Conference Report

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Summary of the 2014 Land O’Lakes Bioanalytical Conference

This Land O’Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the University of Wisconsin-Madison (USA). The purpose of this 3-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program is a mixture of lectures, interactive discussions and a poster session. This report summarized the presentations at the Fifteenth Annual Conference. Keywords: analytical kits • bioanalysis • biomarkers • biosimilars  • flow cytometry • LC–MS technology • microsampling • professional development • regulatory guidance

Background The 15th Annual Land O’Lakes Bioanalytical Conference, titled ‘Adapting to Diverse Bioanalytical Technologies and a New Regulatory Guidance’, was held during 21–24 July 2014. The program represents the second year with several changes to the format used in previous conferences: changing of the venue to Madison and a conference facility on the University of Wisconsin Campus; shortening of the program by one day with educational sessions in the afternoons; and conference material presented in electronic rather than print formats. The conference brings together an international group of bioanalytical scientists with the express intent of discussing cutting-edge science for small and large molecule bioanalysis in a relaxed atmosphere that promotes networking opportunities. It provides an educational forum with presentations by scientific leaders from industry, contract laboratories and regulatory authorities. The success of the program is due to the Planning Committee, which consists of scientists involved with bioanalysis on a daily basis, and their ability to identify timely

10.4155/BIO.14.243 © 2014 Future Science Ltd

topics and presenters who are experts in the field. Specific objectives for the conference and a list of the Planning Committee members can be found at [1] . On Monday evening the conference opened with a science oriented, but not bioanalytical specific presentation on translational biomarkers by Joshua M Lang (University of Wisconsin Carbone Cancer Center, WI, USA), which focused on clinical research with circulating tumor cells and applications for the early detection of prostate cancer.

James E DeMuth*,1, Eric N Fluhler2, Stacy Ho3, Matthew Cyronak4, Douglas J Turk5, R John Stubbs6 & Jeffrey Moran7 1 University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA 2 Pfizer Research, Pearl River, NY, USA 3 Sanofi, Waltham, MA, USA 4 Alliance Pharma, Inc., Malvern, PA, USA 5 Pharma Medica Research Inc., Mississauga, Ontario, Canada 6 Stubbs and Hensel Pharma Consulting, LLC, Blue Bell, PA, USA 7 inVentiv Health, Lincoln, NE, USA *Author for correspondence: [email protected] wisc.edu

Three morning plenary sessions Each day of the conference began with a plenary session consisting of four presentations, each followed by an open discussion. The first plenary session focused on two important themes: development of biosimilars; and the use of biomarkers in drug development. Hazel Gorham (PRA Health Sciences, Berkshire, UK) opened the session with a talk on biosimilars from a clinical and regulatory perspective, and an overview of the current biotherapeutics market (global biologics sales growth, pending patent expirations, potential influence of biosimilars on drug costs).

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Conference Report  DeMuth, Fluhler, Ho et al. Dr Gorham then contrasted the development of the European and US regulations and reviewing comparative testing performed during the development of biosimilar medicines (including immunogenicity). She emphasized that biosimilar development is still somewhat of an art, requiring flexibility in scientific approach, applied technology and robust regulatory interaction. Xun Wang (QPS, DE, USA) continued the discussion on biosimilars presentating bioanalytical assay issues. He discussed the lack of specific regulatory guidance on bioanalytical approaches to development and the unique challenges that biosimilars present to the bioanalyst. Dr Wang discussed development and application of  anti-drug antibody (ADA) assays for biosimilars and critical success factors along with the pros and cons of a one assay vs two assay approach. He shared two case studies where acid dissociation and immuno-depletion steps were required to resolve target interference and drug tolerance issues. Susan Richards (Sanofi, MA, USA) discussed the role of biomarkers in personalized medicine with a detailed look at its utility with respect to patient diagnosis, selection of medications and dosing regimen, and how this approach promises to improve drug efficacy and safety. She described how understanding of biological systems and success of translational medicine relies on appropriate biomeasures and biomarkers. Dr Richards then used Sanofi’s efforts in Pompe Disease to illustrate what a broad clinical and translational biomarker program can look like and demonstrated the potential utility of a potential translational biomarker of enzyme activity. In the final presentation, Binodh DeSilva (Bristol-Myers Squib, NJ, USA) talked about biomarkers and dealing with endogenous analytes. She discussed the common issues that plague the measurement of endogenous biomarkers, including availability and consistency of reference standards (RS) and reagents, analytical selectivity, matrix interference, impact of the disease state, lack of parallelism between RS and endogenous analyte. She discussed the complexities associated with using surrogate matrices when analyte free matrix is not available and the importance of understanding the state of the analyte being measured (free, total or complexed). Dr DeSilva used five case studies to illustrate real life examples of the above issues. The second morning plenary session focused on new applications and challenges associated with LC–MS technology. Jianing Zeng (Bristol-Myers Squibb, NJ, USA) gave a brief overview of traditional techniques for determining concentrations of therapeutic proteins including ligand-binding assays (LBA) and then focused the remainder of the presentation on the advantages of using LC–MS alone or in combination

