Mass Spectrometry & Spectroscopy


Boosting Discovery and Early Development of Drug Candidates with New MALDI Technology


Dr Rohan Thakur, Executive Vice President, Dr Meike Hamester, Director Pharma Small Molecule Business, Dr Dale Shannon Cornett, Applications Development Manager and Dr Jens Fuchser, Product Manager MALDI TOF Mass Spectrometry at Bruker Daltonics


A golden rule of R&D in pharma is if you are to fail, fail early and fail cheap. This mantra has guided scientists for many years and this principle has been shown to drive innovation in a wide range of industries. The practice commonly adopted in pharma – using technology plus automation to rapidly screen compound libraries looking for ‘hits’, together with integrating ADME/TOX (Absorption, Distribution, Metabolism, Excretion/Toxicity) investigations as early as possible in the discovery process, to understand essential details of drug distribution and metabolism – has undoubtedly made a contribution to the rise in productivity in recent years. There has been an estimated 11.5% year-on-year drug pipeline growth (2015 – 2016), with the biggest rise being seen in the pre-clinical phase. Here, the 6,061 compounds reported to be in this phase in 2015 has grown to 6,861 in 2016 [1]. This article will discuss recent innovations in MALDI (Matrix-Assisted Laser Desorption Ionisation) mass spectrometry related to how two detection schemes that utilise these technology advances are being applied to accelerate pre-clinical drug discovery, one in ultra-high throughput screening (uHTS) programmes, the other in drug tissue distribution MALDI-imaging studies.


Context


There has been much written about desirable characteristics of an ideal analytical tool for small molecule R&D, for example, label-free, no probes, and with the ability to measure target analytes directly and quantitatively. Rapid, robust, easy-to-use, cost-effective and automation-ready are also requirements for most companies. Mass Spectrometry (MS) inherently delivers on many of these criteria, and has opened up a new arena for MALDI MS within the last years. MALDI is utilised with various MS analysers, the most common being axial TOF (Time of Flight) detectors, but others, such as orthogonal TOF or FT ICR (Fourier Transform Ion Cyclotron Resonance) are employed, depending on the analytical needs.


Today the technique is established across a range of applications in drug discovery and development. MALDI-MS is helping researchers identify the most promising small molecule leads, and is now expanding into the ultra-high throughput compound screening programmes that ‘big pharma’ relies on. Likewise, MALDI mass spectrometry imaging (MSI) is helping developers understand the spatial distribution and tissue physiology of a candidate drug and related metabolites before quantitative whole body autoradiography (QWBA) experiments, thereby permitting informed decisions whether to move the candidate to the next development stage.


It is also important to note that many candidate compounds generate metabolites which possess biological activity. These active metabolites may have different pharmacology and PK properties than the parent drug. A thorough understanding of the properties of active metabolites is central to estimating toxicity, which is the number 1 reason for withdrawal of a drug. Early information about the enzymes involved in the drug metabolism is very useful in the design of drug-drug interactions studies.


Application Example: Technology in Practice When confi gured for HTS, as with the rapifl eX MALDI PharmaPulseTM


good correlation of results before and after 108 measurements.


They conclude that the technology and approach for uHTS was robust and can deliver very fast analysis times. The group has measured more than 1 million samples a week and found that it has been possible to measure more than 2 million samples without having to clean the instrument lens stack. Finally, looking forward to even higher throughput, the group achieved similar assay performance using 6144 well plates. If adopted into routine, this would cut the time required to screen 2 million compounds to 2.39 days.


In contrast, where MALDI MSI is being used to understand the distribution of a drug and its metabolites in model tissue, features such as those seen on the solariX system are ideal. Traditionally, quantitative whole- body autoradiography (QWBA), and/or liquid chromatography coupled to mass spectrometry LC/MS, have been the methods used to obtain drug distribution and metabolism. Both have challenges. QWBA is a robust technique, and the data generated is accepted by regulatory bodies around the world, however, QWBA presents a composite of the total radioactivity present – it may include any combination of parent drug, metabolites, impurities and degradation products. Thus, it has severe limitations for researchers looking for insight into biochemical pathways and mechanisms.


instrument,


the result is a system that is up to 20 times faster than a traditional instrument, with improved robustness, sensitivity and extended mass range (MS/MS). Dr Peter Marshall et al (GlaxoSmithKline, Stevenage, UK) used the MALDI-TOF MS coupled with nanolitre liquid handling to enable them to screen more than 1 million samples per week [2]. The work was performed on a MALDI-TOF instrument with a 10 kHz laser. Mass spectra were acquired in the range of either m/z 80-400 or 700-3500, with 200 laser shots per sample. Under these experimental conditions, the system processed a 1536 well plate in 7.36 minutes. With a vast in-house collection of candidate compounds, simple scale-up calculations indicate that the analysis of 2 million compounds would require 7.85 days. An assessment of the robustness of the system and the methodology were made, with


LC/MS analysis is performed on extracts from tissue homogenates. The technique can not indicate any spatial information and, equally importantly, can be misleading. For example, if an analyte in the tissue is highly localised, the extraction and homogenisation process will act as a dilution, masking this distribution and giving a relatively low concentration, sometimes even below limit of detection. Localisation of an analyte is often an indication of toxicity, and would be missed in this case. Alternatively, if an analyte is determined to have a high concentration from tissue homogenate, a researcher could draw incorrect conclusions about toxicity because the analyte is presumed to be evenly present throughout the tissue.


Figure 1. Optical scans of kidney tissue sections from PND 7-13 juvenile rats [4].


INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2017


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