HOW qnmr BRINGS SPEED ND CCURCY TO DRUG DEVELOPMENT INTRODUCTION The total cost of bringing a drug to market is now estimated at about $2.5 billion 1, and the pressure to advance new drugs faster is greater than ever. Part of the challenge is the rising expense of late stage drug testing, which now typically costs several hundred million dollars. But developers can speed their drugs to market more easily if they learn more about a candidate drug s chemical characteristics as early in the development as possible. The goal is to invest as little as possible before the drug candidate has proven its potential. Throughout the drug development process, analytical chemists typically develop testing methods to determine potency of compounds and these have long relied heavily on liquid chromatography platforms, which can be time-consuming and expensive. One powerful, though often overlooked, tool is quantitative Nuclear Magnetic Resonance (qnmr). qnmr can be a single point replacement for multiple traditional studies. What s more, although this technology has long required substantial expertise, new software is making it much easier for a wider range of pharmaceutical professionals to more quickly and accurately interpret and apply the data from these analyses. s a result, more professionals in drug discovery and development are becoming aware of this technique and using it for early-stage characterization of new molecular entities (NMEs). BSICS OF qnmr It s been around for decades, with the first literature reference to qnmr published in 1954. 2 The technique uses NMR to highly accurately determine the concentration of chemicals in solution. The NMR signal is directly proportional to the amount of a chemical in the solution it s actually measuring the number of nuclei present. s a result, one of the great strengths of this technology is that it works with the same level of reproducibility for every molecule being studied. It doesn t suffer from variable-inducing features such as compound-specific response factors or relative volatility. Each proton has a universal response, so there is no need for relative response factors. BROUGHT TO YOU BY
2 Naturally, qnmr requires certain experimental requirements be taken into account to be successful. But if the right steps are followed, the main considerations are that the sample under study must be able to dissolve completely and it must contain NMR-active nuclei. qnmr readings are determined based on comparison to known reference compounds (See Table 1). This establishes the NMR response factor per nuclide. Standard approximate chemical shift (ppm) duroquinone 2 dimethylsulfone 3.2 maleic acid 6.2 benzoic acid 7.4 8.2 3,5-dinitrobenzoic acid 9.2 Table 1. Commonly Used qnmr Internal Standards Source: Expanding the nalytical Toolbox: Pharmaceutical pplication of Quantitative NMR. nalytical Chemistry. 2014, 86, page 11476. In early development, a key task is determining purity/potency of a drug, which is usually calculated by taking the amount of active drug and subtracting the sum of inactive substances, process impurities and degradation products. Traditionally, drug developers have used a variety of tests using liquid chromatography to analyze active pharmaceutical ingredients (PIs) in the early stages of drug development. However, besides being expensive and time-consuming, these systems also have issues with variability. For rapid, selective and accurate potency determination without using liquid chromatography, qnmr is increasingly becoming the tool of choice: It is both more cost-effective and now much easier to use. qnmr can be used for potency calculation, purity assessment, identity testing, residual solvent, moisture analysis, relative response factor calculation and more. It can, in fact, be a onestop solution for early chemical characterization, replacing workflows that required multiple experiments and techniques. The majority of analytical techniques have principal strengths as either qualitative or quantitative methods, with NMR being the notable exception, write Pauli et al. 3 qnmr can perform both relative and absolute determinations and is capable of absolute quantitation, akin to (thermo)gravimetry, coulometry and titrimetry, they added. One of the key developments has been the rise of high magnetic field instrumentation. Increasing the magnetic strength provides greater resolution and sensitivity. Some early studies, such as determining the potency of aspirin, used just a 1 H frequency of 60 MHz. 4 Currently, most studies employ instruments with 1 H frequencies greater than 300 MHz. But instruments with frequencies of 400 MHz or more are available.
