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Challenges in Small Molecule Analysis in the Pharmaceutical Industry

Day one (19th May 2022)

Session 1- Small Molecule Analysis – Pharmaceuticals : the application of HTS to finding new drugs.

The ChromSoc spring symposium began with a welcome address from Tony Edge (current president), who introduced our partners LCGC that were filming the event and interviewing attendees / speakers. After a short welcome, Tony introduced keynote presenter Tim Underwood from GlaxoSmithKline. Tim’s presentation ‘Challenging existing screening strategies’ highlighted the importance of continuous optimization of small molecule screening approaches. As speed of synthesis is a high priority driver in the pharmaceutical industry, analysts are continually shouldering the demands of small molecule purification. Tim discussed that chromatographic screening approaches help to reduce process turnaround times and enable efficient scale up to purification. In respect to chiral separations, Tim highlighted that 5-6 years ago, 32 chiral separations could be performed in a 72 hour period using a combination of normal phase and polar organic screens. As demand has increased, GSK have updated their instruments and columns to achieve faster separation times and increase the amount of chiral separations performed. With these updates and the exploitation of further selectivity by including an SFC within their instrumentation, 93 separations could be performed in a 2 hour window. Implementation of secondary screening (for example using aqueous reversed phase conditions) increased this number to 422 in 50 hours, which in a 1200% improvement in chiral recognition systems. Tim also discussed achiral screening approaches and how GSK have also optimised. His take home message was that SFC and HPLC enable an orthogonal screening approach and the most comprehensive strategy for small molecules.

Dr. Lewis Couchman from Analytical Services International was next to present. His talk ‘Turning your LC-MS “up to eleven”: Progress in Ultra-Rapid Bioanalysis by LC-MS’ began with re-emphasizing Tim Underwood’s point on continuous optimization for faster results. ASI must get results back to clinicians quickly to facilitate rapid clinical treatment decisions. Lewis has effectively ditched the chromatographic column to perform flow injection MS in under 60 seconds. The issues Lewis experiences when analyzing biological samples are matrix effects, which can become rate limiting. To overcome this, Lewis and his colleagues re-introduced a 5 mm column and high flow rate to perform high resolution LC-MS within 21 seconds. The result of this approach is decreased matrix effects and minimal delay volume by attaching the LC column straight onto the MS source. Lewis also presented an interesting case study of rapid dissolution profile testing on 25 years’ worth of ecstasy tablets to profile historical dose changes. Rapid LC-MS analysis enabled 1200 samples to be analysed per night. His take home message was that high resolution instrumentation coupled with pushing the separation speed to as fast as you can (turning it up to 11) assists in rapid analytical turnarounds.

The next presentation was ‘Automating analytical data batch-processing in Drug Discovery with MGears’ by Sylvain Demanze from AstraZeneca. Sylvain highlighted the fact that laboratories are fast acquiring a range of robotics to perform high throughput experiments. The high throughput instruments generate a large volume of data and the associated software may not process that data according to the needs of a screen. A level of ‘in-house’ manual processing may be required, which is challenging if you have many data sets to process. ‘Mnova’ software by Mestrelab is a software platform that enables data processing of high data volumes. It originated to process NMR data but can also process LC or MS data in a vendor agnostic manner. ‘MGears’ is a plugin that facilitates the user to set-up automated analytical workflows. Sylvain has been using the software to perform proactive system suitability testing of instruments, reaction screening experiments and large scale solvent screening for solubilization of product impurities. As parameters such as peak shape, mass or retention time can be in-put by the user, there often is no need to interrogate chromatograms or spectra, and the visualization of the output can be graphical or within an automated report.

To conclude the mornings session, Richard Blankley from Agilent presented ‘High- Throughput Mass Spectrometry Analysis of Synthetic Oligonucleotides – A Comparison of Data from Fast LC and RapidFire Methods’. Richard presented Agilent’s RapidFire 400 high throughput system for microfluidic solid phase extraction coupled with MS screening. The instrument can hold 140,000 samples and analytical run times are between 3-15 seconds, which is enabled by specialist seals within the flow path. Richard showed that the data is comparable with fast LC-MS, reproducible and quantitative, as demonstrated by case reports on pharmaceutical metabolomic studies and nitrosamine analysis. If the RapidFire is too out of scope, Richard discussed some of the parameters one would need to employ to enable fast LC-MS separations on a traditional LC and exemplified this with oligonucleotide separations data.

