December 9, 2016 1

Solutions pour l’analyse de routine des petites molécules

Fabrice Moriaud, Ph.D. - Applications Development - Fällanden

30ème Réunion d’Utilisateurs RMN Bruker

At university …

… Academia benefits from

• Free TopSpin 3.5 processing SW!

• Download from website

At the spectrometer …

… In next topspin 3.5 release, an acquisition license allows to run:

• Free CMC-assist 2.5

• Free Fastlane, SmartDriveNMR, qNMR

Brian runs an NMR lab

Brian

• … Works in a hypothetical pharma company / university

• … Runs the NMR service lab

• … Is the “resident NMR expert” in the organization

• … Brian has a solid NMR background

• … He needs help to make the right choices for numerous experiments

Brian needs to select for Kevin, the optimal experiments for QC or Structure verification of diverse samples

Numerous and diverse sample: concentrations, structural complexity, spectra complexity, water suppression in some DMSO samples

Brian thought about handling these numerous sample at night or week-end, without coming to a smart solution:

• Running all samples with a complete set of experiments to ensure optimal data (enough scans, HSQC on everything, maybe even auto water suppression…). That would take the spectrometer time for the other tasks

• Run the 1D’s and let Kevin figure it out on Monday (knowing that several spectra will need to be re-run )

• … Or check back on the progress every few hours, look at the results and setup follow-up experiments where needed. Brian is not at the lab at night and at week-end!

A week in the life of Brian

SmartDriveNMR: – makes life easier for measuring and analysing

Questions asked to the spectrometer are delegated to SmartDriveNMR : • Measure a routine 1H spectrum with optimized parameter sets: number of scans, and

suppressions.

• Measure a set of experiments with optimized information and acquisition time

SmartDriveNMR decision-making algorithm

• Step by step decision for further experiments based on both structure and already acquired spectra: 1H, 1H suppressed, more scans, edited HSQC, 13C, HMBC.

• Saves spectrometer time by automatically running only these experiments that add the most value to solve a given problem – using problem-tailored parameters.

Automatic spectra interpretation done by CMC-assist algorithm

SmartDriveNMR: decides on-the-fly from Proton spectrum to HSQC

• SmartDriveNMR analyses both the structure and the acquired NMR Data. Yields result with

confidence level:

With confidence level:

Auto-result

SmartDriveNMR requests HSQC

Higher confidence result !

Automatic decision: auto-result II

SmartDriveNMR: decision-making analysis

• SmartDriveNMR decisions are based on an analysis of both structure and spectra:

• This data analysis is the same as for CMC-q.

• CMC quantification is tested/proven on Expert analysed Data and integrated in

industry workflows.

Concentration

Deviation < 20% < 30% < 40% < 50%

Consistent (85%) 91% 96% 97% 99%

Inconsistent (15%) 60% 68% 77% 83%

Total 86% 92% 95% 96%

Comparison between CMC-q and expert interpretation (~1’700 samples!)

High reliability of the software’s results ! For concentration AND for “consistent”

Example: Actelion’s analysis of the CMC-q auto results

Courtesy of Julien Grimont, Actelion, Allschwil

SmartDriveNMR: consistency check

In addition to the experiment selection, SmartDriveNMR offers a consistency check with CMC-assist analysis, when Consistent, then a full assignment is given. It is done on top of the CMC-q analysis.

• At least “someone” looks at it and suggests an interpretation which is stored along with the data

• Having the right structure is crucial for further work in pharma industry: medicinal chemistry, bioassays. So FP should be as low as possible.

CMC-assist consistency check is validated on Expert analysed Data

CMC-assist – Industry workflow with Structure Verification

I want to improve on selecting sample with the correct structure within a synthesis library

AND look at as little as possible spectra:

what do I achieve with an auto-analysis algorithm if I only look at the red dots?

Idea and reasoning: John Hollerton @ GSK UK

10000 compounds: 5% expected wrong

CMC-assist – Industry workflow with Structure Verification

Idea and reasoning: John Hollerton @ GSK UK

8235 compounds: 0.65% expected wrong

1765 compounds 75% expected correct

False positives

13%

False negatives

14%

(0.65% = 10000 * 5% * 13%)

These I look at or simply mark

These I take for further testing & synthesis

From 5% to 0.65% -> 7 fold better With HSQC, False Positives=3% -> 30 fold better

Predicted Inconsistent with CMC-assist

Predicted Consistent with CMC-assist

Examples for typical confidence values

13 December 9, 2016

Expert: “Small and simple molecule with a nice and disperse spectrum – assignment straight forward”

No follow-up experiment

SmartDriveNMR:

1D 1H Consistent, Confidence 3.5 Stars

No follow-up experiment

Final: Consistent, Confidence: 3.5 Stars

Examples for typical confidence values

14 December 9, 2016

Expert: “Small and simple molecule with an ok spectrum – assignment possible”

maybe more Scans?

