EBF agenda holds two focus workshops in Brussels, Belgium. At 5–6 February 2014 the workshop is focussed to standardized lab manual for bioanalytical support in clinical studies, and at 17–18 June focussed on the tiered approach, entitled ‘taking the tiered approach to the next level’. A knowledge exchange EBF meeting entitled ‘China Days - Meet the Dragon’ is planned for 11–12 September in Berlin, Germany. The 7th open meeting, with the theme ‘Beyond the Horizon - Painting a new Landscape’ is planned for 19–21 November in Barcelona [14] . Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Meesters RJW, Zhang J, van Huizen RJW, Hooff GP, Gruters RA, Luider TM. Dried matrix on paper disks: the next generation DBS microsampling technique for managing the hematocrit effect in DBS analysis. Bioanalysis 4(16), 2027–2035 (2012).
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Sparrow SS, Robinson S, Bolam S et al. Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: a cross-company review. Regul. Toxicol. Pharmacol. 61(2), 222–229 (2011).
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Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry Bioanalytical Method Validation, DRAFT Guidance. FDA, Rockville, MD, USA (2013).
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US Department of Health and Human Services, US FDA, Center for Devices and Radiological Health, Center for Biologics Evaluation and Research, Center for Drug Evaluation and Research. Guidance for Industry and Food and Drug Administration Staff – In vitro Companion Diagnostic Devices. DRAFT Guidance (2011). FDA, Rockville, MD, USA (2011)
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De Vries R, Barfield M, van de Merbel N et al. The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2147–2160 (2013).
European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Method Validation. European Medicines Agency, London, UK (2011).
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Cobb Z, de Vries R, Spooner N et al. In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2161–2169 (2013).
O’Hara DM, Theobald V, Egan AC et al. Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents. AAPS J. 14(2), 316–328 (2012).
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European Bioanalytical Forum. www.europeanbioanalysisforum.eu
References 1
2
Kudoh S, Timmerman P, Van Amsterdam P et al. Global Harmonization on Regulated Bioanalysis and Global Bioanalysis Consortium: a historic perspective on the evolution of regulated bioanalysis since 1990 and why globalization requires harmonization. Pharm. Tech. Japan 28(3), 489–499 (2012). Van Amsterdam P, Arnold M, Bansal S et al. Global Bioanalysis Consortium: working towards a functional globally acceptable and harmonized guideline on bioanalytical method validation. Bioanalysis 2(11), 1801–1803 (2010).
3
European Bioanalytical Forum. http://bru2011.europeanbioanalysisforum.eu
4
Timmerman P, White S, Globig S, Lüdtke Silke, Brunet Leonarda, Smeraglia John. EBF recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis 3(14), 1567–1575 (2011).
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Timmerman P, White S, Cobb Z, de Vries R, Thomas E, van Baar B. Update of the EBF recommendation for the use of DBS in regulated bioanalysis integrating the conclusions from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2129–2136 (2013).
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For reprint orders, please contact [email protected]
Moving forward together: “we are making progress”
The 6th European Bioanalysis Forum Open Meeting
William D van Dongen* & Luuk C Renfurm
20–22 November 2013, Hesperia Tower Hotel, Barcelona, Spain
TNO Triskelion BV, Utrechtseweg 48, 3704 AV Zeist, The Netherlands *Author for correspondence: [email protected]
At the 6th European Bioanalysis Forum Open Meeting, held from 20–22 November 2013 in Hesperia Tower Hotel, Barcelona, Spain, bioanalytical experts from pharmaceutical industry, academia, contract laboratories and regulatory bodies discussed current topics of interest in bioanalysis. 450 delegates from more than 170 institutes and companies participated in 75 open and stimulating presentations regarding the new US FDA Guidance for industry, technology updates, including liquid chromatography– mass spectrometry of proteins and antibody–drug conjugates, dried blood spots, sampling and extraction and regulatory aspects of, for example, flow cytometry, parallelism, and interferences in ligand-binding assays. This article aims to provide an overview of the highlights discussed at the meeting.