After this, the vendors exhibited over lunch. Representatives from VWR International, Buchi UK, Crawford Scientific, Fortis Technologies, Avantor, Agilent, Elga Veolia, ATG and LCGC were networking with delegates and presenting latest products and technologies available for small molecule analysis or purification.

Session 2 – Small Molecule Analysis – Pharmaceuticals: the analysis of impurities.

Lewis Jones, from Sensient Flavors kicked off session 2 with his presentation ‘Eating Ice Cream and Chemical Analysis – The Joy of Working With Vanilla’. He highlighted that Sensient Flavors wants to bring joy to people through their work and brought vanilla ice cream for the delegates to eat while he presented. He introduced the physiochemical processes surrounding taste and aroma and the chemicals that are involved in our perception of aroma. Vanilla originates from South America, but is primarily grown in Madagascar. The beans are hand pollenated by the farmers, which results in Vanillas’ high expense. Vanilla is extracted from these beans through solvent extraction processes (via ethanol, water and carbon dioxide). This creates a range of different flavors for food or perfume uses at a range of concentrations. The main quality marker in the flavor is vanillin, which is analysed using HPLC, however Sensient Flavors analyze numerous flavor compounds using GC-FID and HPLC-DA for QC of their products. Lewis demonstrated the ‘sensomics’ approach to quality, which focuses on identifying compounds associated with aroma- followed by their quantitation. They perform discrimination testing to validate the compounds in product by a ‘Napping method’, which is a descriptive approach to assess samples. This approach enables discrimination between species of vanilla beans and different extraction processes.

The next presentation was ‘Method Development Strategies for Nitrosamine Analysis’ by Dr. Brian Montgomery, from Avantor. Brian highlighted that nitrosamines are formed between amines and nitrate salts and can potentially form within many different products (food, plastics, pharmaceuticals, cosmetics, environmental products). These small compounds are genotoxic and potentially carcinogenic, which creates a requirement to characterize their presence within different products.  In 2021 the FDA released control guidelines regarding nitrosamines after trace levels of nitrosodimethylamine was found in the pharmaceutical Valsartan in 2018. LC-UV-MS is a sensitive approach for nitrosamine analysis, and Brian presented methods for Valsartan. Chemicalize.org was used to calculate physicochemical properties for a modelling approach to optimize the method. A reversed phase LC- quantitative MRM MS experiment was used with stable isotope internal standards to identify nitrosamines within samples with <3% CV. Brian stipulated that a critical element to optimizing the method is choosing the right internal standard and MRM transitions for each analyte.

The final presentation was by Dr. Andrew Feilden, from Hall Analytical, who presented ‘Extractables and Leachables: The What, The Why, The How’. Andrew began with introducing the concept of safe pharmaceutical products that have not been altered by extractable or leachable compounds from the product container or contain any toxic compounds extracted or leached from the container. A range of tests are performed to analyse volatile, semi-volatile, non-volatile and elemental compounds, including GC headspace analysis, GC-MS, LC-UV, LC-MS, LC-CAD and ICP-MS. Techniques need to be sensitive enough or coupled with concentration approaches to identify extractable or leachable compounds present at low levels. Identification of the compounds is key, so there is a high dependence on using multiple methods to characterize the cohort of extractables and leachables present within the drug formulation.

Session 3 – Small Molecule Analysis- Pharmaceuticals: quality by design.

Professor John Dean, from Northumbria University was the keynote speaker for the final session of day one, presenting ‘Designing an Exogeneous Volatile Organic Compound for Biomedical Analysis’. He described a project he has collaborated within for the past decade with Biomerieux – developing alternatives to bacterial culture analysis of biosamples within hospitals. One way of identifying specific bacteria is via the enzymatic generation of volatile organic compounds (VOCs) that can be identified by GC-MS analysis. John described the QBD approach to developing a pathway to exogeneous VOC generation, identifying the considerations for endogenous enzymes present within specific bacteria, the enzyme substrate, influences on detection and extraction of the analytes. He presented a case study where C.difficile was identified by adding 3-fluoro-4-hydroxyphenylacetic acid substrate to the culture sample to enzymatically react with p-hydroxyphenylacetate decarboxylase within the bacteria and produce VOC 2-fluoro-4-methylphenol. This was sequentially analysed using optimised GC-MS to identify the bacteria within the samples. John highlighted that some bacteria, such as salmonella, will naturally emit hydrogen sulfide that can be analysed using static headspace multi-capillary column GC-ion mobility spectrometry.

