HPLC for Pharmaceutical Scientists 2007 (Part 11)

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Great efficiencies have been achieved in the drug discovery process as a result of technological advances in target identification, high-throughput screening, high-throughput organic synthesis, just-in-time in vitro ADME (absorption, distribution, metabolism, and excretion), and early pharmacokinetic screening of drug leads. These advances, spanning target selection all the way through to clinical candidate selection, have placed greater and greater demands on the analytical community to develop robust high-throughput methods. This review highlights the various roles of high-performance liquid chromatography/mass spectrometry (HPLC/MS) in drug discovery and how the field has evolved over the past several years since the introduction of myriad...

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  1. 11 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Daniel B. Kassel 11.1 INTRODUCTION Great efficiencies have been achieved in the drug discovery process as a result of technological advances in target identification, high-throughput screening, high-throughput organic synthesis, just-in-time in vitro ADME (absorption, distribution, metabolism, and excretion), and early pharmacokinetic screening of drug leads. These advances, spanning target selection all the way through to clinical candidate selection, have placed greater and greater demands on the analytical community to develop robust high-throughput methods. This review highlights the various roles of high-performance liquid chromatogra- phy/mass spectrometry (HPLC/MS) in drug discovery and how the field has evolved over the past several years since the introduction of myriad high- throughput drug discovery technologies. Included are significant develop- ments in HPLC/MS to support target selection (proteomics), biological screening and assay development, high-throughput compound analysis and characterization, UV- and mass-directed fractionation for unattended, automated compound purification, and high-throughput in vitro ADME screening. Focus within the pharmaceutical industry has been to increase the likeli- hood of successfully developing clinical candidates by optimizing the compo- nents of the discovery process (i.e., spanning target identification → chemical HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. 535
  2. 536 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY design → synthesis → compound analysis and purification → registration → biological and ADME screening. By optimizing each step in the iterative dis- covery process, it is expected that the compound attrition rate will be reduced dramatically as compounds advance into preclinical development. Both HPLC and LC/MS enjoy important roles throughout the discovery process, as will be highlighted in detail in this review. Once considered primarily an enabling tool for medicinal chemists, HPLC and LC/MS are now key technologies incorpo- rated at just about every stage of the drug discovery process. Drug discovery programs typically initiate, as follows. Assuming that the relevant therapeutic area (e.g., oncology, metabolic diseases, inflammation, pain, CNS, etc.) has been selected, the next step is to identify a biological target relevant to the disease. As will be discussed shortly, numerous technological advances in the field of analytical chemistry (e.g., nanocolumn HPLC/MS/MS) that have greatly facilitated protein/target identification have been made since the human genome initiative was launched. Following on the heels of target selection is the requirement to establish tools for “just-in-time” high-throughput screen- ing of compound repositories (so-called corporate collections) and synthetic libraries as a means for identifying initial hits/actives. In combination with structure–activity relationship (SAR) data generated from these high- throughput screens, chemists incorporate knowledge of protein three- dimensional structures and utilize computational tools (i.e., in silico methods that measure diversity and “drug-likeness” as well two-dimensional and three- dimensional pharmacophore models [descriptors] that predict biological activ- ity) to support iterative compound design, synthesis, and biological testing. Once the hits or actives have been identified, the process of hit refinement and lead optimization is initiated. At this stage, a chemistry team is established and both parallel synthesis and more traditional medicinal chemistry strategies are incorporated to rapidly converge on qualified leads (so-called hit-to-lead stage). HPLC and LC/MS play an extremely important role in the hit-to-lead stage of discovery, providing key enabling analysis and purification capabili- ties to the medicinal chemist. Furthermore, activities that were traditionally relegated to drug metabolism and pharmacokinetics departments within development organizations are now integrated into early discovery so as to provide early measurements and predictions of in vivo properties. Again, LC/MS has played an extremely important role in enhancing the drug devel- opability of these hits and leads. All of these advances have helped to stream- line the discovery phase of pharmaceutical drug discovery and development and are presented within. 11.2 APPLICATIONS OF HPLC/MS FOR PROTEIN IDENTIFICATION AND CHARACTERIZATION The human genome initiative that took a stronghold on biotechnology com- panies in the early 1990s through the first few years of the twenty-first century
