MRR: A New Tool in Applied Analysis for the Pharmaceutical Industry
Even the most advanced scientific methods can have their challenges. Liquid and gas chromatography (LC and GC, respectively), currently the gold standard for analysis and separations, can be limited in the insights they can provide for difficult solvents. Molecular rotational resonance (MRR) is rapidly coming into routine use with its unique ability to provide clear structural information on compounds and isomers, even within mixtures, without requiring pre-analytical separations. This article will discuss how MRR can minimize separation time, provide high-quality data for and quantitate challenging solvents, provide the sensitivity required to analyze solvents in line with U.S. Pharmacopeia Chapter <467> residual solvents requirements for Class 2 mixture C solvents, and remove the need for consumables. It will also discuss how MRR can support in the structural identification of particularly difficult molecules, and look at future applications of MRR in reaction optimization, before concluding that this technique has a place in the laboratory alongside current chromatography systems.
Developing new drugs is a risky and costly process. Only about 12 percent of drugs entering clinical trials are ultimately approved by the U.S. Food and Drug Administration (FDA), and the expected cost to develop a new drug, including capital investment and spend on drugs that fail to reach the market, is estimated to be about $2 billion (1). As a result, pharmaceutical companies are under consistent pressure to derisk development and reduce cost in production. In addition, regulatory authorities are demanding more, and earlier, information as a candidate moves through the development process, as well as regularly reviewing analytical methodology and permitted levels of ‘contaminants’, for example.
Against this background, the analytical laboratory must play its part by streamlining workflows, adding new capabilities, and improving efficiency. The modern analytical laboratory already contains a suite of high-powered systems to probe and investigate samples of all types. From chromatography, through mass spectrometry (MS), to Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, these instruments answer research questions and provide the data that guides production and quality control.
However, even the most advanced scientific methods have their limitations, and scientists often need to find new ways to overcome long-standing challenges. Sometimes, the answer isn’t a new system, a new method, or a new technique, but rather a reinvigoration and modernization to bring an existing technology to new applications.
Molecular rotational resonance (MRR) spectroscopy is a prime example of how a well-established, trusted, primary analytical technology can find renewed application in the digital age. MRR has long supported basic scientific discovery through its unique ability to provide unambiguous structural information on compounds and isomers within mixtures, without the need for pre-analysis separation.
Until now, having an MRR instrument in the lab meant adapting or building your own. A breakthrough in chirped pulse Fourier-transform microwave spectroscopy (2) gave MRR renewed impetus as a transformative technique for applied analysis, and the subsequent development of the first commercial MRR instrument holds the key to translating the technology into routine use, unlocking its potential to resolve persistent challenges in analytical chemistry.
This article considers the broad range of potential applications of MRR in the pharmaceutical industry, where it shows wide-reaching promise in helping accelerate the drug development process.
The Right Tool for the Job
The unambiguous structural characterization provided by MRR means a molecule that, once analyzed by MRR, is “forever recognizable” (3).
Notably, isomers in a mixture can be definitively characterized without having to first separate the individual components. MRR provides the three-dimensional (3D) structure by determining each unique pattern in the spectrum.
Other techniques offer different strengths and weaknesses; for example, NMR is regarded as the gold standard for structural identification, but lacks sensitivity in the analysis of mixtures and the resulting spectra require expert interpretation. MS tends to be used in tandem with liquid or gas chromatography (LC and GC, respectively), requiring expensive consumables and significant method development time, and, although highly sensitive, it does not generally identify isomers unambiguously. FT-IR spectroscopy analyses are challenging to interpret if reference standards are not available.
MRR spectroscopy can combine the speed of MS with the structural information of NMR. It has remained, until now, the staple of the academic laboratory, and has been used mostly for challenging compounds. MRR’s new commercial availability brings its game-changing potential to routine, high throughput laboratory applications.
