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  • 1.7Applications of CapillaryElectrophoresis Technologyin the Pharmaceutical IndustryCharles J. Shaw and Norberto A. GuzmanThe R. W. Johnson Pharmaceutical Research Institute,Raritan, New JerseyI. INTRODUCTIONPharmaceutical companies are facing new challenges in the starting millennium.Drug discovery is a high-stakes game; therefore, innovative strategies are beingplanned for the research and development of new and novel breakthrough prod-ucts (17). Genomics, high-throughput screening, robotics, combinatorial chem-istry, proteomics, proteinomimetics, informatics, and miniaturization are someof the strategies that are revolutionizing drug discovery efforts (818). In addi-tion, the genome age will change biology forever, providing sequence blueprintsfor numerous bacteria, fungi, plants, and animals, and thus facilitating the under-standing of human behavior, disease, and other health issues. Nevertheless, asthe Human Genome Initiative nears completion, it is becoming increasingly clearthat the behavior of gene products is difcult or impossible to predict from thegene sequence (1921). Genomics, or the analysis of gene sequences, allows identication of novelproteins which are potential drug candidate molecules targeted as therapeuticagents (22,23). The ability to analyze entire genomes is accelerating gene discov-ery (8) and presents unique scientic opportunities for understanding the structureand function of the proteins coded for those genes (2427). The exponentialgrowth of knowledge obtained about genes, from the early experiments of Mendelto the most recent high-throughput sequencing of DNA by capillary array electro-Copyright 2002 by Marcel Dekker. All Rights Reserved.

2. Fig. 1 Genetic discovery timeline. This schematic is a brief chronological outline and essence of the various events that took place for more than a century regarding gene studies. The information covers the period from 1866 to 1871, which describes the pioneer- ing work of Mendel and Miesch in the discovery of genes and DNA, respectively, through the beginning of the massive DNA sequencing that started in the early 1980s. A powerful tool that accelerated the sequencing of the human genome was the use of high-throughput DNA sequencers based on multicapillary-array electrophoresis. (A) Representation of a model system of coupling conventional slow-pace techniques with modern fast-pace and high-throughput techniques for the advancement of science. (B) Representation of some of the key steps that were needed in the genomic era to reach the massive undertaking of sequencing the human genome. (Adapted from Ref. 28.) phoresis, is illustrated in the genetics discovery timeline (Ref. 28 and Fig. 1). A complete understanding of the DNA code, which underlies human physiology, is crucial to unlocking the mystery of normal functioning and disease at the mo- lecular level (2932). Comprehending how that happens is, literally, having the keys to life. Furthermore, it is the gateway to developing better drugs more rap- idly. The entire history of medical research is that once a disease is well under- stood, it is only a matter of time until that disease or its effects are ameliorated. Proteomics is a eld that promises to bridge the gap between sequence and cellular behavior. It aims to study the dynamic protein products of the genomeCopyright 2002 by Marcel Dekker. All Rights Reserved. 3. and their interactions, rather than focusing on the simple static DNA blueprintof a cell. Understanding the proteome poses an even greater challenge than se-quencing the genome (3342). High-throughput screening techniques and combinatorial chemistry havegreatly expanded the compound universe from which to pick our developmentcandidates (43). Combinatorial chemistry produces extensive chemical librariesand screens them for potential pharmacological activity by searching among alarge source of chemical entities for promising lead compounds (1013). Oncea promising lead compound is identied in a preliminary screen, it is turnedover to chemists for complete characterization, modication, and eventually forsynthesis in bulk amounts. Combinatorial chemistry in its various manifestationsenables the generation of new molecules with unprecedented speed. As a result,the supply of lead compounds has never been greater. However, the evolutionfrom promising lead to useful drug requires much optimization, ranging from thereorganization of key structural features on the molecule to the reconguration ofasymmetric centers. Biocatalysis is particularly well suited to these tasks. In fact,researchers must do more work after nding hits from screening combinatoriallibraries of pure compounds. A million-compound library may not be any morevaluable than a ten-thousand-compound library. What is really more importantis the types of compounds in the library, their diversity and physical properties,molecular weight, substructure, and ease of incorporation (44,45). It is noteworthy that a compound with a great receptor afnity and selectiv-ity, but with poor biopharmaceutical properties for formulation or delivery, israrely regarded as ineligible to enter development. Desirable characteristics ofthe well-designed compound can include increase potency, higher selectivity,reduced toxicity, good solubility, excellent stability, effective permeability, andgreater bioavailability. The overall aim of combinatorial chemistry is to makenew molecules faster, more cheaply, and in numbers large enough for high-throughput screening (HTS). High-throughput screening refers to the integrationof technologies to rapidly assay thousands of chemical compounds simulta-neously in search of biological activity and a potential drug (13,43,46). The appli-cation of HTS techniques represents a major increase in automation of the drugdiscovery laboratory. The success of drug development in the pharmaceutical industry, whichuses genomics and combinatorial chemistry strategies as well as proteomics, iscontingent upon the availability of a battery of tools (13,38,39,47,48). Capillaryelectrophoresis (CE), or separations on micromachined planar substrates such asmicrochips (MC), are outstanding state-of-the-art modern analytical tools to assistthese strategies, as well as to provide additional information through the develop-ment of numerous and diverse kind of applications aimed at the diagnostic andpharmaceutical industry (4968). Once a potential useful drug exits the phase of drug discovery, it entersCopyright 2002 by Marcel Dekker. All Rights Reserved. 4. the phase of drug development. From the rst part of drug development until the drug is approved, a series of analytical, chemical, toxicological, and ADME (absorption, distribution, metabolism, and excretion) studies are carried out. The timeline for drug development from preclinical research to postmarketing surveil- lance is illustrated in Ref. 69 and Fig. 2. The entire process from discovery to lead candidate selection, through development, and to registration is highly inte- grated, with overlap and sharing of the informational content across the various phases (the preclinical phase or Phase 0, the three clinical phases 1, 2, and 3, and even the postmarketing surveillance phase or pharmacovigilance phase, which is commonly referred as Phase 4). Currently, pharmaceutical companies are trying to optimize the science of drug development, from production to evaluation, in order to manufacture a successful drug product (70). Physicochemical character- ization studies of the drug start in the early part of the discovery process and continue until completion of the nished product. Stringent requirements by regu- latory agencies (7173) require ne characterization of drug substances, isomeric forms, degradation products, excipients, impurities, contaminants, and possible micromolecular interaction of components in nished pharmaceutical products. Fig. 2 Timeline for drug development. The schematic representation of the time and steps needed in the development of a successful drug to reach the market. The diagram represents the process of manufacturing a drug, starting at the early part of the discovery and screening of a target compound, continuing until reaching the market. The entire process must comply with a series of stringent requirements by regulatory agencies. (Mod- ied from Ref. 69.)Copyright 2002 by Marcel Dekker. All Rights Reserved. 5. Furthermore, the requirements for stability tests, which are usually performed on hundreds of samples, require reliable, rugged, and fast analytical tests that are crucial in the control and research laboratories in order to meet these demands.Sample preparation may not be a glamorous part of chemical analysis, but it is where most of the errors occur and where most of the analytical time is spent. One way of improving sample preparation is to automate these procedures to shorten analysis time and increase throughput by parallel formats. Advances in micro-electro-mechanical systems (MEMS) has afforded accurate and repro- ducible injection volumes by means of microelectronic and micromechanical pumping, even at volumes of less than one microliter. New technology has re- cently emerged that has the potential to take automation and miniaturization to a level unimaginable just a few years ago, drastically reducing costs and sample analysis time. A comparison of analysis time and sample costs utilizing various analytical techniques is presented in Tables 1 and 2.Pharmaceutical analysts rely heavily on separation science to perform the physicochemical characterization required from drug development to stability testing of approved drugs (7480). Many challenges are presented for analytical chemists (see Table 3), since potential pharmaceutical drugs have a wide range of physicochemical properties, and a characterization study may take from one week to several months. For years, gas chromatography (GC) and high- Table 1 Compara