  3. APPLICATIONS OF HPLC/MS 537 spawned a completely new field that had analytical chemistry as its corner- stone. Specifically, high-resolution capillary and nano-column HPLC coupled with tandem mass spectrometry became one of the tools of choice for char- acterizing proteins and identifying potential therapeutic protein targets. Although capillary HPLC/MS/MS was applied as early as 1989–1991 to the characterization of proteins and for identifying sites of post-translational mod- ification [1–4], the field took off in earnest following the genomics boom and became known as proteomics, coined by Wilkins et al. [5]. In essence, the mandate of the proteomics field since its inception has been to identify dif- ferences at the protein level, in cells, tissues, plasma, and so on, between a disease state and control (“normal”). The basic premise is that proteins will be either up- or down-regulated (i.e., over- or underexpressed) in the disease state relative to “normal” state, and these differences can be identified and quantified by mass spectrometry. There have been several analytical advances made in the field of proteomics since its inception, far too numerous to capture in this review. One noteworthy advance in proteomics is the technique of multidimensional protein identification technology (MUDPIT), developed by Yates and co-workers, which has been used widely in place of the more labo- rious, less automated method of 2D-polyacrylamide electrophoresis [6]. MUDPIT is a column chromatography method whereby ion-exchange chro- matography is used in the first dimension of chromatography to simplify the complexity of the complex mixture of peptides by separating them based on charge followed by reversed-phase HPLC for the higher-resolution separation based on molecular weight and hydrophobicity. An equally important devel- opment in the field of proteomics has been isotope-coded affinity tags (ICAT) technology, a method whereby isotopic labeling of peptides containing cys- teine residues is performed so as to facilitate peptide quantitation and identi- fication of putative biological targets [7]. The reader is directed to the following review in the field of proteomics for more information [8]. A wealth of preclinically validated targets has emerged as a result of mouse genetics [9] and siRNA technology [10]. For both techniques, a single gene knockout is performed, and the effect of the deletion is monitored/evaluated. Proteomics, on the other hand, generally takes a shotgun approach to identi- fying the targets that are relevant and specific to the disease. Unfortunately, because many diseases are polygenic in origin and because protein pathways are extremely complex (e.g., intracellular protein signaling pathways [11]), proteomics has been best at identifying a short list of “candidate” protein targets rather than a single protein target completely unique to the disease. The challenge has been to sift through all the proteins that have been identi- fied as altered in a disease state relative to healthy state, and this has proved extremely challenging. The focus of proteomics has turned to identifying potential biomarkers of disease. A biomarker, by definition, is (a) a molecular indicator for a specific biological property or (b) a feature or facet that can be used to measure the progress of disease or the effects of treatment. As an example, a biomarker
  4. 538 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY for Type II diabetes is higher fasting blood glucose levels relative to age- matched controls. Another, more definitive biomarker of type II diabetes is elevated HbA1c levels. For many diseases, however, the relevant biomarkers are less well understood. This is especially true in the fields of oncology and inflammation research. Biomarker research is a particularly intense area of focus for many pharmaceutical companies, with new departments being formed for the purpose of identifying both preclinical and clinical biomarkers to facilitate their drug discovery and development programs. Like the field of proteomics, the field of biomarker research is far too vast to warrant its review here. A very nice review article by the late Wayne Colburn, a pioneer in dia- betes biomarker research, describes this maturing field [12]. 11.3 APPLICATIONS OF HPLC/MS/MS IN SUPPORT OF PROTEIN CHEMISTRY Independent of the tool used to identify the protein target, whether it be mouse genetics, siRNA technology, or proteomics, once a protein has been identified as a suitable target for drug discovery,the next step in the drug discovery process is to express and purify the protein (carried out combining molecular biology and protein chemistry techniques) in sufficient quantities so as to support bio- logical screening, X-ray crystallography, and any other drug discovery studies requiring purified protein material.The traditional method for assessing protein expression and purification has been to use 1D-polyacrylamide gel elec- trophoresis. 1D-PAGE is capable of separating proteins based on molecular weight and charge (pI). However, the technique is unable to provide more than a crude assessment of protein molecular weight. Recently, open-access or walk- up LC/MS has been incorporated into protein chemistry and molecular biology labs and has greatly facilitated confirmation of protein expression [13–15]. Generic gradient LC-MS methods are used to trap and elute expressed, purified proteins by RP-HPLC/ESI/MS. Open-access protein QC is a bit more challenging than its small-molecule counterpart in that not all proteins “fly” by electrospray ionization, identifying a “universal” HPLC method for their sep- aration can be challenging, and instrument calibration and mass accuracy are of paramount importance. We developed a fast, 5-minute protein QC method using a Poroshell 1-mm-i.d. column and found the method to be satisfactory for the vast majority of protein separations and analyses performed in our labora- tory. To achieve adequate mass accuracy for protein molecular weight deter- minations, an external calibration with myoglobin is performed at the beginning and end of each overnight queue of protein samples so as to ensure that the instrument calibration is maintained over the course of the batch analysis. Mol- ecular weights of deconvoluted protein spectra are then compared to the pre- dicted protein molecular weight, and the results are captured graphically (in the form of a microtiter plate view) as well as in tabular format, amenable to data- base uploading, as shown in Figure 11-1.