6th European Bioanalysis Forum Open Meeting The 6th European Bioanalysis Forum Open Meeting was held from 20–22 November 2013 in Hesperia Tower Hotel, Barcelona, Spain. The EBF organization, moving forward under the enthusiastic and energetic leadership of Peter van Amsterdam and Philip Timmerman, assured that all the relevant developments and issues in the comprehensive field of bioanalysis are brought to the footlight. At least 450 bioanalytical experts, lab managers of pharmaceutical companies and contract research organizations shared scientific developments, procedures, opinions, regulatory aspects and global harmonization in the field of bioanalysis. Besides organizing open meetings, EBF also defines the most urgent and relevant bioanalytical themes and assigns them to topic teams, involving bioanalytical experts from pharmaceutical and CRO companies. The topic teams use surveys, its expertise, interviews, literature to provide clarity, produce recommendations and white papers [1,2]
10.4155/BIO.14.53 © 2014 Future Science Ltd
often resulting in improved guidance and regulatory documentation of the EMA. Several topic teams reported their progress on the open meeting, for example, ‘IS variability’, ‘the importance of parallelism during validation of ligand binding assays’ and ‘flow cytometry in regulated bioanalysis’. As always, the EBF meeting in beautiful Barcelona was an excellent opportunity for networking and informal meetings. The exhibition floor was filled with 45 booths and the attendees had intensive interactions with CROs en vendors of equipment. The highlights of the different sessions are discussed below. Scientific & technological developments in bioanalysis Peptide & protein analysis with LC–MS
Due to the large number of requests for presenting the subject of protein LC–MS bioanalysis, the organization took the opportunity to organize a last-minute session in this emerging field. Significant progress was reported in comparison to data reported at the
Bioanalysis (2014) 6(9), 1159–1165
part of
ISSN 1757-6180
1159
Conference report EBF 2011 Focus Meeting ‘Large meets Small’ in Brussels, Belgium [3] . The session was kick-started by Nico van de Merbel (University of Groningen, The Netherlands). He postulated existential bioanalytical questions, such as what is ‘the concentration’ of a protein? Because of its multidimensional complex structure, a one dimensional concentration of a protein cannot be determined, as this depends on the sought information and the applied tools. Ligand-binding assay (LBA), the current standard for therapeutic protein bioanalysis, measures the unbound intact 3D domain of the therapeutic protein binding to the assay’s capture reagent, can be considered as a ‘biological concentration’. In contrast, van de Merbel explained that LC–MS will give a ‘chemical concentration’ by monitoring a unique primary structure piece of the protein. The advantages that LC–MS has over LBA is that no highly selective biological reagents have to be developed, which can take up to six months. He clearly explained that the biological and chemical protein level determined in the same sample with, respectively, LBA and LC–MS may not necessarily be similar. Rand Jenkins (PPD, Richmond, VA, USA) and William van Dongen (TNO Triskelion, Zeist, The Netherlands) continued this session by demonstrating the potential of immunoprecipitation, using protein A capture and LC–MS to support monoclonal antibody (mAb) development. Jenkins reviewed the progress of the development of a nano-LC–MS assay to simultaneously quantify trastuzumab and its soluble sHER2 ECD receptor, associated with HER2-overexpressing cancers. William van Dongen showed the feasibility of nano LC–MS to determine therapeutic mAbs at sub ng/ml levels. For their nano-LC experiments, Jenkins applied the Waters TRIZAIC™ bioanalytical nano-LC tile and Van Dongen, the Thermo EasySpray™ technology. The presented data illustrated that nano techniques have potential for bioanalytical quantitation of therapeutic proteins. Continued developments in DBS
EMA does not yet accept DBS technology to support drug-development studies and the US FDA requires that DBS delivers the same concentration data as conventional techniques. Since, DBS is a tool that has potential for regulated bioanalysis, EBF monitors innovations that overcome current essential limitations of DBS [4,5] . Variation in the blood hematocrit (Ht) level is one of the major issues as it influences spot size and extraction recovery and consequently may cause poor precision when only part of the spot is extracted [6] . Bert Ooms (Spark Holland, Emmen, The Netherlands) gave a potential solution to this issue using the
