SLAS2015 Presentations On-Demand

Recorded On: 02/11/2015

10 Sessions from the SLAS2015 Conference:

Spheroid Culture Screening in Anti-Cancer Drug Discovery
An Automated Open Platform for Exclusion-based Sample Preparation: Getting More Information from Limited Patient Samples
The Phenotypic Screening "Rule of 3": Developing More Predictive Assays
Novel Acoustic Loading of a Mass Spectrometer- Towards Next Generation High Throughput MS Screening
Droplet-Based Three Dimensional Cell Migration Assay with Flow Cytometry Based Automated Analysis
Precise Genome Editing and Stem Cell Technologies - Novel Tools for Novel Medicines
From Brackets to Body Parts - How 3D Printing Is Changing the Way We Work
Screening for Novel Inhibitors of Thymidylate Synthase Using CETSA - a High Throughput Target Engagement Assay
Real Time Predictive Modeling of Clinical Samples in Transit to Ensure Sample Viability
Biomarker Qualification at CDER, FDA

Stephen P. Trowbridge,

Director

Initially working in the agricultural sector then moved into the pharmaceutical industry. For the past 25 years working at GlaxoSmithKline, starting in Safety Assessment then in Sample Management. Specializing in automation, engineering and robotics, especially in the sample storage and liquid handling areas. Increasingly, moving into the 3D printing technology within GSK.

Thomas Lundbäck

Senior Scientist

Thomas Lundbäck works within the Chemical Biology unit at Science for Life Laboratories located at Karolinska Institutet in Stockholm, Sweden. The unit is part of Chemical Biology Consortium Sweden established in 2010 as a government-funded, non-profit strategic resource for academic researchers across Sweden. The organisation coordinates and makes available a framework of infrastructures for the discovery, development and utilization of small-molecules and molecular probes for life-science applications.

Fabien Vincent

Associate Research Fellow

Fabien Vincent studied enzymology and medicinal chemistry during his PhD studies at the University of Houston under Dr Harold Kohn (inventor of Lacosamide). Following a postdoctoral stay at the Genomics Institute of the Novartis Research Foundation (GNF) in San Diego with kinase expert Kavita Shah, he entered the drug discovery field with biotech company Renovis in South San Francisco where he was a biochemistry group leader and project leader before moving to Pfizer in Groton, CT as a laboratory head in the Assay Development and Pharmacology group. Key scientific interests include atypical molecular mechanisms of action as well as the use of phenotypic screening to discover novel targets and drugs.

Jarie Bolander

COO

Jarie Bolander is the Chief Operating Officer of Lab Sensor Solutions a company that provides sample tracking solutions for clinical laboratories. He has over 10 issued and pending patents and has been bring innovative products to market for over 20 years

Jonathan Wingfield

Principal Scientist

Since joining AstraZeneca's Oncology department in 2000 I have worked to build secondary screening capabilities within AZ. In 2006, established a centralized biochemical screening group to support oncology projects and deployed a LIMS to support screening activities. In 2008 the LIMS project was recognised with the Microsoft Innovation in Pharma award. More recently I have focused on landing new technologies such as acoustic dispensing Currently areas of interest include the application of Mass Spectrometry in hit to lead and applications for Nanotechnology in drug discovery.

David J. Guckenberger Jr.

Research Assistant

David J. Guckenberger Jr. is research assistant at the University of Wisconsin - Madison. He obtained a bachelors degree in mechanical engineering and is pursuing a Ph.D. in Biomedical Engineering in the laboratory of Prof. Beebe. During his career at UW-Madison, David has published 9 papers in leading microfabrication, MEMS, and cellular engineering peer-reviewed journals. His work has led to 6 patent applications in the areas of simpler assays and sample preparation technologies. He is a co-inventor of the Kit-On-A-Lid-Assay (KOALA) technology, which was a 2013 SLAS innovation award finalist and provided novel techniques to perform biological assays with limited resources. His current research interests include, cell-biology, micro-technology, microfluidics, micro-fabrication, high-throughput technologies, and automation. David has ownership in Salus Discovery, LLC, and Tasso, Inc, which licenses and commercializes some of the technology disclose.