  5. APPLICATIONS OF HPLC/MS/MS IN SUPPORT 539 Figure 11-1. Automated protein AnalysisOpenLynx LC/MS for protein molecular weight confirmation. 11.4 APPLICATIONS OF HPLC/MS/MS IN SUPPORT OF ASSAY DEVELOPMENT AND SCREENING The overwhelming majority of biological assays have been developed in microtiter plate format (typically 96-well, 384-well, 1536-well) and with paral- lel detection methods such as fluorescence polarization. The vast majority of druggable targets, including enzymes, ligand gated ion channels, and G- protein-coupled receptors, are all amenable to screening in high-throughput microtiter plate format. In general, serial-based chromatographic methods, such as HPLC and HPLC/MS, are unable to compete with the high-throughput screening tech- nologies. However, a small number of targets, such as those involved in medi- ating protein–protein interactions, are not well-suited to HTS methodologies. For this class of targets, HPLC coupled with mass spectrometry has proved to be a very reliable, albeit lower throughput, alternative. The technique that has been used most widely for directly assessing protein–small molecule and protein–protein interactions is affinity chromatography–mass spectrometry. Kassel et al. [16] presented one of the first papers coupling affinity chro- matography with mass spectrometry. In their work, a two-dimensional LC/LC/MS method was developed to assess protein–ligand binding. Affinity chromatography was used in the first dimension of separation, followed by reversed-phase chromatography coupled with mass spectrometry for the identification of binders. Kaur et al. [17] showed the power of size exclusion
  6. 540 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY chromatography (SEC) coupled with reversed-phase HPLC/MS for identify- ing ligands for a receptor derived from a 576-component combinatorial library. Today, size-exclusion columns are available in microtiter plate format, permitting higher-throughput characterization of protein–protein and protein–ligand interactions. Berman et al. [18] pioneered one of the earliest applications of HPLC in support of assay development. They showed the power of HPLC for the deter- mining preferred substrates of the enzyme collagenase, a metalloprotease. Complex mixtures (pools of 100 components each) of probe substrates for collagenase were prepared by combinatorial methods. Each of the pooled libraries was incubated with enzyme. Substrate disappearance (turnover) and product appearance profiles were monitored by HPLC and the optimal sub- strate(s) identified. Recently, Lambert et al. [19] published a two-dimensional LC/LC/MS method for the identification and optimization of substrates for TNF convertase. Scientists at Nanostream, Inc., a company dedicated to high- throughput HPLC, introduced a parallel capillary LC/fluorescence method to support screening for kinase inhibitors. Their method complements the more traditional (and higher-throughput) fluorescence-based screening approach but offers the advantage of chromatographic separation of phosphorylated and unphosphorylated products, thereby reducing background interference. Another emerging role of HPLC/MS is in support of cell-based assays for which no direct measures of drug effect are possible and require indirect methods for detection. A recent publication by Clark et al. highlights the power of LC-MS for screening inhibitors of HMG-CoA reductase (a rate- determining enzyme in the cholesterol biosynthesis) [20]. In addition, Thibodeaux et al. [21] and Xu et al. [22] reported on methods for directly assessing the cell-based activity of inhibitors of the metabolic disease target, 11β-hydroxysteroid dehydrogenase-1 (11β-HSD-1). LC-MS was used to measure the effect of 11β-HSD-1 inhibitors on the intracelleular conversion of cortisol and cortisone using LC/MS/MS. 11.5 SOURCES OF COMPOUNDS FOR BIOLOGICAL SCREENING Once the assay and assay format have been decided upon, the next step in the discovery process is to initiate compound screening for the purpose of identi- fying hits or lead compounds. The fundamental requirement is that the assay results identify a collection of actives or “hits.” The definition of “hit” varies between organizations, but most accept the definition that the compound shows a confirmed structure, shows a confirmed dose response, exhibits an IC50 ≤ 10 µM potency, and is a member of a chemotype that is amenable to analoging and fast follow-on synthesis. What is the source of these initial actives or hits? There is a wide array of compound sources. Generally, pharmaceutical and biotechnology organiza- tions initiate screening by accessing their internal compound repositories (so-
  7. SOURCES OF COMPOUNDS FOR BIOLOGICAL SCREENING 541 called corporate collections or compound archives). Often, the corporate col- lections are not particularly diverse but are biased to the therapeutic focus(es) of the organization. Consequently, the screening libraries are often augmented by addition of commercially available screening libraries that are (a) gene- family focused (e.g., GPCR-targeted libraries, kinase-targeted libraries, etc.) and/or (b) general diversity sets. Further augmentation of the initial screening activities is to include custom synthesis compound libraries (typically pro- duced by automated high-throughput organic synthesis (HTOS) methodolo- gies, such as those described by Nikolaou et al. [23]. One of the challenges with compound collections is that they are historical by nature. For large Pharma, it is not uncommon for corporate collections to include compounds that were synthesized more than 25 years ago. At the time of synthesis, it can be presumed that the compounds met the purity criteria for compound registration. However, it can also be presumed that a high like- lihood exists that the compounds have degraded over extended storage time. Another reason for poorer quality of compound collections is attributable to the fact that most compounds are stored as DMSO stock solutions as opposed to storage as solid materials. Storage of compounds in DMSO is done primarily for the reason that (a) DMSO is considered a “universal” solvent and (b) solu- tions are much easier to