1160
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fully automated DBS system of Spark Holland. This system automatically punches the whole bloodspot of DBS card followed by temperature-enhanced flowthrough desorption, on-line solid-phase extraction and mass spectrometry. Ooms demonstrated that hematocrit-variations can be handled by this system as near 100% recovery was obtained independent of HT values ranging from 0.3–0.7. Another major concern is the PK/TK measuring in blood, rather than plasma, can affect quantitation accuracy [7] . As a result of that plasma microsampling strategies have been reported in the literature. [8] . Jack Henion (Quintiles Bioanalytical and ADME laboratories, NY, USA) presented a device utilizing a dual sampling card where the upper layer filters the red blood cells (RBC) from applied low volumes of whole blood, so plasma micro samples are collected on the lower layer generating dried plasma spots. This device was inspired on a commercially available device, originally designed by Yorktest for at-home (pointof-care) measurement of homocysteine development. Ynze Mengerink (Chemelot, Geleen, The Netherlands) presented promising data about the use of new card material available that generates blood spot sizes independent of the Ht value. Innovative applications in sampling & extraction
Kathryn Chapman (NC3Rs, London, UK) introduced this session. She explained that the largest driver for the number of rodents in regulatory toxicology studies is the collection of samples for TK analysis [9] . The use of capillary microsampling techniques allows the reduction of the use of satellite animals for TK analysis. Kathryn summarized the conclusions of the NC3Rs workshop held in May 2013 all 80 delegates showed an interest in the use of microsampling for GLP regulatory toxicology studies at their companies. Nevertheless, there was still some hesitation to apply microsampling for main study animals, as its impact on the clinical pathology, pathology and other readouts as CNS effects is not known. During the workshop it was agreed that an evidence-base is needed to demonstrate that microsampling from main study samples does not compromise study end-points. In general, the conclusion of this session was that capillary microsampling is becoming more and more important, especially when samples have to be collected from children or small animals. A number of microsampling capillaries are commercially available and several methods using capillary microsampling have been validated in accordance with the FDA Guidance.
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Analytical challenges of antibody–drug conjugates
Antibody–drug conjugates (ADCs) are complex drugs consisting of cytotoxic drugs covalently attached to mAbs via a chemical linker, to achieve targeted delivery. The degree of complexity of ADCs is increased as it represents a heterogeneous mixture with different drug antibody ratios (DARs). It is important to assure the stability of the ADC, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk due to its high potency. Bernard Beckermann (Bayer Pharma, Berlin, Germany) explained that the above-mentioned complexity, heterogeneity and required stability is reflected in the desired bioanalytical methods for ADC development programs, for example, total amount of cytotoxic drug containing antibodies, drug-free mAbs, the released cytotoxic drug, the drug to antibody ratio (DAR) and the potentially formed antibodies against the ADC (ADA). Depending on the specific molecular design and the endpoints the bioanalytical strategy needs to be defined and usually includes ELISA formats for the antibody part, LC–MS for the released cytotoxic drug, high-resolution accurate mass for characterization of DAR during circulation and cell based assays for ADA testing. Steffen Gross (Paul-Ehrlich-Institut, Langen, Germany) discussed the challenges of the analytical characterization of ADC drug substance, where he also pointed out that special emphasis should be taken on the stability of the ADC. Jasja Wolthoorn (TNO Triskelion, Zeist, The Netherlands) gave a nice overview of the bioanalytical strategies that are recommended during the different stages of development of the ADC development program. Depending on the tools available and the (pre-) clinical phase, initial analysis might focus on the analysis of the antibody part (DAR≥0) of the ADC, the toxin, anti-drug antibodies and the average DAR. Later on it is recommended to use techniques that enables to get information on DAR distribution of the ADC.