Volatile Organic Solvent Analysis Made Simple
Solvents are widely used in the pharmaceutical production process, and, unless properly removed, can compromise the quality or safety of a drug product. Residual solvents in pharmaceuticals are defined as “organic volatile chemicals that are used or produced in the manufacturing of drug substances, excipients, or dietary ingredients” (4). They are a potential toxic risk for pharmaceutic products and have been a concern for manufacturers for many years (5).
Residual solvent analysis of bulk drug substances and finished pharmaceutical products is necessary, as the solvents not only represent a risk to human health, but also play a role in the physiochemical properties of the substance. The main purpose for residual solvent testing is to provide a monitoring check for further drying of bulk pharmaceuticals or as a final check of a finished product. Changes in the dissolution properties and formulation problems in the finished product can be caused by differences in the crystal structure of the bulk drug substance. Furthermore, odor and color changes can be created, which can lead to customer complaints (5).
Testing is essential to avoid potential quality issues that would affect the production of a safe, regulatory-approved product, and is governed in the United States by the U.S. Pharmacopeia “<467> Residual Solvents” (USP <467>) (4) and by the FDA guidance “Q3C Impurities: Residual Solvents.” (6).
Headspace GC coupled either to a flame ionization detector (FID) or MS is the current standard methodology (noted in USP <467>) for residual solvent testing. This has its limitations; some sample matrices may contain non-volatile substances which can reduce analyte peak response, or generate volatiles that would give false responses for other volatiles (5).
Furthermore, analysis of some solvents requires more complex procedures, a process that can delay decision making. USP <467> states that solvents in Class 2, Procedure C should be analyzed using an alternative procedure to headspace GC, as this technique falls short in terms of the sensitivity required.
Scientists can find the answer in MRR, as it offers dramatically simplified method development by removing the need for consumables and solvents. MRR also reduces analysis times compared to chromatography-based systems, and provides the identity of in-process samples with equivalent sensitivity and better quantitative performance compared to a GC system.
Characterizing Impurities
Impurities in raw materials used in drug synthesis can lead to significant variations in the quality of the final drug formulation, and the reliable identification and quantification of raw material impurities can present a challenge. MRR can be useful in this critical step of validating the purity of pharmaceutical raw materials, especially since, unlike most other critical process parameters, manufacturers have little direct control over raw material impurities. These impurities often have similar reactivity to the desired compound, which can introduce potentially toxic by-products with structures similar to that of the desired drug, in turn requiring additional purification steps.
The most common approaches incorporate chromatographic separation techniques, which require the time-consuming development of effective methods for each raw material. Direct spectroscopic techniques are preferable for their simplicity and speed, but these often do not perform as well as chromatography in mixture characterization. However, because MRR can be applied directly to mixtures, it can clearly and accurately distinguish molecules with highly similar structures, including chiral impurities.
A study (7) used MRR to quantify regioisomeric, dehalogenated, and enantiomeric impurities in two raw materials used in the synthesis of cabotegravir, a HIV integrase inhibitor. This is important, because these raw materials can introduce structurally similar impurities into the final drug. Notably, this is the first application of MRR to the rapid quantitative monitoring of isometric and dehalogenated impurities in pharmaceutical and chemical raw materials (in both chiral and achiral analyses) in studying compounds that are challenging for GC. The high resolution and selectivity to small changes in molecular structure of MRR perform these measurements rapidly and without the need for developing a chromatographic separation method. It was noted that MRR was “an extraordinarily selective technique applicable to the direct analysis of mixtures, including isomers.” The same study also suggests that “MRR can have unique value in pharmaceutical process analytical technology (PAT) and quality by design (QbD) programs.”
Another study (8) used MRR in online reaction monitoring to quickly characterize the yield, specificity, and impurities generated in a chemical reaction, and is the first demonstration of MRR for the characterization of the purity of a pharmaceutical synthetic process. Online instrumentation can run continuously during a chemical reaction, saving valuable time. The key advantage of MRR over other process monitoring techniques is its high resolution and specificity, where different species can be unambiguously resolved and quantified in a reaction mixture. MRR was used to monitor the product composition of an asymmetric continuous flow reaction, in the hydrogenation of artemisinic acid needed for the semisynthesis of the antimalarial drug artemisinin, where MRR is highly sensitive to small changes in molecular structure and can rapidly quantify isomers and other impurities in a complex mixture without first requiring chromatographic separation.