handle in plate-based high-throughput biological screening systems. However, the drawback to DMSO is that it is a very hydro- scopic solvent and unless the compounds are stored under inert conditions, they are prone to hydrolysis. Kozikowski et al. [24] evaluated the effect of freeze/thaw cycles on stability of compounds stored in DMSO. Until very recently, with the introduction of high-throughput analytical technology, these compound sources were far too large to merit re-analysis and/or re-purification and hence were screened “as is.” The result was (and has been observed frequently) that hits could not be reconfirmed during follow- on bioassay screening, and subsequent evaluation of the compounds by tech- niques such as HPLC/MS and NMR showed that the expected compound was not pure and, in some cases, was completely absent! The adage “garbage in, garbage out” became a mantra of many high-throughput screening laborato- ries and forced companies to take a much more serious look at the quality of their compound collections. Morand et al. [25] from Proctor and Gamble set out to fully assess the quality of their >500,000 compound corporate collec- tion.They achieved this goal through incorporation of a massively parallel flow injection–mass spectrometry system, capable of analyzing a plate of samples in less than 2 minutes.The throughput of their technique was one to two orders of magnitude faster than typical flow injection–mass spectrometry systems used for reaction monitoring [26]. In addition to quality control over compound collections, the issue of purity of synthetic libraries derived using combinatorial chemistry quickly came under the microscope. In the early to mid-1990s, “combichem” became a household word throughout the pharmaceutical industry and was believed to be a key technology that would revolutionize drug discovery. The basis of
  8. 542 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY combinatorial chemistry was the ability to perform split-mix synthesis on solid support and to take advantage of the combinatorial nature of the process to generate vast arrays of compounds. Combinatorial libraries were purported to be pure, owing to the fact that they were synthesized on solid support and amenable to extensive washing to remove excess reagents and, therefore, directly amenable to high-throughput screening. However, these combinator- ial libraries synthesized on solid support suffered from the same problems that have long plague solution-phase synthesis—that is, the generation of unex- pected and unwanted by-products. Due to the shear size of these compound libraries and the relatively small amounts available following resin cleavage, it was not possible to either characterize or purify the expected products. Con- ventional split-mix combinatorial methods, though still popular with some bench chemists, have been replaced largely by the technique of directed par- allel solution and parallel solid-phase organic synthesis. 11.6 HPLC/MS ANALYSIS TO SUPPORT COMPOUND CHARACTERIZATION Combinatorial chemistry paved the way for high-throughput, parallel organic synthesis techniques, now mainstream in the pharmaceutical and biotechnol- ogy industries for lead generation activities. The ability to synthesize com- pound libraries rapidly using automated solution-phase and solid-phase parallel synthesis has led to a dramatic increase in the number of compounds now available for high-throughput screening. The unprecedented rate by which compound libraries are now being generated has forced the analytical community to implement high-throughput methods for their analysis and characterization. As early as 1994, groups adopted high-speed, spatially addressable auto- mated parallel solid-phase and solution-phase synthesis of discretes [27–31]. Both solution-phase and solid-phase parallel synthesis permits the production of large numbers as well as large quantities of these discrete compounds, eliminating the need for extensive decoding of mixtures and re-synthesis following identification of “active” compounds in high-throughput screening of combinatorial libraries. Importantly, parallel synthesis is performed readily in microtiter plate format amenable to direct biological screening, as was touched upon earlier. The relative ease of automation of parallel synthesis led to a tremendous in flux of compounds for lead discovery and lead optimization. Almost all of the analytical characterization tools (e.g., HPLC, NMR, FTIR, and LC/MS) are serial-based techniques, and parallel synthesis is inherently parallel. Consequently, this led rapidly to a new bottleneck in the discovery process (i.e., the analysis and purification of compound libraries). Parallel syn- thesis suffers from some of the same shortcomings of split and mix synthesis (e.g., the expected compound may not be pure, or even synthesized in suffi-
  9. HPLC/MS ANALYSIS TO SUPPORT COMPOUND CHARACTERIZATION 543 cient quantities). The analytical community was faced with the decision of how to analyze these parallel synthesis libraries. The traditional method for assessing compound purity has been to perform the following: Purify the desired product to homogeneity by crystallization or column chromatography (e.g., RP- or NP-HPLC), acquire a 1D-NMR and 2D- NMR spectrum on the isolated product, obtain confirmatory molecular weight information by mass spectrometry, perform a C, H, and N combustion analy- sis, generate an exact mass measurement (to within 5 ppm of the expected mass) by high-resolution mass spectrometry, and determine the amount of iso- lated product by weight—all prior to compound submission and biological screening. In the era of high-throughput compound library synthesis, however, this extensive characterization is simply not possible. Therefore, groups have focused principally on a limited number of analytical measurements for compound identity and purity—in particular, LC/MS analysis incorporating orthogonal detection methods, such as UV and evaporative light scattering detection (ELSD) and flow-probe 1D-NMR [32]. The most commonly employed technique for characterizing compound libraries is to incorporate LC/MS with electrospray or atmospheric pressure chemical