David Evans

Lab Head In Vitro Screening Group

David has been with FNCLR for 18months and is responsible for the NCI60 lab and the Target Validation and Screening labs. David has more than 20 years experience in small molecule drug discovery in cancer, reproductive health, infectious diseases and CNS disorders. Most of David's experience has been in Biotechnology companies where he was responsible for building out and management of HTS capabilities and driving preclinical development of lead candidates including small molecules and siRNAs. David has previously worked at Millennium Pharmaceuticals, Serono Pharmaceuticals, Psychiatric Genomics and Thermo Fisher (Dharmacon products). He has also cofounded several companies including Sirnaomics - a clinical stage siRNA therapeutics company.

Shashi G. Amur

Biomarker Qualification Scientific Coordinator

Shashi Amur, Ph.D. is the Biomarker Qualification Scientific Coordinator in the Office of Translational Sciences at the Center for Drug Evaluation and Research, FDA. Dr. Amur received her Ph.D. in biochemistry from Indian Institute of Science, India and completed post-doctoral fellowship at Temple University and at UCLA. She then joined Specialty Laboratories in Santa Monica, CA as a Molecular Biologist. Dr. Amur moved to Applied Biosystems in Foster City, CA, and applied DNA sequencing and PCR technologies to transplantation and toxicogenomics research. Prior to joining FDA, she worked as the Associate Director of Assay Development at Immune Tolerance Network and Neotropix, Inc. Dr. Amur is the Past-Chair of the Pharmacogenomics Focus Group at the American Association of Pharmaceutical Scientists (AAPS). Her current research interests include pharmacogenomics, HLA-associated adverse events and biomarkers in Autoimmune Diseases and in Alzheimer's disease.

Marie-Elena Brett

Post Doctoral Associate

Microfluidic techniques have become a valuable tool to study individual cells as well as whole tissues. Biological problems can be can be elucidated in the micro-scale to attain fast and high throughput data collection. My research is focused on creating microfluidic tools to solve biological problems, specifically, applying the high throughput capabilities of microfluidic droplet generation and flow cytometry to develop a high throughput migration assay that will be used to identify the key components of the microenvironment that influence cell migration. The assay will be used to screen a targeted siRNA library as well a library of currently used cancer therapies. In addition, I am developing a tissue on a chip model that will first be used to model the extravasation that occurs during metastasis and ultimately be used to study the effects of lung fibrosis.

Lorenz M. Mayr

Vice President & Global Head

Lorenz is working since 09/2012 as Vice President, Reagents & Assay Development with global responsibility for generation of biological reagents and assay development activities at AstraZeneca. This includes generation of proteins and cell lines for hit finding, hit-to-lead and lead optimisation activities including structure & biophysics for all therapeutic areas, generation of tool antibodies, transgenic animals, stem/primary cells as tools for target validation and lead optimisation. His department is responsible for the development of biochemical, cell-based and phenotypic assays at AstraZeneca. Before that, he was working as Executive Director at Novartis in Basel/Switzerland, at Bayer in Leverkusen& Wuppertal/Germany and at the MIT/Whitehead Institute in Cambridge/MA (U.S.A). He has published more than 50 papers in peer-reviewed journals and serves on several editorial and scientific advisory boards, including two terms at the Board of Directors for SBS (2004-2011) and is working as the Conference Chair of the MipTec Conference, Europe's largest drug discovery event.

Although 3D printing has been around for many years, with the advances of Materials and Technology it has become increasingly more viable. GSK have been using this technology to produce many items from simple Laboratory brackets and automation enhancements, through to human body parts. The presentation will explain the Printing technology used and some of the areas where the technology has been successfully implemented , focussing on business benefit and ROI. Finally looking at potential future applications and areas in GSK that could be influenced by this technology.

Phenotypic Screening is enjoying a Renaissance since Swinney and Anthony (Nat. Rev. Drug Discov., 2011) documented its positive impact on the translation of pre-clinical discoveries to the clinic. Nonetheless, it is to be expected that not all phenotypic screens will offer the same potential in that regard. A critical question then follows: what are the characteristics of the best phenotypic screens? This presentation will cover an analysis of this question conducted by a team of Pfizer scientists as well as propose 3 specific criteria (physiological relevance of assay system, stimulus and assay endpoint) to help identify and design the most promising screens. The rationale behind these proposed criteria and their reduction to practice will be shared.

