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Introduction Guidance for Assay Development & HTS March 2007 Version 5 Section I: Introduction Introduction Copyright © 2005, Eli Lilly and Company and the National Institutes of Health Chemical Genomics Center. All Rights Reserved. For more information, please review the Privacy Policy and Site Usage and Agreement. Table of Contents A. INTRODUCTION This document is written to provide guidance to investigators that are interested in developing assays useful for the evaluation of compound collections to identify chemical probes that modulate the activity of biological targets. Originally written as a guide for therapeutic projects teams within a major pharmaceutical company, this manual has been adapted to provide guidelines for: a. Identifying potential assay formats compatible with High Throughput Screen (HTS), and Structure Activity Relationship (SAR) b. Developing optimal assay reagents c. Optimizing assay protocol with respect to sensitivity, dynamic range, signal intensity and stability d. Adopting screening assays to automation and scale up in microtiter plate formats e. Statistical validation of the assay performance parameters f. Secondary follow up assays for chemical probe validation and SAR refinement g. Data standards to be followed in reporting screening and SAR assay results. General definition of biological assays This manual is intended to provide guidance in the area of biological assay development, screening and compound evaluation. In this regard an assay is defined by a set of reagents that produce a detectable signal allowing a biological process to be quantified. In general, the quality of an assay is defined by the robustness and reproducibility of this signal in the absence of any test compounds or in the presence of inactive compounds. This robustness will depend on the type of signal measured (absorbance, fluorescence, radioactivity etc), reagents, reaction conditions and analytical and automation instrumentation employed. The quality of the HTS is then defined by the behavior of this assay system when screened against a collection of compounds. These two general concepts, assay quality and screen quality, are discussed with specific examples in the chapters of this manual. Assays developed for HTS can be roughly characterized as cell-free or cell-based in nature. The choice of either biochemical or cell-based assay design and the particular assay format is a balancing act between two broad areas. On one side of the fulcrum is the need to ensure that the measured signal is providing relevant data to the desired biological process. For assays that are to be used in HTS, this must be balanced with the ability of these assays to support reagents that yield robust data in microtiter plate formats where typically 105 to 106 samples are screened in the assay. General Concepts in Method (Assay) Development and Validation The investigator must validate the assay methodology by proceeding through a series of steps along the pathway to HTS. The overall objective of any method validation procedure is to demonstrate that the method is acceptable for its intended purpose. As mentioned above, the purpose can be to determine the biological and or pharmacological activity of new chemical entities. The acceptability of a measurement procedure or bioassay method begins with its design and construction, which can significantly affect its performance and robustness. Guidance for Assay Development & HTS March 2007 Version 5 Section I: Introduction This process originates during method development and continues throughout the assay life cycle (Figure 1). Successful completion of validation at an earlier stage increases the likelihood of success at later stages. During method development, assay conditions and procedures are selected that minimize the impact of potential sources of invalidity (e.g. so-called false positives or false negatives) on the measurement of analyte or the biological end point (eg. Gene expression, protein phosphorylation ) in targeted sample matrices or test solutions. There are three fundamental general areas in method development and validation: (a) Pre-study (Pre- screen) validation (b) In-study (In-screen) validation, and (c) Cross-validation or method transfer validation. These stages encompass the systematic scientific steps in assay development and validation cycle. Figure 1: The Assay Development Cycle Pre-study validation: The investigator is faced with a number of choices with respect to the assay design and format. For many well characterized target classes there are a number of methods and kits available. At this stage the choice of an assay format is made. Close attention must be paid at this early stage to factors such as the selection of reagents with appropriate specificity and stability. Validation of assay performance at this stage should proceed smoothly if high quality procedures are chosen during method development. This requires the generation and statistical analysis of confirmatory data from planned experiments to document that analytical results satisfy pre-defined acceptance