ionization with UV and ELSD and, more recently, photoionization [33]. LC/MS emerged as the method of choice for the quality control assessment to support parallel synthesis because the technique, unlike flow injection mass spectrometry, provides the added measure of purity (and quantity) of the com- pound under investigation. In addition, “universal-like” HPLC gradients (e.g., 10% to 90% acetonitrile in water in 5 minutes) have been found to satisfy the separation requirements for the vast majority of combinatorial and parallel synthesis libraries. Fast HPLC/MS has been found to serve as good surrogate to conventional HPLC for assessing library quantity and purity [34–37]. Fast HPLC/MS is simple in concept. It involves the use of short columns (typically 4.6 mm i.d. × 30 mm in length) operated at elevated flow rates (typically 3– 5 mL/min). Typically, short columns are used for compound analysis because they allow for fast separations to be carried out at ultrahigh flow rates. Also, these columns tend to be more robust than narrow bore columns (1-mm and 2-mm i.d.) (i.e., less clogging is experienced and longer lifetimes are observed when these columns are subjected to unfiltered chemical libraries). A typical LC/MS analysis consists of injection a small aliquot (10–30 µL) of the reaction mixture (total concentration of 0.1–1.0 mg/mL) and performing the separation using a “universal” gradient of 10–90% Buffer B in 2–5 minutes. Buffer A is typically H2O containing 0.05% trifluoroacetic acid (or formic acid), and Buffer B is typically acetonitrile containing 0.035% trifluoroacetic (or formic acid). HPLC columns are operated typically at flow rates of 3–5 mL/min (depending on their dimensions), and the cycle time between injections is 3–5 minutes. An example of a fast LC/MS analysis of a combinatorial library compo- nent is shown in Figure 11-2. Fast LC/MS run times incorporating these short columns is typically between 3 and 5 minutes including re-equilibration.
  10. 544 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-2. (A) A 4-minute HPLC/MS separation of a solution-phase parallel synthesis library. The gradient profile for fast HPLC/MS was 10–90% acetonitrile in H2O in 4 minutes with a 1-minute equilibration time. (B) A 1-minute, total cycle time chromatographic separation of the same crude product. (Reprinted from reference 42, with permission.) Recent reports by Kyranos et al. [38] suggest that “pseudo-chromatography” (in essence, step elution chromatography) provides a more rapid and reliable assessment of the quality of library synthesis than methods such as flow injec- tion mass spectrometry. 11.6.1 Purity Assessment of Compound Libraries The issue of compound purity has received a great deal of attention over the last several years as more and more chemists have adopted high-throughput organic synthetic protocols but are unwilling to compromise the quality of the molecules submitted for biological evaluation. The general consensus target purity of a compound library compound before it is to be archived or screened for biological activity is between 90% and 95% pure. This purity criterion is more stringent than in the past, where 85–90% (based on UV detection) was considered acceptable. This may be attributed primarily to a shift toward smaller, focused (or biased) libraries than larger, diverse collections of com- pounds. The majority of mass spectrometry manufacturers now offer software packages that aid in the automatic determination of purity. UV chromatograms are typically used, rather than the total ion current chromatogram, to assess purity. This is because the total ion current chro- matogram is a measure of a compound’s “ionizability,” which is well known to vary dramatically from one compound to the next. Orthogonal detection methods, such as chemiluminescence nitrogen detection (CLND) [39] and ELSD [40, 41], have been proposed to be more universal detection methods than UV and hence are being used with increasing frequency to assess reac- tion yields and purity. CLND, as indicated from its name, measures the amount of nitrogen in a sample. In this method, a compound is transferred to a high-
  11. HPLC/MS ANALYSIS TO SUPPORT COMPOUND CHARACTERIZATION 545 temperature oxygen reaction chamber (set to 1000°C) whereby the compound undergoes rapid decomposition to form nitrous oxide (NO). The liberated NO reacts with ozone (O3) to form metastable NO2, which is selectively detected by release of a photon. CLND has been demonstrated to be a valuable tool for quantifying low quantities of material and has been shown to be particularly well-suited to NP- HPLC and SFC-MS, for the principal reason that separations are carried out using solvents that do not contain nitrogen (i.e., CO2 and CH3OH). ELSD measures the mass (quantity) of the material directly, is often presented as being a molecular-weight-independent detector, and is a tool that has gained wide-scale acceptance for on-line quantification of compound libraries. An example of a separation of a four-component library incorporating UV, ELSD, CLND, and MS detection is shown in Figure 11-3. Using these various detec- tors, the chemist is able to obtain measures of purity of their library with greater confidence than when relying solely upon LC/UV/MS data. An example of automated purity assessment of a compound analyzed by LC/UV/MS is shown in Figure 11-4. In this example, purity is assessed at two different wavelengths, λ220 and λ254. Excel macros are used for automated Figure 11-3. Column flow rate was 5 mL/min. A portion of the column effluent was split to each of the three detectors (CLND, 200 µL/min; ELSD, 200 µL/min; MS, 100 µL/min). A make-up flow of 50/50 MeOH/H2O (300 µL/min) was added to the flow stream diverted to the mass spectrometer ion source. Mass spectra were acquired using electrospray ionization with no special modifications to the ion source. (A) Total ion current chromatogram showing two of the four components ionize efficiently under electrospray ionization conditions. (B) ELSD chromatogram of the four components, all showing comparable response. (C) UV chromatogram (254 nm) shows some selec- tivity in detection as does. (D) CLND detection.