Effective binding of a ligand to a protein can be monitored by the ligand induced shift in thermal stability of the protein. Here we present the implementation of a high throughput screening protocol for thermal shift assays in a cellular context, i.e. the ligand induced stabilization is not studied on isolated proteins but instead on endogenous proteins in their normal cellular environment. We refer to the approach as the cellular thermal shift assay (CETSA) and use it for studies of target engagement of drug candidates in a cellular context. The original methodology is based on quantitative detection of remaining soluble intracellular protein after a heat challenge using Western blots (Martinez Molina, Science, 2013). Herein we present and discuss challenges associated with high throughput adaptation of the Western blot protocol and will exemplify this work using human kinases p38α and Erk1/2 and lately also human thymidylate synthase (TS). Whereas the kinase work relies on commercially available detection reagents, the development of a screen compatible CETSA assay for TS first required a careful selection and validation of suitable detection reagents from several mouse and rabbit derived antibodies. The final protocol involves cell treatment, sample handling and detection in low µl volumes in 384-well plates. Results from a screening campaign of a diverse small molecule library of 10.500 compounds on TYMS will be presented together with confirmation of further validation of identified hit compounds. The hit list contains a number of known antimetabolites and folates as well as some novel classes of inhibitors.

The high throughput direct measurement of substrate-to-product conversion by label-free detection has been considered the “Holy Grail” of drug discovery screening. Mass spectrometry as a detection system has the potential to be part of the ultimate screening solution. However, MS with existing sample introduction modes, despite being widely used in drug discovery typically cannot meet the throughput requirements of HTS. We propose a unique, innovative solution to the problem of throughput by using acoustic droplet ejection (ADE) to transfer femtoliter samples from microliter assays rapidly, precisely and accurately directly into a mass spectrometer. Acoustic technology has been widely used to support compound management activities within the pharmaceutical industry. The speed, accuracy, precision and robustness of acoustic dispensers have been proven. In principle, the integration of an acoustic source with a MS detector would result in a system capable of delivering ~4000 data points per hour into a high sensitivity label-free detector. It would enable sampling from 1536-well plates and reduce the total required assay volume to <5µL. The rapidity of sampling would enable real-time kinetic studies to capture multiple data points within the first minute of initiating a reaction. Together Labcyte Inc, Waters Corp and AstraZeneca have built a prototype acoustic source linked to a mass spectrometer. Droplets in the range of 50-200fL are acoustically ejected directly into the MS through a charge field. The ion beam is detected in the single quad MS where the typical signal has a very sharp attack profile and instant stop when the acoustic spray is turned on and off. This process produces a square wave signal which is simple to integrate for quantitative assays, and generates reliable and reproducible spectra. The system works in both positive and negative ion modes. The process is capable of producing singly or multiply charged species. The ability to load samples into a MS detector at such a high rate from much reduced assay volumes has significant potential not only within drug discovery but other areas of industry. Dynamic fluid analysis, the ability of the acoustic injector to adjust automatically to varying viscosities and surface tensions of the sample, allows the generation of droplets from a wide range of fluids including blood, plasma, cell culture medium, acid digests, and chemical syntheses. In principle, the simplicity of the acoustic source enables it to be fitted to any type of MS detector with an atmospheric pressure interface (single quad, triple quad, ToF), extending the range of applications into the “omics” field.

Clinical samples in transit are required to be maintained at precise temperatures to ensure sample viability. Traditional methods for ensuring temperature stability of samples in transit have relied on labor intensive processes that cannot predict when samples may become unviable and furthermore, these traditional processes are error prone and inconsistent. We have developed a system and method to monitor the conditions of clinical samples as they get transported from a collection center to a core laboratory facility. This system, called T-Tracks ™, allows laboratory staff to monitor the temperature a sample has been kept at and any reported transportation issues. The system also predicts when a sample container may go out of temperature compliance and sends a warning to laboratory staff. These predictive warnings allow staff to preempt any potential issues before the sample deteriorates. The system eliminates the manual process of recording the temperature of clinical sample containers while also allowing laboratory staff to be confident that their samples were held at the proper temperature during transportation. This confidence ensures that the tests performed on the collected samples are of the highest quality. The collected temperature and location data also allows the system to learn of potential hazards beforehand and warn staff to take preventive action. Such warnings are impossible with the common manual systems of temperature and location recording presently in use. The real time nature of the system gives laboratory staff the ability to plan the laboratory work load since bottlenecks can be identified based on conditions enroute to the laboratory.