SmartDriveNMR:

1D 1H Consistent, Confidence 2.8 Stars

More Scans

Final: Consistent, Confidence: 3.2 Stars

Examples for typical confidence values

15 December 9, 2016

Expert: “Spectrum dominated by Water (and Solvent DMSO)”

Suppression

SmartDriveNMR:

1D 1H Consistent, Confidence 0.3 Stars

Suppression

Final: Consistent, Confidence: 3.1 Stars

SmartDriveNMR: spectrum and structure

Expert: “Structure is complex and 1H spectrum is not easy to assign”

HSQC

SmartDriveNMR:

1D 1H Consistent, Confidence 2.1 Stars

HSQC

Final: Consistent, Confidence: 3.9 Stars

Expert: “Even though structure looks complex and worth an HSQC, the 1H spectrum is fairly easy to assign”

No HSQC

SmartDriveNMR:

1D 1H Consistent, Confidence 3.5 Stars

No HSQC

Final: Consistent, Confidence: 3.5 Stars

M = 422 g mol-1

M = 520 g mol-1

Examples for typical confidence values

17 December 9, 2016

Expert: “Structure is complex and 1H spectrum is not easy to assign”

HSQC

SmartDriveNMR:

1D 1H Inconsistent, Confidence 2.9 Stars

HSQC

Final: Consistent, Confidence: 3.9 Stars

Courtesy of Valentin Poirier, Structuralis, Romainville

SmartDriveNMR: HSQC statistics

When are HSQCs suggested:

• In 32% of > 1000 1D test cases (Ø 300 – 500 g/mol)

• More likely for high MW compounds

0%

20%

40%

60%

80%

100%

Ratio of suggested HSQCs on top of 1D by MW

Summary SmartDriveNMR

19 December 9, 2016

• SmartDriveNMR carries out experiments automatically in an optimized way based on the previous experiment

• Optimization of spectrometer’s time while keeping best quality in results - saving user time “in front of the spectrometer”, e.g. day, night, week ends

• Both NMR experts and non expert benefit

• Interactive / Manual Structure Verification using CMC-assist 2.5

• SmartDriveNMR is new and available in TopSpin 3.5 fully integrated in IconNMR - just a few clicks to try it !

Meet - Rachel

Rachel

• … Works in drug development, Quality Control, Galenic Formulation

• … In charge of determining the amount of active drug in a formulation

• … Needs a rapid and cost effective solution

• … She needs qNMR in automation

qNMR Internal Standard

IS

IS

IS

IS

Mw

Mw

Wt

Wt

I

IPP

a

a

aa

Accuracy Eliminates errors introduced by inherent sample differences

Pa, PIs = potency of analyte/standard Ia, IIs = integral area of analyte/standard from NMR spectrum normalized by number of nuclei Mwa, MwIs = molecular weight of analyte/standard Wta, WtIs = weight of analyte/standard.

e.g. Potency = 90 % means that out of 100mg, only 90 mg are from analyte. Remaining 10% can be anything, moisture, solvent, impurities.

Quantification en solution avec reference externe et interne (qNMR)

Meet - Kim

Kim

• … Works in a hypothetical analytical lab: pharma, forensic

• … In charge of providing consistency check using HRAM MS and NMR

• … Has expertise in NMR or HRAM MS

• … Needs to deliver consistency checks with a high confidence

• … She needs Fusion-SV

Kim’s full verification…

•She wants to be sure, not to file the wrong structure

•Orthogonal data is needed to minimize the chance for false positives

•MS can do things NMR can not (Cl/Br, S/O: totally different mass and small change in chemical shift …)

•Nominal mass leaves 1’000s of potential false positives

•HRAM-MS verifies the molecular formula

NMR can do things MS can not

•100’s of possible structures per molecular formula potential false positives

•1D1H

•HSQC

•HMBC

•13C

Further reduction of

potential wrong passes

Bruker Fusion-SV 1.1

HRAM-MS and NMR data

Details

info in a nutshell

one unified result

shared projects

accurate mass and

isotopic pattern information

automated analysis and

assignment of NMR data

Bruker Fusion-SV 1.1

Automatic analysis of combined 1D1H, HSQC, HMBC and 1D13C data for

Automatic processing of NUS data (HSQC, HMBC)

Spectra interpretation easily understandable

13C assignment HMBC correlations

New: Bruker Fusion-SV 1.1

• License

• TopSpin 3.5pl7: acquisition license allows to use SmartDriveNMR and qNMR (No need of CMC-assist license anymore at the spectrometer)

• SmartDriveNMR

• automatically provides an optimal set of optimized experiments

• Delivers pre-interpreted spectra by CMC-assist

• Qnmr

• Rapid and cost effective solution to determine the amount of active drug in a formulation

• Integrated in IconNMR and CMC-assist, side by side with external reference quantification

• Fusion-SV

• High confidence Structure Verification combining High Resolution Accurate Mass Spectrometry and NMR

December 9, 2016 28