A further study (9) demonstrates the use of MRR as a reaction characterization tool to simplify analytical workflows. This study involved the identification and quantification of regioisomeric impurities in crude reaction mixtures. Identifying and quantifying products arising from novel synthetic chemical reactions can be difficult, as reactions can yield complex mixtures with structurally similar analytes. A standardized method for reaction characterization is sought, as this can be a highly laborious process requiring many manual steps to obtain pure products for analysis, and MRR offers a technique that allows reaction yield and byproduct content to be determined directly on crude materials without reference standards, resolving numerous mixture compounds without purification.
Chiral Analysis in Action
The rapid analysis of chiral purity is an unmet need in the pharmaceutical sector. MRR offers a new route to definitive chiral analysis based on structure. Chiral tag MRR provides a new routine way to measure the enantiomeric excess (EE) and to establish the absolute configuration of chiral molecules (10). The ability of MRR to measure EE without chromatography enables high-throughput reaction optimization.
A recent study (11) using targeted MRR spectrometry measured the EE of pantolactone without the need for reference samples with known enantiopurity. Pantolactone is a chiral lactone used as a central intermediate in the synthesis of panthenol and pantothenic acid (vitamin B5), that is marketed globally in personal care products and over-the-counter medication. Complexes with small chiral tag molecules were measured using broadband MRR spectroscopy and the chiral tag methodology to determine the EE.
Because complexes of pantolactone have distinct moments of inertia, their spectra are resolved by MRR. Further investigation (11) of reference samples prepared from mixtures of (R)-pantolactone and (S)-pantolactone broadband used a chirped pulse spectrometer to characterize the structure of the complexes. MRR spectrometry was also used in EE analysis, in a rapid 15-min sample-to-sample cycle time, with equivalent analytical specifications and much faster analysis time than a chiral gas chromatography method.
The results were clear—the measurements using broadband MRR spectroscopy show quantitative agreement with the results from chiral GC and offer “significant reductions in measurement time and sample consumption.”
Supporting the Deuterium Switch
Recently, there has been a trend to use selectively deuterated small molecules as novel drug candidates. These new active pharmaceutical ingredients have, in turn, driven demand for highly selective deuteration reaction methodologies (10). The strategic replacement of hydrogen with deuterium can affect both the rate of metabolism and the distribution of metabolites for a compound, improving the efficacy and safety of a drug (12).
This “deuterium switch” has attracted considerable interest in the pharmaceutical industry. Deutetrabenazine, for example, is an FDA-approved deuterated drug that shows promise in the treatment of Huntingdon’s disease (12). Site-specific deuteration can also create chiral molecules called enantioisotopomers, which are chiral solely because of the deuterium substitution.
When used as an API in a drug, chiral molecules will likely react differently to achiral molecules. Because chiral raw materials are a major contributor to the materials cost of pharmaceutical manufacturing, there is a strong incentive to find effective, lower-cost synthetic alternatives (7).
Direct On-Line Reaction Monitoring
As pharmaceutical manufacturers work to improve development success rates, optimizing synthetic routes is critical, and forms an integral part of the United States PAT initiative that aims to improve pharmaceutical manufacturing processes in real-time.
MRR is an attractive technique here. Rather than sampling from a reactor and measuring at-line or close-to-line, MRR can, with the appropriate sample feeding system, identify and quantify individual components directly from of reaction mixtures without the need for chromatographic separation (13).
The Future Looks Bright
MRR is heralding a new era in the application of spectroscopy. While chromatography will retain its place as a mainstay of routine analytical chemistry, and other techniques will remain the best choice for certain applications, the benefits of MRR in definitive structural analysis for mixtures, chiral compounds, and in the high throughput and process control environment offer analysts exciting new potential.