  12. 546 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-4. Purity assessment is a critical component in the decision process by the chemist as to whether their isolated compound is of sufficient quality to be submitted for compound registration and biological testing. To facilitate automated and rapid purity assessment of compound libraries, applescripts and visual basic scripts are used. (A) Total ion current chromatogram shows two components. (B) Extracted ion chro- matogram for the expected product identifies its retention time. (C) Mass spectrum observed for the expected product. (D) UV 220-nm chromatogram indicates the expected product is approximately 75% pure. (E) UV 254-nm chromatogram indicates the expected product is approximately 66% pure. post-data acquisition processing with associated graphical representations of data to facilitate analysis. For libraries generated in microtiter plate format, the results of each individual well may be color-coded to reflect relative degrees of purity. More often, as described earlier, compound purity is reported taking into account the purities determined from the UV, ELSD, and CLND detectors. In some instances, purity assessment has been made based on the intensity of the expected ion in the mass spectrum relative to the sum of the intensities of all ions in the spectrum. This method, however, is only a very crude estimate of purity, because ionization efficiencies for compounds can vary widely within and between classes of compounds. Though LC/MS (with UV and/or ELSD detection) has been adopted as the method of choice for assessing the quality and quantity of material prepared by parallel synthesis techniques, a decision still needs to be made by each respective organization as to what constitutes acceptable quality before submitting a sample for biological testing.
  13. PURIFICATION TECHNOLOGIES FOR DRUG DISCOVERY 547 Some groups have evaluated ultra-fast chromatography separations (so- called ballistic, pseudo-chromatography) in order to provide a snapshot of the sample purity [42, 43]. The major drawback to the ballistic chromatography technique is that column resolution is reduced when operating at these sub- optimal linear velocities. Also, the pseudo-chromatography approach is best suited to applications where purity assessment is secondary to rapid com- pound profiling. 11.7 PURIFICATION TECHNOLOGIES FOR DRUG DISCOVERY Historically, it was believed that solid-phase synthesis protocols eliminate the need for purification because excess reagents are removed readily by exten- sive washing. Unfortunately, even for solid phase peptide synthesis, final prod- ucts, acid-cleaved from the resin are found to be far from pure. Furthermore, parallel solution-phase synthesis has found greater popularity, because it is readily automated and extends the “portfolio” of reactions available to the chemist for high-throughput parallel synthesis. The limitations with solid- phase synthesis and the movement toward parallel solution phase synthesis are forcing numerous groups to evaluate and implement a variety of purifica- tion strategies. A prevailing assumption is that if the chemistry is sufficiently high-yielding during the process development phase of synthesis, then it is reasonable to expect comparably high yields during the production phase of synthesis. In process development, a subset of the total library to be synthesized is rigor- ously optimized to maximize reaction yield. During production, it is assumed that the vast majority of members of the library will behave similarly and that the desired product will be the major component in the well. The reality is that far too often, the biological activity cannot be tracked to a single component or, in some instances, to the expected product in the well. Groups attempting to elucidate the active component(s) of the well have expended significant effort, only to find that the activity does not correlate with a single component within the sample. Consequently, more and more groups have embraced the value of “quality in, quality out” and are now applying the same analytical rigor to parallel synthesis chemical products as they have for more classical medicinal chemistry synthesis. These activities have enhanced the quality of structure–activity relationships (SAR) and structure–inactivity relationships (SIR) that can be derived from the assaying of these compounds for biologi- cal activity. Numerous techniques are available to the organic chemist to support library purification. Zhao et al. [44] published an extensive review on com- pound library purification strategies, including HPLC, liquid–liquid extraction, solid-supported liquid–liquid extraction, solid-phase extraction, ion-exchange chromatography, and countercurrent chromatography, among others. This
  14. 548 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY review focuses exclusively on HPLC- and HPLC/MS-based purification methods. 11.7.1 UV-Directed Purification Both activity and inactivity data are being used increasingly to generate SAR and direct subsequent synthetic efforts. Consequently, organizations have rec- ognized the importance of confirming the purity of compounds prior to screen- ing, and not only those compounds for which activity is observed. In order to minimize false positives and false negatives, it is advantageous to assay only high-quality compounds. Therefore, great effort has been devoted to the devel- opment of automated purification technology designed to keep pace with the output of high-throughput combinatorial/parallel synthesis. Automated methods are now available to the chemist to perform high- throughput purification. Although HPLC has long been a method available to the chemist for product purification, only recently have these systems been designed for unattended and high-throughput operation.Weller et al. [45] were one of the first groups to demonstrate “walk-up” high-throughput purification of parallel synthesis