Conference report
(TQMS) (Sciex APl4000) and a HRMS (Waters Synapt G2), under identical UPLC conditions. She demonstrated that for bioanalysis of a small molecule in bile, plasma and urine, the quantitative performance was comparable for HRMS and TQMS systems. The qualitative output of the HRMS was valuable, major excretion pathways have been identified as well as a circulating metabolite. However, as the UPLC conditions were optimized for quantitation, the metabolites did largely co-elute. Therefore, optimization with respect to the separation of parent and metabolites is in most cases required. ln practice, Lieve recommended to apply Quan/Qual bioanalysis only for selected cases, as it is a compromise between Quan efficiency and Qual data quality, and workflows are not as simple as promised. Mohammed Abrar (Unilabs York Bioanalytical Solutions, York, UK) compared TQMS (Waters TQS) and HRMS (Thermo QExactive) for bioanalysis of a 5 kDa therapeutic oligonucleotide in human plasma. Mohammed explained that LC–MS bioanalysis of oligonucleotides is challenging due to the presence of the multiple negatively charged phosphate groups in the analyte. For LC separation, the necessary ion pair chromatography is challenging to optimize and electrospray generates multiple charged states that are further complicated by the exchange of H+ for Na+, K+ at the phosphate groups. As a result bioanalytical LC–MS methods for oligonucleotides are relatively insensitive and subject to background ion interferences. Mohammed demonstrated that the very high resolving power of the Q-Exactive was able to give clean chromatograms of the 5 kDa therapeutic oligonucleotide at 0.4 ng/ml, whereas the TQ-MS gave significant background signal at this level. The overall outcome of this session was: apply HR-MS only for certain studies where metabolite lD is needed and in cases where the resolving power is required to obtain clean SRM chromatograms. Bioanalytical regulation & guidance updates
High-resolution MS
New FDA Guidance for Industry
The availability of well-priced accurate high-resolution mass spectrometers (HRMS), featuring enhanced sensitivity, resolving power, mass accuracy, acquisition speed and dynamic range makes this technique affordable for the bioanalytical laboratories. HRMS is capable of, besides target quantification of drugs and their anticipated metabolites, qualification of still undisclosed metabolites, endogenous compounds responsible for matrix effects, and/or potential biomarkers accessible in post-dose preclinical samples. Lieve Dillen (Janssen, Beerse, Belgium) made a bioanalytical comparison between a triple quadrupole
The FDA draft Guideline on bioanalytical method validation is available [10] , and as part of the feedback process, AAPS/FDA organized a meeting scheduled for December 2013 referred to as Crystal City–V (Baltimore). The EBF program of the 6th Open Symposium (20–22 November 2013, Barcelona, Spain) included a ‘Preparation session’. In this session feedback was received and collected from the EBF member companies. There were many comments. For example, for the ligand-binding assay part, many criteria have been copied directly from the chromatographic assays and modifications of the text are highly recommended. It was
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Conference report agreed that all feedback will be summarized and the EBF steering committee will represent all EBF companies at the Crystal City meeting in December 2013. Challenges for flow cytometry in regulated bioanalysis
• Discuss the project as early as possible with the CRO and assure that technical, operational, cultural differences between the laboratories are identified/understood;
Flow cytometry is increasingly used for safety/ exploratory purposes in (pre-) clinical trials, for example for immunogenicity, PK, potency testing, receptor occupancy testing. There are numerous advantages of flow cytometry such as quick quantitative analysis since multiple markers can be analyzed simultaneously. The main issue discussed in this session, organized by Topic Team 32, was the lack of guidance on how to perform flow analysis under regulated regime. Representatives from the FDA, AAPS, ICCS and ICSH request for clear guidelines. Topic Team 32 will soon publish a White Paper with the EBF’s view.
• Ensure noncommercial reagent early supply and continuity;
Assay transfer-expectations and practicalities
• Blame the CRO, ensure that solved issues go through a closing process.