The commercialization of MRR in a robust, reliable, well-supported, widely-accessible instrument allows, for the first time, laboratories to add this powerful technique to their analytical armory.
References
(1) “Research and Development in the Pharmaceutical Industry.” Congressional Budget Office. https://www.cbo.gov/publication/57126 (accessed 2024-04-08)
(2) Park, G. B.; Field, R. W. Perspective: The First Ten Years of Broadband Chirped Pulse Fourier Transform Microwave Spectroscopy. J. Chem. Phys. 2016, 144 (20), 200901–200901. DOI: 10.1063/1.4952762
(3) Wilson, E. B. Microwave Spectroscopy in Chemistry. Science 1968, 162 (3849), 59–66. DOI: 10.1126/science.162.3849.59
(4) The United States Pharmacopeial Convention: <467> Residual Solvents, Interim Revision Announcement Official November 1, 2019; Official December 1, 2020. https://www.uspnf.com/sites/default/files/usp_pdf/EN/USPNF/revisions/gc-467-residual-solvents-ira-20190927.pdf (accessed 2024-04-09)
(5) B’Hymer, C. Residual Solvent Testing: A Review of Gas-Chromatographic and Alternative Techniques. Pharm. Res. 2003, 20 (3), 337–344. DOI: 10.1023/a:1022693516409
(6) “Impurities in New Drug Substances (Q3A),” International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Geneva, 1997. https://database.ich.org/sites/default/files/Q3A%28R2%29%20Guideline.pdf (accessed 2024-04-08).
(7) Neill, J. L.; Mikhonin, A. V.; Chen, T.; et al. Rapid Quantification of Isomeric and Dehalogenated Impurities in Pharmaceutical Raw Materials Using MRR Spectroscopy. J. Pharm. Biomed. Anal. 2020, 189, 113474. DOI: 10.1016/j.jpba.2020.113474
(8) Neill, J. L.; Yang, Y.; Muckle, M. T. et al. Online Stereochemical Process Monitoring by Molecular Rotational Resonance Spectroscopy. Org. Process Res. Dev. 2019, 23 (5), 1046–1051. DOI: 10.1021/acs.oprd.9b00089
(9) Joyce, L. A.; Schultz, D. M.; Sherer, E. C. et al. Direct Regioisomer Analysis of Crude Reaction Mixtures Via Molecular Rotational Resonance (MRR) Spectroscopy. Chem. Sci. 2020, 11 (24), 6332–6338. DOI: 10.1039/d0sc01853h
(10) Sonstrom, R. E.; Zoua Pa Vang; Scolati, H. et al. Rapid Enantiomeric Excess Measurements of Enantioisotopomers by Molecular Rotational Resonance Spectroscopy. Org. Process Res. Dev. 2023, 27 (7), 1185–1197. DOI: 10.1021/acs.oprd.3c00028
(11) Sonstrom, R. E.; Neill, J. L.; Mikhonin, A. V.; et al. Chiral Analysis of Pantolactone with Molecular Rotational Resonance Spectroscopy. Chirality 2021, 34 (1), 114–125. DOI: 10.1002/chir.23379
(12) Smith, J. A.; Wilson, K. B.; Sonstrom, R. E.; et al. Preparation of Cyclohexene Isotopologues and Stereoisotopomers from Benzene. Nature 2020, 581, 288–293. DOI: 10.1038/s41586-020-2268-y
(13) Byars, A.A,; Kompally, K. R.; Mechnick, E. et al. An Automated, Highly Selective Reaction Monitoring Instrument Using Molecular Rotational Resonance Spectroscopy. Precis. Chem. 2024, 2 (2), 57–62. DOI: 10.1021/prechem.3c00098
About the Author
Christopher Thompson obtained his PhD in Physical Chemistry at the University of Massachusetts Amherst. He spent 17 years at Bruker Daltonics in various scientific roles, ultimately as Global Business Development Manager for the FT-ICR business unit. Chris moved to BrightSpec in late 2020 to lead the commercialization of MRR, and is currently the Vice President of Commercial Development.