libraries based on HPLC and UV detection. An open- architecture software interface enabled chemists to select the appropriate separation method from a pull-down menu and initiate an unattended automated reversed-phase UV-based fraction collection. Fractionation was achieved using a predetermined UV threshold. Multiple fraction collectors were daisy-chained in order to provide a sufficient footprint for fraction col- lection. Since the early work of Weller et al., a number of commercial systems have been introduced for walk-up preparative LC/UV purification (including Gilson, Hitachi, and Shimadzu, to name a few). One of the challenges associated with UV-based purification systems is that multiple fractions are collected for every sample injected. Although user- defined adjustable triggering parameters (e.g., UV thresholds for initiating and terminating fraction collection) can be used to reduce the total number of frac- tions, all, to some extent, will contain impurities. The exact number of chro- matographic peaks for a given sample will be hard to predict, and therefore the footprint for fraction collection will be difficult to predict. Experience has shown that it is not uncommon for 5–10 fractions to be collected per injection. When purifying only a small number of samples (
  15. PURIFICATION TECHNOLOGIES FOR DRUG DISCOVERY 549 particularly well-suited to semipreparative purifications (using smaller- inner-diameter columns to support low milligram quantities). In order to gain further efficiencies into UV-based purification of com- pound libraries, numerous groups have developed automated high through- put UV-based purification systems coupled with on-line mass spectrometric detection. Kibbey [47] was one of the first scientists to implement a fully automated preparative LC/MS system for combinatorial library purifi- cation. His approach was to perform a scouting analytical run prior to purification so as to optimize chromatographic method and fraction collection parameters. Fraction location and molecular weight information were captured through a custom LIMS system. The added mass spectrometric information greatly facilitated deconvoluting of collected fractions and streamlined their purification process. Hochlowski [48] describes a service- based purification factory incorporating UV and ELSD detection coupled with mass spectrometry that supports purification of over 200 compounds per day. More recently, intelligent UV-based systems for preparative scale purifica- tion of combinatorial libraries have been introduced, utilizing knowledge of retention time of the expected product based on a pre-analytical evaluation followed by preparative HPLC with UV-based fractionation using a narrow time collection window. Yan et al. [49] coined the “accelerated retention window” method as a tool for improving high-throughput purification effi- ciency. In their method, a high-throughput parallel LC/MS analysis is per- formed prior to preparative purification to confirm that the expected product is indeed contained within the well and to identify the approximate retention time of the expected synthetic product. Only those compounds found to be ≥10% pure based on the analytical run are candidates for final product purifi- cation. Furthermore, the information from the high-throughput parallel analy- sis was uploaded to a stand-alone preparative LC system for final product purification. Fraction collection was initiated using an accelerated retention time window method so as to accelerate the preparative HPLC analysis. Addi- tional refinements of UV-based purification strategies have been made recently, allowing for further simplification of the fraction collection and post- purification analysis step. In one embodiment, Karancsi et al. [50] implemented a “main component” fraction collection method based on UV-triggering that supports the “holy grail” of high-throughput purification, that being the one compound/one fraction concept [50]. 11.7.2 Mass-Directed Preparative Purification The technique of preparative LC/MS, introduced in the late 1990s was the first technique to greatly simplify the purification process. For the first time, preparative LC/MS (PrepLCMS) methods allowed the concept of one com- pound/one fraction to be realized [51–55]. In the Prep LC-MS mode, the mass
  16. 550 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-5. Preparative LC/MS systems on the market consist of a binary HPLC system, a combined autosampler/fraction collector (footprint of a Gilson 215 inject/collect liquid handler shown in the figure), and a single quadrupole mass spectrometer. spectrometer is used in this mode as a highly selective detector for mass- directed fractionation and isolation. This technique provides a means for reducing dramatically the number of HPLC fractions collected per sample and virtually eliminates the need for post-purification analysis to determine the mass of the UV-fractionated compound. Preparative LC/MS is now widely incorporated in the pharmaceutical industry. Systems for preparative LC/MS are configured in numerous ways and are operated in numerous ways, includ- ing an expert user mode, walk-up or open access mode, or a project team setting, supporting small teams of chemists working on similar chemistries. All components of the system are under computer control and are hence truly automated. Components of these systems are nearly identical to stand-alone HPLC systems with the addition of a flow splitter device to divert a small portion of the column flow to the mass spectrometer for on-line detection and fraction collector triggering. Typical systems are configured in an automated analytical/preparative mode of operation. In this configuration, the chemist is able to select between a variety of column sizes for either analytical, semi- preparative, or preparative separations. The HPLC, switching valves, mass spectrometer, and fraction collector are under complete computer control, as shown in Figure 11-5. In some instances, a solvent pump is added to deliver a methanol make-up flow to the mass spectrometer. The flow splitter and extra solvent pump serve the primary purpose of reducing the potential for over- loading of sample into the ion source. An advantage of the flow splitter and make-up pump is that it reduces the trifluoroacetic acid (ion pairing) in the