The final presentation of the day was ‘Development of Oligonucleotide Impurity Methods within an AQbD Framework’ by Dr. Chris Gripton, from GlaxoSmithKline. Chris described an analytical target profile approach to optimizing ion-pair reversed-phase chromatography assays for oligonucleotide therapeutics. These molecules are highly chemically modified and this leads to a large range of oligonucleotide impurities from synthesis or degradation pathways. The current assay methods employ LC-UV-MS analysis and Chris described that GSK are developing simpler methods for oligonucleotides. He highlighted that along with the analytical target profile, in silico LC method development tools are used for method optimization. The amine and counterion choice (or combinations thereof) is critical for chromatographic and mass spectrometry performance, and to avoid on-column degradation of the oligonucleotide therapeutic. A DOE approach was applied to method parameters such as source settings, temperature and flow rate to optimize accuracy, selectivity and sensitivity. Chris demonstrated that optimisation of methods for oligonucleotides creates simpler, complementary approaches to assay impurities or API by LC-UV or LC-MS, and an AQbD Framework is a key tool in focusing on development strategy.

The evening was concluded with a barbeque within the racecourse and evening networking among the vendors, delegates and speakers.

Day 2 (20th May 2022)

Session 4- Challenges in Small Molecule Analysis: polar and chiral compounds.

Prof John Lough from The University of Sunderland was the keynote speaker for the first session of day 2. His talk on ’The Role of chiral Screening in the Analysis of Small Molecules; Asymptotic Growth?’, was an in depth evaluation of chiral screening over the past two decades. By asymptotic, John means a steady growth in throughput over time. John evaluated the current state of the art in chiral stationary phases and how columns and instrumentation has changed over the years. John discussed SFC and how it became implemented in chiral screening strategies. John demonstrated the plethora of columns available for screening and different methods he has utilized within his research. He added that even simple lean screens can be appropriate methods as he demonstrated with his work screening anti-cancer compounds. John highlighted some future challenges for growth in chiral separations, such as analysis of multi-chiral drugs, improvements in SFC technology, development of tiered screens and increasing throughput.

The next presentation was ‘The Dark Art of Chiral SFC’ by Keira Bailey from Reach Separations. Kiera explained that supercritical fluid chromatography harnesses supercritical fluids for separations as they have the low viscosity of a gas and the solubilizing power of a liquid. The high diffusivity value of CO2 leads to increased column efficiencies, which Reach Separations utilize for chiral separations. Extensive screening approaches use 88 chromatographic conditions to find the optimal separation and often, SFC is used over HPLC due to its higher throughput. Keira presented some case studies of chiral and achiral separations and introduced the sustainability aspect of SFC: lower waste generation, usage of safer solvents and renewable feedstocks, which will be potential analytical drivers in tomorrow’s world.

The final presentation of the session was from Dr Luisa Pereira from Unilever, ‘Porous Graphitic Carbon- a Unique Stationary Phase for Challenging Separations. Luisa presented an in depth overview of the chemistry and retention mechanisms associated with this type of stationary phase. PGC is very pH stable and highly retains polar analytes by inducing strong dipole-dipole interactions. She presented some case studies of separations, such as the separation of purines and pyrimidines using reversed-phase conditions and commented on the complementarity between porous graphitic carbon separations and hydrophilic interaction chromatography. Luisa also discussed high temperature liquid chromatography using PGC stationary phase and just water for mobile phase. High temperature separations demonstrate high efficiency, faster analysis times and higher resolutions.

Session 5- Challenges in Small Molecule Analysis: environmental analysis.