The process of assay transfer requires intensive communication such as clear definition of final goal of the project, a mutually understood and accepted plan, and regular communication between sending and receiving lab. Especially assays for biopharmaceutical, due to their size and complexity, and the inability to characterize biopharmaceuticals fully, their instability, makes assay transfer comparably more complex than for small molecules. Cheryl McAlpine (Merck Millipore, Oxford, UK) discussed the highly challenging transfer of neutralizing anti-drug antibodies (nAb) assays. For a receiving lab to be successful it is critical to establish at the set-up stage of any assay transfer, the assay status at the transferring lab. This can vary from merely conceptual to the transfer of a fully validated protocol. In either case, it is essential that the transferring partner fully understands the mechanism of action, and that the most relevant of multiple signaling pathways of the in vivo effect is used. Also one should aim that, after implementation at the receiving laboratory, target expression levels are adequate, stable and specific, the assay’s dynamic range is known and cell growth conditions/seeding density are optimized. Matrix interference effects from human plasma/serum of different populations must also be assessed and the final assay must meet the requirements of clinical sample analysis. Fabienne Deckert-Salva (Novartis Pharma, Mulhouse Area, France) presented a useful do/do not list with respect to outsourcing of biological assay transfer: Do: • Outsourcing is a win–win collaboration, both parties have the same goals;
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• Build mutual trust;
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• Ask for regular feedback on methods, coordination and monitoring processes and so on. Do not: • Underestimate CRO expertise (CROs are exposed to other external expertise and opinions); • Hide any weakness/complexity of the transferred method;
From biomarker to diagnostics or personalized medicine
John Mills (AstraZeneca, Stockport, UK) introduced this session by stating “more than 90% of drugs only work in 30–50% of the people.” Personalized healthcare (PHC) can significantly improve this number and has the potential to benefit prescribers, payers, regulators, and obviously patients by aiming to deliver the right medicine, to the right patient at the right dose and time. The potential to use biomarkers for classifying patients into groups that are less or more likely to benefit from a certain therapy, is anticipated to have a major impact on the integrated development of new drugs and their companion diagnostic method. John presented some illustrative examples in which therapies (e.g., Vemurafenib, Crizotinib) are matched with specific patient population characteristics using companion diagnostics, and discussed the challenges and implications of this approach to a drug development program and the involved bioanalysis and regulatory requirements. John made it clear that companion diagnostics are considered to be high risk and therefore likely to need to meet the highest regulatory hurdles in the US, for example, Class III, Pre Market Approval (PMA) required [11] . Suntje Sander-Struckmeier (Abbott, Weesp, The Netherlands) and Peter van Amsterdam (Abbott, Weesp, The Netherlands) presented an example of optimization treatment efficacy diagnosis of pancreatic exocrine insufficiency (PEI). They discussed the use of faster, better and less laborious tools for determine the efficacy of
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pancreatic enzyme replacement therapy (PERT). Instead of complete stool fat excretion measurement during 72-h collection, measurement in only sparse stool samples was evaluated. Potentially more efficient alternatives under investigation are 13C-mixed triglyceride breath testing and the malabsorption blood test. Interpretation & implementation of guidance on haemolyzed/hyperlipidemic plasma, & co-administered drugs
Benno Ingelse (MSD, Oss, The Netherlands) presented the progress of EBF’s topic team 15 sorting out “how to deal with haemolysed and hyperlipidemic samples for LC–MS bioanalysis”. The EMA guidelines [12] mention that hemolyzed and hyperlipidemic matrices must be included as part of the selectivity assessment. Hemolyzed plasma contains hemoglobin from damaged red blood cells. Hyperlipidemic plasma, originating from humans having a higher risk for heart disease, has higher triglyceride and cholesterol levels. Plasma samples with >240 mg/dL cholesterol, corresponding to high heart disease risk are considered to be hyperlipidemic. Hemolyzed and hyperlipidemic matrix samples can result in ion-suppression or enhancement during LC–MS analysis. The stable isotope-labeled (SIL) IS do not always compensate sufficiently for these effects especially close to LLOQ. Benno explained how to prepare suitable hemolyzed and hyperlipidemic matrices for matrix effect testing. Haemolysed plasma can be self-prepared by spiking plasma with 2% (V/V) hemolyzed blood to plasma. Hemolyzed blood is typically prepared by subjecting control whole blood to three freeze-thaw cycles. There is still discussion how hyperlipidemic samples should be obtained; one can either use plasma with a predefined minimum (>240 mg/dL) cholesterol level or triglyceride level, or selfprepare samples by spiking plasma with Intralipid. Based on the outcome of a recently held EBF survey an EBF recommendation on haemolysed/hyperlipidemic samples will be published soon. Progress was also reported in the issue of co-administered drugs. The EBF survey held on co-medication testing will soon result in a risk-based tiered approach recommendation, where also scheduled or nonscheduled co-medication will be taken into account. Interpretation & implementation of Guidance expectations IS variability
As also discussed in the previous section, SIL IS do not always compensate sufficiently during LC–MS bioanalysis. Based on an EBF survey, an EBF recommendation will be issued soon. The recommendation will address both individual irregularities as well as systematic variability.