3 With qnmr today, there is also no need for a fully characterized reference standard for the analyte, commercially available reference standards are used. It s faster, there is no need to calculate response factors or calibration curves. It s a one-stop solution: Potency and structure are confirmed in a single experiment. It s highly accurate because internal standards eliminate errors introduced by inherent sample differences. It s highly reproducible, because an automated workflow from acquisition to analysis decreases human error and variability. It s also intuitive and flexible: Straightforward manual interaction can be used when needed. When characterizing NMEs, it is essential to be able to detect even very small structure for impurities. Figure 1 1 H NMR spectrum of a hypothetical mix of three similar compounds. If analyzed using liquid chromatography, these compounds would all have similar elution characteristics. Compound 1 and 2 only differ by a methyl substitution on the phenolic hydrogen. Compound 3 is a combined product of the other two. Figure 1 shows that a mixture of these compounds would be easily resolved in a qnmr experiment using signals such as the methyl of the methoxy group of compound 2. This demonstrates that the resolution of NMR is ideal for quantification of structurally similar compounds in a mixture. 46 40 39 41 H 3 C 45 38 36 44 37 CI H C 48 CH 3 47 42 43 O 14 6 5 4 NH 1 2 N O 3 7 8 13 9 H 3 C 19 12 10 18 11 CI H C 21 CH 3 15 20 16 17 3 33 26 25 27 H 3 C 32 24 22 31 23 CI 28 H 3 C 35 CH 3 34 29 O 30 CH 3 Group nh Shift Conf. Limits ve. Exp. Neural Net. 1 1 9.94 1.86 8.78..11.39 11.01 4 1 7.48 0.23 7.43 7.67 5 1 5.74 0.18 5.81 5.52 9 1 7.86 0.22 7.67 7.49 13 1 7.3 0.62 6.42..7.45 7.48 17 3 3.74 0.09 3.78..3.87 3.82 19,20,21 9 1.41 0.15 1.22..1.52 1.35 25 1 7.58 0.2 7.40..7.80 7.47 27 1 7.60 0.04 7.55 7.54 30 3 3.79 0.07 3.78..3.87 3.84 32,34,35 9 1.42 0.15 1.22..1.52 1.36 39 1 7.63 0.20 7.40..7.80 7.42 41 1 7.49 0.09 7.55 7.37 43 1 5.85 0.68 5.85 4.57 45,47,48 9 1.44 0.15 1.22..1.52 1.35 Figure 1. Resolving a mixture of three similar compounds using NMR. Source: Expanding the nalytical Toolbox: Pharmaceutical pplication of Quantitative NMR), nalytical Chemistry, October 27, 2014 page 11477 In the past, the downside of qnmr was the fact that it required specialized expertise to analyze the data. But with new streamlined workflows and analysis solutions, this powerful technology can now be used by even non-specialists.
4 qnmr is accepted by the International Conference on Harmonization, (ICH). Maniara et al. demonstrated that the technology is accurate for both determining the major component and the impurities in a drug substance, by showing that it could quantify impurities at the 0.1% level or higher with sensitivity, speed, precision and accuracy similar to what is obtained with HPLC. 5 DETERMINING POTENCY ND PURITY USING POTENCYMR Bruker s new potency determination tool PotencyMR has made it possible for both experts and non-experts to reap the benefits of NMR-based analysis. There is no need to have a fully characterized reference standard for the analyte. It uses commercially available reference standards. There is also no need to calculate response factors or calibration curves: NMR is inherently quantitative. The system does potency determination and structure confirmation in a single experiment. It is accurate, intuitive, flexible and reproducible: The automated workflow, which goes from acquisition to analysis, decreases human error and variability (See Figure 2). Sample Preparation Weighing of analyte and internal standard, dissolution and transfer to the NMR tube Sample Submission Experiment and internal standard selection, weights input Results Spectra, potency, Excel table and PDF Report Wt analyte [mg] Wt [mg] N * [mmol] Potency [%] CH Region 1 Region 2 Region 3 veraged rea analyte SD rea analyte N analyte [mmol] Potency [%] SD Potency [%] 10.30 5.10 0.04 99.00 1.03 1.00 0.99 0.98 0.99 0.01 0.04 99.11 13.10 5.60 0.05 99.00 0.88 1.00 0.98 0.97 0.98 0.01 0.05 99.19 11.50 22.50 0.19 99.00 4.05 1.00 0.99 0.97 0.99 0.01 0.05 99.31 99.20 0.08 Figure 2: Potency determination workflow Source: www.bruker.com/products/mr/nmr/nmr-software/software/qnmr/overview.html
5 The process begins by submitting the sample using the IconNMR automation software. default qnmr experiment is already provided and parameters for maleic acid internal standard. It features internal standard peak identification and integration with sophisticated peak snapping algorithm. Error analysis is also automatically carried out. Multiple analyte peaks are integrated and averaged, and the RSD is provided. The software also calculates error analysis between the samples. Replicate samples can be submitted, and the potency for each replicate calculated. In the end, the software delivers a final averaged result and associate error. These results can be presented in a variety of formats, including a PDF report with spectral information and data in excel tables. The data analysis uses established algorithms for NMR quantification (See Figure 3). The results can be obtained automatically from the acquisition module or manually executed. In fact, all the processing features and the analysis can be manually executed if desired. P = P I I Wt Wt MW MW P, P = potency of analyte/standard; I, I S = integral area of analyte/standard from the NMR spectrum normalized by number of nuclei; MW, MW = molecular weight of analyte/standard; Wt, Wt = weight of analyte/standard. Figure 3. Equation to determine the qnmr potency Source: Potency Determination by qnmr Drug developers need molecules that meet specific structural, biological and intellectual property requirements. Once candidate molecules are synthetized, they must be characterized to determine their structure and purity. Using liquid chromatography, this is usually done by subtraction: The area of percent of liquid chromatographic analysis is organic and inorganic impurities (via Karl Fisher titration), residual solvents (gas chromatography or thermogravimetric analysis, residue on ignition, and elemental analysis. The equation is shown in Figure 4. % related % enantiomer potency = 100 100 % water % others MW 100 MW active salt 100 Figure 4: Equation for calculating potency by subtraction. Source: Expanding the nalytical Toolbox: Pharmaceutical pplication of Quantitative NMR. nalytical Chemistry. 2014, 86.