  17. PURIFICATION TECHNOLOGIES FOR DRUG DISCOVERY 551 Figure 11-6. Fifty milligrams of a crude reaction product was solubilized in 1 mL of 50/50 MeOH/DMSO and injected onto a 20-mm × 50-mm-i.d. reversed-phase column. Separation was achieved using a gradient of 10–90% ACN in 7 minutes. (A) TIC chro- matogram shows five components well-separated. (B) Extracted ion chromatogram (XIC) for expected product shows a single, prominent peak at 6.49 minutes. Fraction collection was initiated and terminated, as indicated by the arrows directly below the XIC peak. (C) Post-purification analysis of the isolated component shows that the com- pound was purified to approximately 90% level. ion source, which can affect the sensitivity of detection for acidic library components. An example of a mass-guided fractionation of a combinatorial library is shown in Figure 11-6. In this example the crude reaction product is only about 30% pure. The component of interest shows a prominent single chromato- graphic peak when monitoring specifically for its corresponding mass. Post- purification analysis of this singly isolated fraction (based on mass-directed fractionation) demonstrates that the compound of interest was purified to greater than 90%. Had a UV-based fractionation system been used in this par- ticular example, at least five individual fractions would have been isolated. Extending this to a 96-component library synthesized in microtiter plate format (and assuming this compound was representative of the quality of the members of the library), a UV-based approach would have led to approxi- mately 400–500 fractions requiring reanalysis to pinpoint the desired product. This not only would be a time-consuming reanalysis process but also would require significant time to transfer the appropriate fractions to a screening plate for biological assessment. Debate exists as to whether UV-based or mass-based fraction collection is the more appropriate tool for purifying compound libraries. The choice of
  18. 552 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY technique probably should be governed by the relative importance of any given sample and the purification throughput requirements at any moment in time. As a simple rule of thumb, during the earlier stages of the discovery process, where larger numbers and smaller quantities (
  19. HIGHER-THROUGHPUT PURIFICATION STRATEGIES 553 Figure 11-7. A five-component fluorous split-mix crude reaction mixture was injected onto a 20-mm × 50-mm-i.d. reversed-phase column. (A) UV chromatogram and (B) Total ion current chromatogram. Compounds were purified using mass-directed fraction collection (peaks highlighted). expected [M + H]+ ion for each of the tagged products exceeded the pre-set ion intensity threshold, and fraction collection was terminated when the ion intensity for the expected product(s) dropped below a second pre-set ion intensity threshold value. Combining split-mix fluorous synthesis with high- speed chromatography provides a means for rapidly generating large numbers and large quantities of highly purified druggable molecules. 11.8.2 Parallel Analysis and Parallel Purification The synthetic throughput achievable by the medicinal chemist (having adopted parallel synthesis strategies) has rendered analysis and purification one of the key (and possibly rate-limiting) steps in the discovery process. Although advances in sample analysis throughput have been clearly demon- strated, there is a limit as to how fast a separation and analysis can be achieved
  20. 554 THE EXPANDING ROLE OF HPLC IN DRUG DISCOVERY Figure 11-8. Schematic representation of a column switching configuration to support analysis from one column while the second column is equilibrating. while maintaining good separation efficiency and quality analysis. Two techniques that have been developed to increase throughput without com- promising column chromatography are (a) rapid column switching and regen- eration systems for enhanced-throughput serial-based analysis and (b) parallel chromatography methods. A simple and elegant modification of the LC/MS method is to incorporate a set of switching valves and a third pump to reduce cycle time between injections, as shown in Figure 11-8. While one column is being used to perform the LC/MS analysis, the other column is being regen- erated.An alternative use of 10-port switching valves is to allow for rapid serial sampling between columns. This technique works well for samples that are amenable to either isocratic or step elution. While one sample is being loaded onto one column, the contents of the other column are eluted into the ion source. In order to increase sample throughput while maintaining high-quality analytical data, groups have begun to perform separations in parallel [59–63]. Numerous groups have independently developed parallel sample introduction techniques, although the MUX ion source from Micromass/Waters is the only one commercially available. By performing analyses in parallel, chromato- graphic integrity can be maintained while effectively addressing sample throughput. Di Biasi et al. [59] and Wang et al. [60] presented novel ion source interfaces enabling four to eight samples to be processed in parallel, thereby increasing the sample analysis throughput dramatically over conventional, serial-based LC/MS analyses. Commercially available parallel spray interfaces consist of a multiple spray head assembly and a blocking device (e.g., rotating plate), enabling individual sprayers to be sampled at specific and defined time
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