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Conference report
Analysis of parallelism for biomarkers & therapeutic proteins
Parallelism is defined by the lack of an increase or decrease in concentration along the standard curve after correction of the dilution factor of a sample. During the assessment of parallelism a plot of the determined concentrations of the analyte in an incurred sample at multiple dilutions against the expected concentrations at each dilution should have a slope near 1.0. Incurred samples are recommended, since it is an intrinsic sample issue, and not related to the applied method. The incurred samples should obviously be individual and not pooled to avoid averaging out effects. Also preferably later time point samples should be tested for parallelism to determine the binding protein effects. Issues that were discussed during this session were: • What to do if samples do not dilute parallel? A potential solution might be to use a quasi-quantitative approach by comparing pre- with post-dose samples. Factors to consider in case of non-parallelism: drug aggregation, drug stability in vivo, antidrug antibody presence, endogenous binding partners, and different 3D structural forms of the drug; • Often no access to study samples during validation; • GCP issue: the use of incurred samples required for parallelism testing is often not covered by an informed consent; • Global view on parallelism by the GBC: parallelism testing should be science based and testing on a routine basis is not recommended. Some examples of parallelism were presented, for example, a bridging ELISA for PK analysis of a mAb, where the coating concentration was critical for good assay performance. Bruno Boulanger (Arlenda, Liège, Belgium) discussed the assessment of parallelism in bioassays from a statistical point of view. There are two approaches. The four-parameter logistic curve approach was based on measuring the ratio of two slopes and determination of the EC50 differences between the lines, which should be close to one. The other approach, the linear assay approach, takes the linear range of the S-curve into account. Boulanger recommended the “4-parameter logistic curve approach”, as it is easy to implement on the laboratory and there are less biases in the method. Interferences & surprises in ligand-binding assay method development
Critical reagents (CR) in ligand binding assays (LBAs) has become a topic of attention with the publication by the AAPS Ligand binding in the 21st century:
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Conference report recommendations for characterization and supply of critical reagents [13] . Critical reagents are the backbone of all LBAs, and appropriate quality management of CRs minimizes assay performance issues caused by declining reagent activity throughout all stages of the drug development process. The first speaker in this session, Helen Young (Quotient Bio Analytical Sciences, Teddington, Middlesex, UK), discussed the practical issues around monitoring of critical reagents in a clinical laboratory. She gave an extensive list of CRs: reference standards, antibodies, in-house conjugated reagents, cell lines, matrix (e.g. serum, especially for biomarker assays), precoated assay plates, commercial kits reagents, blocking reagents, assay buffers, substrate, etc. Young recommends to implement a procedure that defines what CRs are, how to monitor the performance of the CRs, what steps to take when the performance of the CRs change, and how to store, treat, and document CRs. Martin Nemansky (PRA Bioanalytical, Zuidlaren, The Netherlands) zoomed in on the practical interpretation of the guidelines or white papers for immunogenicity of anti-drug antibody (ADA) testing. He mentioned that “guideline interpretation is challenging when there is an assay issue” and fit-for-purpose solutions are required. He illustrated this by several examples, e.g. a low positive-control sample was screened positive for ADAs, but could not be confirmed in the confirmatory assay due to low noise. A practical solution was to increase the screening cut point more close to the confirmatory cut point. Bioanalysis Young Investigator Award Each year, Bioanalysis run the Young Investigator Award to identify and reward promising early-career researchers in bioanalysis. Peter van Amsterdam presented the 2013 Bioanalysis Young Investigator Award. This year 18 young scientists were nominated for the Award. This year’s winner of the Award was Anthony O’Donoghue (University of California, CA, USA) for his work on the development of a technique to simultaneously assay all proteases in a biological sample at exquisitely low concentrations. In continuation, Anthony presented his award winning research on the development of affinity microcolumns for high throughput bio interaction analysis. This global identification of protease specificity allowed him to determine the proteolytic signatures of cancer cells and