6 s Webster and Kumar note in their 2014 article for nalytical Chemistry, rapid determination of drug candidate potency requires three key features: It must obviously be rapid, it must be selective for the drug being characterized and it must be quantitative even if a characterized standard of the specific drug being studied is not available. Only 1 H NMR readily fits all three of these criteria and is available in most pharmaceutical laboratories, they write. 6 One of the earliest applications of qnmr, they note, was to enable testing of sufficient batches of candidate drugs to complete both early safety GLP studies and later GMP-compliant clinical trials. qnmr offered an ideal solution to this challenge. By using qnmr for the GLP release, the scientist had a rapid determination of potency and spectroscopic confirmation of structure, they wrote. The qnmr method does not conflict with the GMP release for it is not a method employed in GMP testing so no duplicate testing of the drug batch occurs. s a result, manufacturers realized they could save money by producing a single lot for the initial GLP and GMP testing. fter the GMP methods were finalized and validated, the batch could be tested according to those specifications. THE KEY TO WIDER DOPTION OF NMR New software solutions from Bruker are making it possible for drug developers to get even better use out of a powerful tool NMR. While this technology was once reserved for experts, it can now be much more widely adopted and help drug developers greatly accelerate their early development programs. For example, Bruker s latest release of its TopSpin NMR software, T3.5pl7, features a new, user-friendly graphical user interface (GUI) that provides easy access to vast experiment libraries including standard Bruker pulse sequences and user-generated libraries of data for both industrial and academic use. The GUI lets users build and organize their own customized experiment libraries via simple drag and drop, provides multiple options for experiment setup and optimization, enables script-based parameter adjustment, and makes the setup of sophisticated experiments simple and efficient. TopSpin offers a fully workflow-oriented user interface and exploits the latest features of modern Windows / CentOS / MacOS operating systems for optimum memory usage. Both its new workflow-oriented and intuitive user interface and optimized sample data management deliver many additional innovative features, all designed to speed up operation and sample analysis throughput for higher cost efficiency. TopSpin is also available in a student free version enabling institutions in academia and education to enhance the curricula of their students. qnmr has many potential applications. For example, it can be used to analyze natural products for potential active compounds. Such products typically contain a variety of known and unknown compounds as complex mixtures. qnmr can determine the potency of either known or unknown compounds, as long as at least one NMR peak of the active material and one peak of the internal standard are isolated in the spectrum. This type of analysis is also sometimes used for analyzing plants in the food industry and wine. It has also been employed to
7 analyze such varied substances as bird repellants, marine matrixes of shellfish, and supplements such as Ginko biloba. 7 In conclusion, key features of PotencyMR include: convenient sample submission and processing through new automation software. Parameters are available for one of the most commonly used internal standards. The new software provides internal standard peak identification and integration with a sophisticated peak snapping algorithm, analyte quantification, consistency analysis and potency calculation. It s also easy to calculate error analysis within the sample multiple peaks are integrated, averaged and the RSD given, and between samples. s a result, a broader range of professionals are now turning to qnmr for compound characterization. s Pauli et al. have written Compared with chromatography and elemental analysis, quantitative NMR (qnmr) uses nearly universal detection and provides a versatile and orthogonal mean of purity evaluation. 8 References 1. http://csdd.tufts.edu/news/complete_story/tufts_csdd_rd_cost_study_now_publishedz 2. Shoolery, JN. nal. Chem. 1954, 26, 1400-14003. 3. Pauli FG et al. Importance of purity evaluation and the potential of quantitative 1 H NMR as a Purity ssay. Journal of Medicinal Chemistry, 2014, 57, 9220-9231 4. Vinson, J and Kozak, DM. m J. Pharm. Educ. 1978, 42, 290-291. 5. Maniara G, Rajamoorthi K, Raja S, Stockton GW. nal. Chem. 1998, 70, 4921-4928. 6. Webster KG and Kumar S. Expanding the nalytical Toolbox: Pharmaceutical pplication of Quantitative NMR. nalytical Chemistry. 2014, 86, 11474-11480 7. Webster KG and Kumar S. Expanding the nalytical Toolbox: Pharmaceutical pplication of Quantitative NMR. nalytical Chemistry. 2014, 86, 11474-11480. 8. Pauli FG et al. Importance of purity evaluation and the potential of quantitative 1 H NMR as a Purity ssay. Journal of Medicinal Chemistry, 2014, 57, 9220-9231. C&EN MEDI GROUP 1155 Sixteenth Street, NW, Washington, DC 20036