Mass Spectrometry in Small Molecule Drug Development

• Genentech, Inc.

Abstract

Drug discovery and development is a labor-intensive and timeconsuming process that comes with a significant price tag. Mass spectrometry (MS) technology has evolved to the point where it is used throughout the drug development process, and now plays a key role in advancing the production of pharmaceuticals. In particular, when MS is coupled with a chromatographic separation technology, it becomes a powerful analytical tool, which adds an orthogonal detection function for sample analysis, and provides information-rich assessment of pharmaceutical compounds. This review describes the strategies and current approaches for MS and hyphenated MS in supporting of small molecule drug development. It also highlights the latest developed instrumentation and software that has great potential to expand the utility of MS for pharmaceutical development.

Introduction

In spite of the great progress made in research and development to combat severe diseases such as cancer, rheumatoid arthritis, high blood pressure, and aging-associated diseases, the drug development process itself has become increasingly complex and expensive. On average, it takes approximately ten to twelve years and $1.4 billion to bring a new drug to market^1,2. It is estimated that only one drug reaches market approval for every 5000 new chemical entities evaluated in a discovery program. Drug development generally includes four major stages: drug discovery, preclinical development, clinical development, and commercial manufacturing. The longest stage is typically clinical development, which encompasses the testing done in humans (i.e. Phase I to Phase III). One crucial step is the proof of concept study for efficacy, which is performed early in drug development and is a key decision point and can lead to termination of a drug discovery program of five to seven years’ duration1. Compared to ADME/DMPK, the use of mass spectrometry (MS) in early phase drug development is not well documented. This in part can be attributed to the regulatory requirements in drug development, which limits the development and acceptance of novel methods³. With the recent development in both software and instrumentation, MS techniques have been well adapted and are now the preferred choice for many applications in pharmaceutical development^4,5. Furthermore, new technology is needed to support novel therapies and more stringent regulatory requirements, which requires highly sensitive methods providing full profiles of drug and impurities during development. MS technology has evolved to meet this need and is emerging as the tool of choice for many applications in drug development.

MS is often considered the most sensitive detector and is typically coupled with other technologies, most commonly gas chromatography (GC) and high-performance liquid chromatography (HPLC), but also with supercritical fluid chromatography (SFC), inductively coupled plasma (ICP), ion chromatography (IC), ion mobility spectrometry (IMS) and capillary electrophoresis (CE). This type of orthogonalmass spectrometric methodology has facilitated drug development enormously, primarily due to the superior speed, sensitivity, and selectivity of such “hyphenated” techniques.

This review provides an overview of various applications of MS and hyphenated MS techniques in support of small molecule qualitative and quantitative analysis. It also describes the established workflows during small molecule drug discovery and development that utilize MS for high-throughput pharmaceutical compounds characterization, and impurity and degradant identification. In addition, some newly developed technologies in MS are discussed for their future application within pharmaceutical development.

General Applications of Mass Spectrometry in Small Molecule Drug Development

Figure 1. Common ionization techniques and application areas

MS is an essential tool in determining the molecular mass information of interest by ionizing chemical compounds to generate charged molecules or molecule fragments. The most common forms of ionization in small molecule research are electron ionization (EI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI). EI and APCI have a limited upper mass ranges (< m/z of 1,000), while ESI, and matrix-assisted laser desorption ionization (MALDI) have a high practical mass range. As illustrated in Figure 1, ESI is better suited to higher-molecular-weight and polar compounds, while APCI is best suited for low- to medium-polarity compounds. EI is typically used in GC/MS for small, volatile molecules.

Ambient ionization technologies, a terminology coined by professor R. Graham Cooks at Purdue University⁶, refers to a class of sampling ionization techniques for direct ionization of chemicals from samples in their raw or unprocessed “ambient” state using either spray, heat, plasma, high electric field, or laser impact. The potential value of ambient ionization was demonstrated with desorption electrospray ionization (DESI)6 and direct analysis in real time (DART)⁷, as well as another 30-plus ambient ionization methods developed thereafter^8,9. All these technologies have shown that ambient MS can be used as a rapid tool to provide efficient desorption and ionization with minimal sample preparation in various areas, from pesticides identification on the surface of fruit¹⁰, to residual illicit drugs detection on the surface of paper currency¹¹. Impressive results also have been achieved for chemical reaction monitoring to elucidate reaction mechanisms by MS coupled with DART¹² and DESI^13,14 ionization. Ambient ionization is also a powerful analytical tool for the rapid identification of APIs on the surface of tablets, which is important for analysis of diverted pharmaceuticals or counterfeit products¹⁵.