Over the past 20+ years, many screening labs including ours (the NCI60 screening lab), have performed small molecule screens using cells cultured in plates as adherent or suspension cultures. While these assays have resulted in the identification of several highly potent candidates for progression to in vivo studies, there is a higher failure rate in hollow fiber/xenograft models than the primary data would suggest. While there are many reasons for this failure we have to question whether one of the contributing factors is the method of screening and whether a 2D homogeneous culture system provides the best model for oncology drug screening. We therefore set out to examine whether 3D (spheroid) cultures of tumor cells provide a more robust response for prediction of in vivo efficacy for these agents. In this presentation I will discuss our experience with various methods of making 3D spheroids and the technical hurdles we faced in assay development and compound screening in these models. Results from select cell lines within the NCI60 panel grown in 2D will be compared with those in spheroid cultures and I will discuss the use of Patient Derived Xenograft (PDX) cells in 3D cultures. Funded by NCI Contract No. HHSN261200800001E. This research was supported in part by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.

Sample preparation is increasingly a bottleneck across genomics, proteomics, and other fields. Exclusion-based sample preparation (ESP) is a method of isolating analytes where analytes are bound to paramagnetic particles (PMPs) and drawn across an immiscible interface (e.g. air-liquid) to achieve single-step purification. Advantages of ESP over traditional isolation protocols include: (1) Simplicity (i.e., a single ESP step is equivalent to several traditional “washes”) streamlines and automates workflows (often cutting the time to perform sample preparation by >50%). (2) Moving the analyte instead of moving fluids makes ESP non-dilutive and non-destructive to the sample, enabling extraction of multiple analytes in series from a single small sample. (3) Almost instantaneous isolation maintains analyte equilibrium, enabling capture of weakly bound interactions (protein-protein, protein-cell) that are lost using traditional wash-based isolation methods. (4) Open-environment automated systems can be reconfigured (often via software only) to perform a variety of assays, on a variety of samples, yielding a variety of endpoints. While the simplifying workflows is important, it is advantages 2-4 that are fundamentally new and enable transformative advances in sample preparation that enable basic science and clinical analysis that are currently difficult if not impossible. We highlight advantages 2-4 using a novel open automated platform built on a commercially available instrument (Pipetmax, Gilson). We isolated circulating tumor cells (CTCs) from patient blood samples and performed multi-analyte isolation and multi-omic analysis. CTCs represent a rare (e.g., often <10 CTCs per ml of blood), and precious (emerging studies show predictive and prognostic value) sample. Yet most current analyte isolation methods impose a one analyte, one sample constraint. Automated ESP breaks through that barrier allowing multi-analyte and multi-omic analysis from a single sample. Using automated ESP, we performed parallelized isolation of CTCs with >95% capture efficiency and <1% carryover contamination of white blood cells. Importantly, ESP captures 70% of cells with low surface expression, which is a 50% improvement over typical cell isolation assays. After isolation, cells are automatically fixed, stained (intra- and extracellular), and PMPs are removed, highlighting the ability to easily link multiple processes with ESP. Additionally, this platform enables automated sequential extraction of mRNA and DNA for matched genomic and transcriptional endpoints. All isolation steps are automated, no manual intervention is required. While CTCs serve as an example of the enabling power of ESP, we are also using the platform to analyze lung lavage samples in order to diagnose lung cancer, and to isolate T cell fractions from blood and then extract DNA to monitor establishment of the proviral reservoir in HIV patients. ESP is an easy-to-use and easy-to-adapt method of sample preparation that allows isolation of multiple (and orthogonal) analytes from a single sample enabling multi-omic readouts from a plethora sample types.