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with LBA to achieve greater selectivity and sensitivity. He presenting eight case studies that demonstrated the power of LC–MS in solving challenging problems from measuring picomolar concentrations of soluble targets to combo assays for monoclonal antibodies to the use of LC–MS for  neutralizing antibody (nAb) and ADA analysis. Dr Zheng stated it is critical to integrate LBAs with LC–MS techniques to aid in fast and informative decision making. Kevin Bateman, (Merck & Co., PA, USA) spoke on the use of highresolution MS (HRMS) to aid in identifying metabolites during early drug development. He described the benefits of accurate mass data from HRMS in combination with fast high-resolution chromatography, and how all-in-one data collection and mass defect filtering can provide structural information for discovery stage metabolite ID. Several examples were presented to demonstrate the usefulness. Dr Bateman shared his view on how metabolite ID workflow might appear in the future with HRMS and on how software will be key to success by taking advantage of the large amount of data generated. Troy Voelker (Tandem Labs, UT, USA) presented use of LC–MS in antibody–drug conjugates (ADCs) unconjugated payload analysis. He gave a basic introduction to the chemistry and use of ADCs and the many challenges encountered when attempting to measure the payload. He then presented case studies where LC–MS methods were validated and used in quantifying the payload and understanding its stability under various conditions. He emphasized the importance of LC–MS in achieving high sensitivity. The final speaker was Shane Needham (Alturas Analytics, ID, USA) who presented the use of nanoflow and microflow LC–MS/MS in the bioanalytical laboratory. Dr Needham posed the question, can MFLC-MS/MS be used routinely in the bioanalytical laboratory? Through examples he clearly argued that the technique can be used for both small molecules and protein therapeutics. He described through examples the validation of the technique in regulated bioanalysis and the impact of the technique on instrumentation. He concluded that microflow LC–MS/MS works as an efficient, productive, convenient and useful tool in bioanalysis. The final morning plenary session focused on regulatory issues affecting bioanalysis, especially related to the US FDA’s recent Draft Guidance for Industry: Bioanalytical Method Validation (2013). Suresh B Naraharisetti (US FDA, MD, USA) began with an overview of the draft Guidance, highlighting what was new, what has changed, and how public comments may influence the final document. Douglas M Fast (Covance, WI, USA) presented a perspective on the draft Guidance as it relates to small molecule bioanalysis. Dr Fast

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Summary of the 2014 Land O’Lakes Bioanalytical Conference

discussed a practical concern: should laboratory procedures be updated based on this draft document? His opinion agreed with the consensus of attendees; that laboratory procedures should be updated to conform with current industry standards, but not necessarily the draft Guidance. Fast highlighted some issues where FDA and industry reached consensus and discussed some additional industry concerns related to incurred sample reanalysis (ISR), reference powder expiry, and key differences between draft Guidance and that of the European Medicines Agency (2012). Marian Kelley (MKelley Consultant LLC, PA, USA) presented perspectives of the draft Guidance related to large molecule bioanalysis. She described discussions between FDA and industry, noting that consensus was reached across a variety of large molecules topics; though this consensus will require significant modification of the draft Guidance. Areas of agreement included selectivity, validation parameters, parallelism, inclusion of total error in the validation statistics, and range of the calibration curve. Kelley discussed areas of partial agreement, including ULOQ acceptance criteria and placement of quality controls. Elisabeth Groeber and Jenifer Vila (WIL Research, OH, USA) provided the final presentation by focusing on the quality of biomarker data as it relates to drug development decision making and regulatory requirements. Groeber delved into the implications of the draft Guidance for biomarkers being analyzed with conventional binding assays and diagnostic kits. She provided a history of biomarkers from bioanalysis-focused regulatory meetings and White Papers. Vila ended the session with case studies of biomarker bioanalysis supporting development in specific therapeutic areas, describing how expectations for the quality of biomarker data may vary from early to late development depending on decisions being supported by the data and regulatory implications. Afternoon sessions Because of the change in venue and shorter schedule, traditional afternoon workshops were replaced with educational sessions. Tuesday afternoon’s program focuses on Timely Analytical Topics. Jian Chen (Celgene Coropration, NJ, USA) shared Celgene’s experience on using dried blood spot (DBS) to support the toxicology and first-in-human studies. After evaluating the DBS technologies using various compounds from different chemical classes, one compound was chosen for comprehensive evaluation. Rat, dog, and human DBS methods of the compound were successfully validated. It was found that recoveries of samples above limit of quantification were solvent dependent and the composition of extraction solvent needs to be