For the analysis of complex mixtures, hyphenated techniques, such as HPLC-MS and GC-MS, are used and provide a wealth of analytical information. GC-MS is commonly used to analyze volatile compounds. GC-EI-MS produces reproducible spectra across instruments and labs, and the spectra can be readily searched against commercial libraries for identification of unknown compounds. When MS is coupled with HPLC/UHPLC, it is added as an orthogonal detection technique to UV detection to provide both mass information and quality assessment of pharmaceutical compounds.

Supercritical fluid chromatography (SFC) coupled with MS has provided a valuable tool in a wide range of applications¹⁶, including chiral separation, achiral separation, and mass-directed fraction collection in preparative SFC¹⁷. As the SFC technology matures, there has been an increase in SFC-MS applications for both analytical and preparative areas, in relative to traditional normal phase methods, due to the speed and reduced waste¹⁸.

Other more specialized methodologies have been evaluated for the separation of structural isomers and chiral compounds. Dwivedi et al. has demonstrated that by coupling ion mobility spectrometry (IMS) with MS and employing a chiral modifier to the buffer gas, enantiomers can be isolated in the gas phase¹⁹. In another study, Rudaz et al. demonstrated that chiral separations and identification of enantiomers could be achieved by utilizing Capillary Electrophoresis Electrospray Interface for MS (CESI-MS)²⁰.

Ion chromatography (IC) has been extensively used as a complimentary separation technique to HPLC. It provides efficient separation of charged ions and polar molecules based on their affinity to an ion exchanger²¹. Recent applications include coupling to MS for inorganic ion analysis²² to identify ions such as fluoride, chloride, nitrite, nitrate, bromide, sulphate and phosphate. Burgess et al. demonstrated that IC-MS provides sensitive detection of polar molecules, including nucleosides and nucleotides, which were typically separated by MSincompatible ion-exchange chromatography or ion-pair reversephase HPLC²³.

The identification and quantitation of potential metal contamination in active pharmaceutical ingredients (APIs) is essential in drug development. Inductively coupled plasma mass spectrometry (ICP-MS) is the technique of choice for elemental determination, especially for heavy metal analysis in APIs²⁴. It offers many advantages including small sample size, element specific information, rapid sample throughput, and higher sensitivity for catalyst metals such as Pd when compared to ICP optical emission spectrometry (ICP-OES). As of December 1, 2015, the United States Pharmacopeia (USP) endorses the application of ICP-MS for identifying and quantifying elemental impurities in API in chapters <232> and <233>^25,26. The coupling of ICP-MS with HPLC solves even more complex separation problems²⁷, providing valuable information for unambiguous species identification.

Mass Spectrometry Analysis in Drug Discovery Chemistry

Figure 2. Workfl ow and processes for QC and characterization (blue boxes) in support of small molecule drug discovery in a pharmaceutical company. Reproduced with permission from Lin et al. (2015).
HT= high-throughput, CRO = Contract Research Organization, PK=pharmacokinetics, ADME = Absorption, distribution, metabolism and excretion, SAR = Structure-Activity relationship.

Drug discovery involves rapid testing of compound ideas and requires short cycle times from compound design to synthesis to testing, with the testing results being used for the next compound design. Typically many compounds are synthesized and tested for each discovery project until a suitable clinical candidate is selected. Analytical chemistry plays a key role in ensuring that each compound of interest (COI) has the correct structure and meets purity requirements. It is essential that analytical chemistry not be a bottleneck in the drug discovery process, so analytical labs typically employ high throughput analysis with automated data processing and reporting. Figure 2 shows a schematic diagram of a sample workflow in discovery analytical chemistry laboratory where LCMS provide essential measurement for accurate sample identification and purity assessment. A more detailed discussion can be found in the review paper by Lin et al.²⁸.

Large pharmaceutical companies routinely test tens of thousands of compounds that possess a wide range of properties to meet the requirements of different disease indications. A challenging area of high throughput analysis is selecting an appropriate method for each type of molecule. Samples can be small polar fragments, organic synthetic intermediates, racemic mixtures or single stereoisomers, organometallic complexes, peptides, or linkers and payloads of antibody-drug conjugates.

Table 1. Summary of MS methodologies for purity determination and identity confi rmation. Reproduced with permission from Lin et al. (2015).