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parasitic organisms and to subsequently identify the major proteolytic enzymes involved. Conclusion & future agenda Again, the open meeting successfully connected the bioanalytical community and accomplished this year’s mission of ‘moving forward together’. Almost 75 interesting presentations were shared in 18 different sessions. Significant progress was reported in the new emerging field of bioanalytical LC–MS of therapeutic proteins. Developments in DBS, sampling and extraction, the possibilities and limitations of HRMS for bioanalysis were all very well covered in their respective sessions. In addition, the contours of feasible bioanalytical strategies for ADCs, currently the biggest challenge for the bioanalytical specialist, are becoming distinct. Regulation and guidance updates considering flow cytometry, hemolyzed/hyperlipidemic plasma, IS variability, assay transfer-expectations and practicalities, parallelism for biomarkers and therapeutic proteins, ligand-binding assay interferences and biomarkers and diagnostics for personalized medicine were all comprehensively covered. Again, the EBF organizers succeeded very well in their mission to share, discuss, optimize and seek alignment on a broad array of bioanalytical topics including science, technology and regulatory issues. For 2014, the EBF agenda holds two focus workshops in Brussels, Belgium. At 5–6 February 2014 the workshop is focussed to standardized lab manual for bioanalytical support in clinical studies, and at 17–18 June focussed on the tiered approach, entitled ‘taking the tiered approach to the next level’. A knowledge exchange EBF meeting entitled ‘China Days - Meet the Dragon’ is planned for 11–12 September in Berlin, Germany. The 7th open meeting, with the theme ‘Beyond the Horizon - Painting a new Landscape’ is planned for 19–21 November in Barcelona [14] . Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Meesters RJW, Zhang J, van Huizen RJW, Hooff GP, Gruters RA, Luider TM. Dried matrix on paper disks: the next generation DBS microsampling technique for managing the hematocrit effect in DBS analysis. Bioanalysis 4(16), 2027–2035 (2012).
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Sparrow SS, Robinson S, Bolam S et al. Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: a cross-company review. Regul. Toxicol. Pharmacol. 61(2), 222–229 (2011).
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Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry Bioanalytical Method Validation, DRAFT Guidance. FDA, Rockville, MD, USA (2013).
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US Department of Health and Human Services, US FDA, Center for Devices and Radiological Health, Center for Biologics Evaluation and Research, Center for Drug Evaluation and Research. Guidance for Industry and Food and Drug Administration Staff – In vitro Companion Diagnostic Devices. DRAFT Guidance (2011). FDA, Rockville, MD, USA (2011)
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De Vries R, Barfield M, van de Merbel N et al. The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2147–2160 (2013).
European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Method Validation. European Medicines Agency, London, UK (2011).
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Cobb Z, de Vries R, Spooner N et al. In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2161–2169 (2013).
O’Hara DM, Theobald V, Egan AC et al. Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents. AAPS J. 14(2), 316–328 (2012).
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European Bioanalytical Forum. www.europeanbioanalysisforum.eu
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Kudoh S, Timmerman P, Van Amsterdam P et al. Global Harmonization on Regulated Bioanalysis and Global Bioanalysis Consortium: a historic perspective on the evolution of regulated bioanalysis since 1990 and why globalization requires harmonization. Pharm. Tech. Japan 28(3), 489–499 (2012). Van Amsterdam P, Arnold M, Bansal S et al. Global Bioanalysis Consortium: working towards a functional globally acceptable and harmonized guideline on bioanalytical method validation. Bioanalysis 2(11), 1801–1803 (2010).
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European Bioanalytical Forum. http://bru2011.europeanbioanalysisforum.eu
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Timmerman P, White S, Globig S, Lüdtke Silke, Brunet Leonarda, Smeraglia John. EBF recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis 3(14), 1567–1575 (2011).
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Timmerman P, White S, Cobb Z, de Vries R, Thomas E, van Baar B. Update of the EBF recommendation for the use of DBS in regulated bioanalysis integrating the conclusions from the EBF DBS-microsampling consortium. Bioanalysis 5(17), 2129–2136 (2013).
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