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optimized to obtain accurate results. The toxicokinetic and pharmacokinetic data from DBS were comparable to plasma results. Blood area under the curve (AUC) was parallel with plasma AUC and the AUC blood/ plasma ratio was close to the blood-to-plasma partition coefficient. The second timely analytical topic discussed was analytical kits, presented by Paul Rhyne (Quintile, GA, USA). He explained the considerations for commercializing analytical kits which usually undergoes five stages: • Define requirements, the right market for the kit must be defined and market plan created accordingly; • Ensuring reagent access and if necessary commercial and diagnostic rights have to be obtained; • A method has to be established and optimized to achieve acceptable assay performance; • Produce a kit where every detail is considered, including reagent lot consistency, packaging, and instructions; • Have support that ensures kit performance consistency and quality control. The session ended with an open discuss involving the audience on daily challenges and opportunities that exist in daily practice. The highly interactive session was facilitated by Chad Briscoe (PRA Health Sciences, KS, USA) and Qin C. Ji, (Bristol-Myers Squibb Company, NJ, USA). The focus of the Wednesday afternoon presentations were on applications of cytometry in bioanalysis. Devangi Mehta (Biogen Idec, MA, USA) discussed the relevance of flow cytometry analysis for guiding drug development. Discussed were qualitative and quasiquantitative biomarker assays to assess toxicity and tolerability of drug candidates, evaluate target engagement and support dose selection. Dr Mehta reviewed the difficulties with cytometry based approaches; including sources of analytical variability, inherent complexity of the assays and technical/logistical hurdles of utilizing cytometry in large scale clinical trials. Challenges with gating subjectivity, stability (cell functionality), reagent evaluation and the need for real time sample processing were also identified as potential hurdles. She concluded with several case studies to support clinical development of Daclizumab High Yield Process (DAC HYP) in multiple sclerosis including CD25 receptor occupancy and expression for measuring regulatory T-cells and enumeration of CD56 NK cells. In the second presentation Yongliang Sun (Pfizer, CT, USA) described the combination of flow

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Conference Report  DeMuth, Fluher, Ho et al. cytometry with microscopy (Amnis) for quantitative image analysis. The technique utilizes the statistical power and high speed of flow cytometry combined with the imaging power and information content of microscopy. Specific regions of interest are defined by ‘masks’ that are used to evaluate a variety of commonly assigned features such as object size, shape, texture, signal strength and many others. These features are associated with pre-defined applications indicative of many cell based processes, including cell signaling, cell cycle and DNA damage. The utility of the technique for biomarker analysis was illustrated through mechanistic PK/PD modeling studies, examples of cell signaling analysis in drug discovery and applications with apoptosis. The second part of Wednesday afternoon was the Analytical Investigators Forum which provided a platform for scientists to present research building on the major themes of the conference. John Mehl (Bristol-Myers Squibb, NJ, USA) discussed on the use of highly sensitive and versatile quantitative immunocapture MS to support in vivo studies in early discovery. Mehl compared the results with an ELISA assay of an anti-drug antibody where the later time points showed interference. The LC–MS/MS assay was found to be more specific for measuring tissue levels than free concentrations in the ELISA assay. He discussed the use of immunocapture beads in automated systems for sample preparation of human monoclonal antibody (mAb) drugs in a 96-well plate format which was used successfully to measure ustekinumab and trastuzumab concentrations in a variety of tissue matrices in combination with DBS sample collection. Haixing Wang (Sanofi, MA, USA) gave the second presentation on the development of a DBS LC–MS/MS assay to quantitate a panel of sphingolipids biomarkers for enzyme replacement therapy. Sphingomyelin and Ceramide are biomarkers used in the treatment of Niemann-Pick A/B diseases. Glucosylcermaide (GS-1) is an efficacy marker for Gaucher disease. These biomarkers show poor solubility in blood resulting in high assay variability. Blood volume, hematocrit values, and DBS size were also optimized to give adequate LLOQ and accuracy and precision data and assays that met FDA and European Medicines Agency assay validation

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criteria. Tao Ye (Biogen Idec, MA, USA) concluded the forum with a presentation on the development of LC–MS/MS assays for peptide biomarkers. The assay requirements were high specificity, five orders of magnitude dynamic range, multiplexed detection, no antibodies required, complimentary results to ligand binding assays, and cost effective. Ye discussed the assay of amyloid peptides in the treatment of Alzheimers disease using LC–MS/MS assays with an LOQ of 10 pg/ml. Substance P was also successfully assayed using LC–MS/MS with an LOQ of 1 pg/ml compared with 60 pg/ml using a ligand binding assay (LBA). This proved to be challenging due to the instability of Substance P in human plasma that required the addition of an enzyme inhibitor. Ye also discussed the digestion of large peptides/proteins prior to LC–MS/MS assay and the problems of ion suppression and interference that were encountered. Conclusion This conference contrasts to larger international conventions, because of the informal, non-commercial and informal setting, and because the attendees had ample time to meet each other, discuss their organizations, bioanalytical sciences and challenges they face in daily practice. Feedback was very positive and a summary of the evaluation results are presented on the website for the 2015 program. Next year’s conference is currently being developed and will be posted on the University’s website when finished [1] . 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.

Reference 1

15th Annual Land O’Lakes Bioanalytical Conference. http://ce.pharmacy.wisc.edu/2014JulyLOLInfo 

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Summary of the 2014 Land O'Lakes Bioanalytical Conference.

This Land O'Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the Universi...
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