Table 1 summarizes the high-throughput analytical methodologies used to assess compound purity and identity. The purity profile for COIs is determined by UHPLC chromatography coupled with a diode array detector. Structure confirmation for COIs often includes highresolution mass spectrometry using both ESI positive and negative ion detection modes. Compound quantification from solutions, needed for quality control of compound DMSO stock solutions as well as physicochemical assays, is determined by LC-MS coupled with one or more universal detectors, such as a charged aerosol detector (CAD) or chemiluminescent nitrogen detector (CLND).

Identification and Characterization of Impurity and Degradant for Product Development

Figure 3. Strategies for identifi cation of impurity and degradant from drug substrates and products.

Mass spectrometry is widely used for analysis of impurities and degradation products due to its high sensitivity and selectivity. A general MS-based strategy to analyze small molecule impurity and degradant is shown in Figure 3.

At the early stages of the drug development, rapid analysis methods that provide nominal molecular weight data are commonly used. Nominal mass information, along with the process chemist’s knowledge of the synthetic scheme and associated chemistry, is usually adequate to propose structures of impurities.

As a project progresses through clinical development, the structures of unknown impurities are required and nominal mass measurements are no longer sufficient to elucidate these structures with sufficient confidence. Accurate mass is used to determine the elemental compositions of impurity structures, an essential step in elucidating the structures of unknown compounds. There are several different types of mass spectrometers capable of providing accurate masses, including magnetic sector, time-of-flight (TOF), orbital trap, and fourier transform-ion cyclotron resonance (FT-ICR) systems. In addition to advanced instrumentation, software can also help extend nominal mass data to high-resolution data by using a post-acquisition approach to calibrate mass spectral accuracy developed by Wang et al.²⁹.

Additional structural information can be obtained from tandem MS instruments, such as ion trap, triple-quadrupole, and Qtrap systems. The molecular ions are fragmented in space or time within the mass spectrometer, and the resulting neutral losses by MSⁿ processes are informative for structure elucidation of various chemical/functional groups on target molecules. This greatly facilitates the understanding of the ion fragmentation pathway for an unknown species and enables the identification of unknown compounds. Moreover, accurate mass data on fragment ions can provide additional evidence to support structural assignments.

One challenge in elucidating the structure of unknown compounds using MS is that non-volatile buffers, which are not amenable to MS ionization, are often required for isolation of the COI. In this case, the two dimension (2D)-LC-MS can be used to overcome this issue and has the added advantage of improved chromatographic resolution^30,31. The first LC dimension utilizes the original LC isolation method and the analytes of interest are stored in loops/vials. The second dimension then uses LC-MS compatible solvents to deliver the isolated analytes from the first dimension to the MS for analysis.

To support proposed structural assignments, some straightforward chemical derivatization experiments can be performed, such as TiCl₃ reduction. TiCl₃ is typically used to reduce N-oxides degradant back into the parent molecule^32,33 and is commonly used during drug metabolites identification. It can also be used to reduce other oxidative degradants such as peroxides. Another structurally useful experiment is the hydrogen/deuterium (H/D) exchange reaction which can be used to measure the difference in MW of a compound before and after deuterium exchange. It confirms the number of solvent-exposed, exchangeable hydrogen atoms in a molecule, further confirming a proposed structural assignment.

Normally, LC-MS data alone does not provide a definitive structure assignment. NMR spectroscopy is needed to unambiguously identify unknown and novel compounds. However, NMR is relatively insensitive (~ 1,000x less than MS) and it can be time consuming and expensive, if not impossible, to obtain enough compound for complete NMR analysis. It is for this reason that advanced MS techniques are essential to provide as much confidence as possible for every structural assignment.

Quantitative Analysis by Mass Spectrometry

Coupled with HPLC or GC, mass spectrometry has become the detector of choice for superior sensitivity and selectivity in pharmaceutical compound quantification analysis. The combination of superior performance and ease of use has led to widespread adoption of LC/ GC-single-quadrupole MS systems in regulated laboratories.

