HOW qNMR BRINGS SPEED AND ACCURACY TO DRUG …

HOW qNMR BRINGS SPEED AND ACCURACY TO DRUG DEVELOPMENT

AMERICAN CHEMICAL SOCIETY

INTRODUCTION The total cost of bringing a drug to market is now estimated at about $2.5 billion1, 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. As 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).

BASICS 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. As 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.

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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 Analytical Toolbox: Pharmaceutical Application of Quantitative NMR. Analytical 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 (APIs) 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 1H frequency of 60 MHz.4 Currently, most studies employ instruments with 1H frequencies greater than 300 MHz. But instruments with frequencies of 400 MHz or more are available.

HOW Q-NMR BRINGS SPEED AND ACCURACY TO DRUG DEVELOPMENT

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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 A 1H 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

H3C45 38

36

44

37

CI

H3C48

C47H3

OH 43

42

O 14

6

5

NH 1

4 N 3

8 13

2 O 7

9

H3C19 12 18

H3C21 C20H3

10

11

CI

15

O 16 1C7H3

33

26

25

27

H3C32 24 31

H3C35 C34H3

22

23

CI

28

O 29 3C0H3

Group nH

1

1

4

1

5

1

9

1

13

1

17

3

19,20,21 9

25

1

27

1

30

3

32,34,35 9

39

1

41

1

43

1

45,47,48 9

Shift 9.94 7.48 5.74 7.86 7.3 3.74 1.41 7.58 7.60 3.79 1.42 7.63 7.49 5.85 1.44

Conf. Limits 1.86 0.23 0.18 0.22 0.62 0.09 0.15 0.2 0.04 0.07 0.15 0.20 0.09 0.68 0.15

Ave. Exp. 8.78..11.39

7.43 5.81 7.67 6.42..7.45 3.78..3.87 1.22..1.52 7.40..7.80 7.55 3.78..3.87 1.22..1.52 7.40..7.80 7.55 5.85 1.22..1.52

Neural Net. 11.01 7.67 5.52 7.49 7.48 3.82 1.35 7.47 7.54 3.84 1.36 7.42 7.37 4.57 1.35

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Figure 1. Resolving a mixture of three similar compounds using NMR. Source: Expanding the Analytical Toolbox: Pharmaceutical Application of Quantitative NMR), Analytical 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.

HOW Q-NMR BRINGS SPEED AND ACCURACY TO DRUG DEVELOPMENT

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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 AND 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

Averaged

SD

SD

analyte Wt IS

N IS* Potency

Area

Area N analyte Potency Potency

[mg]

[mg]

[mmol]

IS [%]

CH

Region 1 Region 2 Region 3 analyte analyte [mmol]

[%]

[%]

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: products/mr/nmr/nmr-software/software/qnmr/overview.html

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HOW Q-NMR BRINGS SPEED AND ACCURACY TO DRUG DEVELOPMENT

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The process begins by submitting the sample using the IconNMR automation software. A 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 egxuerceu3ted. In fact, all the processing features and the analysis can be

manually executed if desired.

PA = PIS gure3

IA IIS

WtIS WtA

MWA MWIS

PA, PIS = potency of analyte/standard; IA, IS = integral area of analyte/standard

from the NMR spectrum normalized by number of nuclei; MWA, MWIS =

Figmuorele3c.uElqaurawtioenigthotdoeftearnmailnyeteth/sePtAqaNn=MdaRrPdpISo; WtentIIAcAIS,yWtIS

W= wtISeighMt oWf aAnalyte/standard. WtA MWIS

Source: "Potency Determination by qNMR"

Drug developers need molecules that meet specific structural, biological and

intellectual property requirements. Once candidate molecules are synthetized,

they mgusutrbee4characterized to determine their structure and purity. Using liquid

chromatography, this is usually done by subtraction: The area of percent of liquid

ctihtrraotmioapnt)oo, grteerasnpidchuyical=asnoa%lvlyesrniestslias(gtoeargsdacnh?i1rco%0am0neadtnoinagornartgpiaohnmyicoerimrthpeurrmitoiegsra(vviiamKeatrrlicFiasnhaelrysis,

residue

on1i0g0ngit?uior%ne,

a4wnadteelerm?e%ntaol tahnealryssis. 100

TMMheWWesaqacluttivaetion1i0s0shown

in

Figure

4.

potency

=

%

related

? % enantiomer 100

100 ? % water ? % others 100

MWactive MWsalt

100

Figure 4: Equation for calculating potency by subtraction. Source: Expanding the Analytical Toolbox: Pharmaceutical Application of Quantitative NMR. Analytical Chemistry. 2014, 86.

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HOW Q-NMR BRINGS SPEED AND ACCURACY TO DRUG DEVELOPMENT

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