The keynote speaker for session 5 was Dr Helena Rapp-Wright, from Imperial College who presented a talk on ‘Analysing Small Molecules in the Environment: Challenges and Opportunities’. She changed the title of the presentation to fit better to the theme and presented an engaging talk on characterising contaminants of emerging concern (CEC). These CECs have the potential to be bioactive, enter the food chain, induce antimicrobial resistance and other toxic effects. Helena’s work was to sample water routes around specific locations and analyse CECs using direct injection LC-MS/MS and a 5 mm biphenyl stationary phase. Helena explained the method optimisation strategy that facilitated the analysis of over 100 CECs in a 4 minute run, allowing the analysis of 261 injections per day for high throughput analysis. The case examples that she presented covered analysis of pesticides and other toxic CECs, as well as illicit drugs within water effluent streams. Helena mentioned that matrix effects can impede the sensitivity integrity of analytical data, therefore her group developed 3D printed passive sampling systems that capture CECs over a longer period and across a range of seasons to concentrate low level analytes and increase sensitivity. The increase in ability to characterise CDCs will be complemented by implantation of machine learning to large data sets in future and biomonitoring remains challenging, she concluded.

Following Helena, Dr Simon Hird, from Waters presented ‘Bringing Reliable Determination of Anionic Polar Pesticides in Food to the Routine Laboratory’. Simon introduced his talk with discussing food analysis and the challenges of extracting pesticides in food samples, such as getting the extraction technique correct to facilitate detection. The quick polar pesticides method (QuPPe) can extract multiple pesticides, however can be affected by LC-MS/MS matrix effects due to there being no sample cleanup step. Waters perform LC-MS/MS analysis of pesticides found in food using a HILIC/WAX mixed mode column originally designated for SFC and obtained great results from the phase chemistry. Simon presented a tool to investigate matrix effects and enables acquisition or MRM and full scan data simultaneously. He concluded that flexibility is key for method optimization and that there remains the need to investigate improved sample clean-up options.

The final presentation was ‘Exploiting Flat Plate Heating to double VOC Analysis Throughput, whilst Improving GC Peak Shape for the Most Volatile Components’, by Bryan White from Agilent. Bryan quickly polled the audience to investigate how many of them use or have used GC and went on to argue how GC is not as challenging to approach as many might think. Cartridge type GC columns make installation much easier, and couple that with more user friendly software can help reduce operational errors. He discussed VOC analysis using flat plate heating technology, which can rapidly heat and cool to facilitate shorter analysis times. Faster temperature ramps can lead to band broadening, so shorter columns can overcome this and smaller column diameters can overcome resolution limits.

Session five was followed by the Chromatographic Society annual general meeting where the society activities and expenditures throughout 2021 were described by Paul Ferguson, Tony Edge and Greg Jonas.

Session 6- The Future of Small Molecule Analysis.

Prof Peter Myers from the University of Liverpool was the keynote speaker for the final session. His presentation ‘A World Without Chromatography’ highlighted a vision to move away from the need to separate molecules. Peter talked about historical events such as witnessing the birth of new detector types and how people would attend symposia to witness technological breakthroughs. He argued that we haven’t moved on much from the observation of peaks in recent years and there is still so much we have the opportunity to do with technology. Peter introduced the idea of transitioning to pattern recognition and sensor technology for the future. He used many examples to highlight this concept, one of which was the ‘electronic nose’, that senses odors and identifies the pattern that is created. This technology is currently limited by a requirement for 1000s of sensors to match what we have naturally in the nose. He discussed the need to couple powerful computing with high resolution sensors to understand complex patterns in the future, as well as alternative technologies for sensors in the future, such as electrical pore wetting and de-wetting or nanowires. His key message was that if we can identify a sample pattern, then we would not need to separate the mixture using chromatography.

The final presentation was a closing discussion from Prof Tony Edge, from Avantor discussing ‘Chromatography of the Future’. Tony argued that chromatography is not dead and that we are on the cusp of something very exciting in the future. He discussed technological advancements, such as the development of sub-micron particles, and the challenges they introduce to the field, such as increases in back pressure. We need faster chromatography and greater selectivity for complex samples through alternative approaches to separations. Tony identified that there is a need for in-situ analysis and instantaneous results, which may be facilitated by advancements in chromatography. He discussed future opportunities in modelling approaches to separations, improved stationary phase chemistries with defined pore structures to improve selectivity of complex mixtures. Printed or etched columns, respective to our separation needs may be a product of the future, which might also further in situ analysis approaches. Tony also discussed high temperature analysis, which makes water behave more like an organic solvent and improves sustainability. He concluded with the idea that chromatography is a world full of opportunity!