Gaining greater understanding of cell migration is important to determine the underlying mechanisms of many important biological processes such as cancer metastasis and wound healing. There is a critical need to create a cell migration assay that is both high throughput and physiologically relevant. Such an assay could be used for drug screening and exploration of microenvironmental factors affecting cell migration. To determine drug candidates able to modulate migration, the ability to screen for cell migration as a functional readout in a high throughput assay is essential. Current methods, such as the scratch assay and Boyden chamber, are throughput limited and do not recapitulate physiological conditions. To overcome these obstacles, we combine rapid 3D tissue generation with automated high throughput flow analysis to quantify cell migration. A systematic method to quantify cell migration has been created in which cell aggregates are encapsulated within microscale 3D tissue constructs suitable for cell migration. Cell migration from aggregates into surrounding tissue is measured via rapid imaging of tissue constructs in flow and automated analysis. To construct cell aggregates, a droplet generator with a nozzle size of 50 µm is used to create a droplet of poly(ethylene glycol) prepolymer, containing fluorescently labeled cancer cells. The 50 µm gels are cross-linked using UV radiation to form cell aggregates then washed and mixed with unpolymerized gel. This solution is perfused into a second droplet generator with a 150 µm nozzle to encapsulate cell aggregates in a larger gel, which is cross-linked to form the final microtissue structure. The use of microfluidic technologies allows for rapid generation of microtissue constructs. Constructs are analyzed in a manner similar to flow cytometry where fluorescent signal area and intensity within the microtissue is measured to quantify cell migration and proliferation respectively. We have successfully generated microtissue constructs using a set of microfluidic droplet generators and shown that cell migration can be measured in these constructs. Additionally, we demonstrated the ability of our fluorescent readout to distinguish tissues with fluorescent objects either concentrated in aggregates or dispersed in a manner similar to cell migration. Because microtissue generation and migration analysis are both high throughput, this assay is well suited to elucidating the microenvironmental factors that are important to cell migration. Ongoing experiments are focused on measuring the effects of microenvironmental cues, such as ECM protein composition, chemical modulators of migration, and stiffness within a 3D microenvironment. Determining the modulators of cell migration is a key step in identifying what drives many disease pathologies such as breast cancer growth and metastasis. The biological information gleaned from this platform is highly likely to uncover new therapeutic targets by characterizing signaling event/pathways operant in cellular microenvironments that are faithful to the native tissue environment.

The Drug Development Tools (DDTs) Qualification Program was created by CDER as part of the FDA's Critical Path Initiative (CPI) to provide a framework for development and regulatory acceptance of scientific tools for use in drug development programs. Currently, CDER has three DDT qualification programs for -- clinical outcome assessments (COAs), animal models for use under the Animal Rule, and biomarkers. “Qualification” can be defined as a conclusion that within a stated context of use, assessment of a DDT can be relied on to have a specific interpretation and application in drug development and regulatory review. The goals of CDER's Biomarker Qualification Program are to 1) provide a framework for scientific development and regulatory acceptance of biomarkers for use in drug development, 2) facilitate integration of qualified biomarkers in the regulatory review process, 3) encourage the identification of new and emerging biomarkers for evaluation and utilization in regulatory decision-making, and 4) support outreach to commercial and noncommercial parties to foster biomarker development. The Biomarker Qualification process is intended to provide a framework for scientific development and regulatory acceptance of biomarkers for broad use in drug development. Biomarkers can be employed in multiple ways in drug development: for example, for stratified randomization or in patient selection (selecting more likely to respond- or eliminate less-likely to respond), and for evaluation of treatment response (including surrogate biomarkers and safety response biomarkers) in clinical trials. Qualified biomarkers can be utilized in the agreed upon context of use in multiple drug development programs without a need to submit additional data. The Biomarker Qualification process has three stages: 1) Initiation stage, 2) Consultation and Advice stage, and, 3) Review stage. If it is determined in the review stage that the data adequately support the proposed use of the biomarker, a qualification recommendation is issued and made publicly available as draft guidance. Draft qualification recommendations are announced in the Federal Register and are posted on the FDA Guidance web page.

Recent advances in Precise Genome Editing (PGE) and stem cell technologies have enormous potential to revolutionise the drug discovery process at all stages, from target identification through to toxicological studies. The ability to generate physiologically relevant cells in limitless supply makes induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC) an attractive alternative to currently used recombinant cell lines or primary cells in all processes from hit finding/screening to lead optimisation and safety testing/toxicology/DMPK. Recent advances with precise genome editing of stem cells have further accelerated the use of these cells towards personalised medicines. This presentation will describe the use of cell lines derived from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) for drug discovery at AstraZeneca.
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