criteria. The choice of detection is made here. If fluorescent labels are chosen, careful attention must be paid to the wavelength to ensure low interference by compounds, compatibility with microtiter plate plastics and that appropriate filters are available on high-throughput plate readers. If available, the assay sensitivity and pharmacology is evaluated using control compounds. Section IV illustrates procedures common to compound evaluation using dose-response curves. Several examples of assay design and optimization are given in the additional sections of this manual for well-studied target classes (Sections V-XI). A complete discussion of design of experiment procedures will be a topic for a future chapter in this manual. Guidance for Assay Development & HTS March 2007 Version 5 Section I: Introduction In-study validation: These procedures are needed to verify that a method remains acceptable during its routine use. For assays to be run in HTS the assay must be adapted to microtiter plate volumes. Therefore, plate acceptance testing is required where the assay is run in several microtiter plates (at least 96-well plates). From this data, statistical measures of assay performance such as Z-factors are calculated. Some methods may require additional experiments to validate the automation and scale up of an assay that may not have been addressed in earlier stages. The plates should contain appropriate maximum and minimum control samples to serve as quality controls of each run to check the performance. This will allow the investigator to check for procedural errors and to evaluate stability of the method over time. Assaying a randomly selected subset of test samples at multiple dilution levels monitors parallelism of test and standard curve samples. Sections II and III illustrate the procedures typically used to evaluate assay performance in microtiter plates and some of the common artifacts that are observed. Cross validation: This portion includes the assay hand-off from the individual investigator’s team to the high-throughput screening center. More broadly, this procedure is used at any stage to verify that an acceptable level of agreement exists in analytical results before and after procedural changes in a method as well as between results from two or more methods or laboratories. Typically, each laboratory assays a subset of compounds and the agreement in results is compared to predefined criteria that specify the allowable performance for HTS. Considerations in adapting assays to automated robotic liquid handling and plate screening protocols are also discussed in the sections of this manual. REFERENCES: 1. Findlay JWC, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, Bowsher RR: Validation of Immunoassays for Bioanalysis: A Pharmaceutical Industry Perspective. J. Phamac. Biomed. Analysis, 21, 1249-73, 2000. 2. Smith WC, Sittampalam GS: Conceptual and statistical Issues in the validation of analytical dilution assays for pharmaceutical applications. J. Biopharm. Stat., 8, 509- 532, 1998. Guidance for Assay Development & HTS March 2007 Version 5 Section II : Assay Validation SECTION II ASSAY VALIDATION Authors Brian J Eastwood Stephen J Iturria Philip W Iversen Chahrzad Montrose Roger Moore G Sitta Sittampalam Previous Edition Authors Brian J Eastwood Benoit Beck Yun-Fei Chen Viswanath Devanarayan Walthere Dewe Mark Farmen Stephen Iturria Phil Iversen Roger Moore Jeffrey W Weidner 1 Guidance for Assay Development & HTS March 2007 Version 5 Section II : Assay Validation Table of Contents SECTION II 1 TABLE OF CONTENTS 2 A. OVERVIEW 4 B. STABILITY AND PROCESS STUDIES 5 B.1. Reagent Stability and Storage Requirements 5 B.2. Reaction Stability Over Projected Assay Time 5 B.3. DMSO Compatibility 5 C. PLATE UNIFORMITY AND SIGNAL VARIABILITY ASSESSMENT 7 C.1. Overview 7 C.2. Interleaved-Signal Format 8 C.2.a. Procedure 8 C.2.b. Summary Signal Calculations and Plate Acceptance Criteria 9 C.2.c. Spatial Uniformity Assessment 10 C.2.d. Inter-Plate and Inter-Day Tests 13 C.2.e. Summary of Acceptance Criteria 14 C.2.f. 384-well Plate Uniformity Studies 14 C.3. Uniform-Signal Plate Layouts 16 C.3.a. Procedure 16 C.3.b. Summary Calculations and Plate Acceptance Criterion 17 C.3.c. Spatial Uniformity Assessment 17 C.3.d. Inter-Plate and Inter-Day Tests 18 C.3.e. Impact of Plate Variation on Validation Results 18 D. REPLICATE-EXPERIMENT STUDY 21 D.1. Overview 21 D.2. Procedure (Steps) 22 D.3. Analysis (Potency) 22 D.4. Diagnostic Tests and Acceptance Criterion (Potency) 24 D.5. Analysis (Efficacy) 25 D.6. Diagnostic Tests (Efficacy) 26 D.7. Summary of Acceptance Criteria 26 D.8. Notes 26 E. HOW TO DEAL WITH HIGH ASSAY VARIABILITY 28 E.1. High Variation in Single Concentration Determinations 28 E.2. Excess Variation in Concentration-Response Outcomes (EC50, IC50, Ki, or Kb) 28 F. BRIDGING STUDIES FOR ASSAY UPGRADES AND MINOR CHANGES 31 F.1. Overview 31 F.2. Tier I: Single Step Changes to the Assay 31 Protocol 32 Analysis 32 Example 32 2 Guidance for Assay Development & HTS March 2007 Version 5 Section II : Assay Validation F.3.
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