Triple-quadrupole MS instruments are prevalent in small molecule bioanalytical labs due to their high sensitivity. The most common method used in MS quantitation is multiple reactions monitoring (MRM), which selects a parent ion in Q1 and monitors its unique fragment ion in Q3. The latest triple-quadrupole LC-MS system can detect impurities well below the limits required by regulatory authorities for potential genotoxic impurities (PGIs). This is illustrated in Figure 4A where simultaneous analysis of four PGIs for one pharmaceutical compound was achieved by using HPLC-MS/MS in MRM mode. Cleaning verification (CV) also demands highly sensitive analytical methods. HPLC-MS/MS method is well established as a versatile tool for quantifying known compounds in the solvent rinsates or swabbing extracts from manufacturing equipment³⁴. This is especially useful when dealing with cleanout testing for high potency drugs, i.e. human health criteria (HHC) category 3 and 4 compounds, where the acceptance criteria requires low ng/mL detection.

Figure 4. Chromatograms of 4 ng/mL of PGIs spiked into 4mg/mL of API. (A) The data was acquired on QqQ-MS instrument. (B) The data was acquired on high-resolution MS instrument.

Although LC-MS/MS has long been recognized as a state-of-art, high-sensitivity tool for quantitation, HRMS is showing promise^35-37, particularly where efficiency and fit-for-purpose quality are critical. In full scan HRMS experiments for small molecule quantification, selectivity is achieved by creation of extracted ion chromatograms (EIC) of quasimolecular ions of the compound of interest, with a narrow mass-extraction window. The more narrow the setting of the mass-extraction window, the higher the selectivity. This is illustrated in Figure 4B where the chromatogram of four PGIs was acquired on a high-resolution MS instrument at full scan mode and the data were processed by extraction of the signal from compounds with a protonated mass-to-charge ratio within a 5 ppm (part-per-million) mass accuracy window. Compared with traditional QqQ-MS, there is no significant drop in sensitivity or selectivity observed with the HRMS system, and the response is linear which enables reliable quantitation (see Table 2).

Table 2. Quantitative results on QqQ-MS and HR-MS at 4 ng/mL of PGIs in the presence of 4 mg/mL API.

Future Perspectives in Drug Development

The recent advent of miniature/portable MS systems enables the use of MS detection beyond the analytical laboratory. A common deployment is portable GC/MS systems, where there is a need for rapid, on-site analysis of volatile and semi-volatile species important to human health, homeland security, and environmental monitoring. Miniaturized systems have also been developed to target semi- and nonvolatile species using ionization methods such as ESI and APCI. It provides a simple-to-use mass detector that can be added as an orthogonal detection technique to routine UV detection. This system has also been implemented in continuous reaction monitoring by coupling it to flow chemistry systems, allowing real-time observation of reaction intermediates at the chemists’ bench³⁸. Ambient MS methods, as mentioned above, when coupled with portable MS platforms³⁹, reduce the need for chromatographic separation and associated sample preparation.

The most common approach for identification of impurity is carried out using HPLC coupled with UV detection and mass spectrometry. However, this approach is challenging when the impurities of interest are below the UV detection limits, or low concentrations impurities are buried in the chemical noise of a mass spectrum. Advance datamining software, predominantly used in metabolomics studies, has great potential for the discovery of chemical signatures in impurity profiling. This software is able to identify unknown impurities from noisy mass spectrograms of complex samples^40,41 Combined with powerful statistical tools, such as t-test and principle component analysis (PCA), the data analysis is relatively straightforward and manageable. The combination of this type of chemometrics software with mass spectrometry provides a powerful tool for impurity profiling during small molecule drug development.

Mass spectrometry is also showing great potential in surface analysis. MS imaging (MSI) generally refers to the use of MS for detecting the distribution of drugs and their metabolites in tissue slices⁴². It is also emerging as a technique that can provide insight into the molecular entities within cells, tissues and whole-body samples and lead to better understanding of the inherent complexities within biological metabolomes. In terms of drug development, a recent paper by Earnshaw et al. demonstrated the use of MALDI to directly image tablets⁴³ and the potential of this method to be used to assess the homogeneity of API in tablets during formulation development. DESI also has promise for analyzing drug tablet surfaces and has an advantage over MALDI in that no additional sample preparation is required, which could significantly eliminate potential low molecular weight MALDI matrix mass interference.

Summary

This review highlights the advantages of utilizing MS for performing qualitative and quantitative analysis of small molecules. The combination of high sensitivity, selectivity, and informationrich technology has led to MS becoming an essential tool for the analytical chemists in all stages of pharmaceutical drug discovery and development. As MS technology continues to advance and evolve, MS systems will see even wider applicability in the pharmaceutical industry.

Acknowledgements

The authors would like to thank Alan Deese for his support over the years. We also thank David Russell and Michael Dong for insightful discussion and suggestions on this paper.

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