Steve Hollis1, Dave Strand2, Patrick Wheeler3

1Amgen, Inc., Cambridge, MA, 2Protasis, Corp., Marlboro, MA, 3Advanced

Chemistry Development Inc., Toronto, ON, Canada

Integrating Automation and Data Management

in Open-Access NMR

Better tools are always required to smooth the pathway from sample submission to

data acquisition, to knowledge, while integrating as many legacy systems as

possible. Amgen, Protasis and ACD present here a web-centric, enterprise-wide

automation software - NMR Visor, which supports complete NMR scheduling and

acquisition using IconNMR and standard Bruker auto-sampler hardware. NMR

data is automatically passed to ACD Automation Server where it is processed

using automated structure verification, and the results are distributed directly to the

submitter. Structures can be included with sample submission in standard

commercial formats and metadata is accessible through Active Directory or LDAP

services. Processed data, NMR report documents and structure searchable

databases are constructed for easy access to the chemists using web based

software tools.

Making Life Easier for Our Chemists

Chemists can submit crude reaction mixtures directly to the separations

group for automated purification and LC/MS/NMR characterization

Can submit up to 250 mg/sample dissolved in up to 2.5 mL of DMSO

Desired component should be > 50% purity by initial HPLC screen

Can submit single samples or entire library of up to 24 compounds

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3

Automating NMR Data Collection and Analysis

for HTPP and Sample Registration at Amgen

HTPP Process is an automated mass-directed purification procedure that aids

chemists in their efforts to design and synthesize new therapeutic agents.

Chemists employ parallel synthesis to make focused libraries which are purified

and characterized using LC-MS and 1H NMR using Flow-NMR techniques.

Purified materials are provided in Sample-Bank vials and processed NMR data is

distributed to chemists in a convenient electronic format for e-Notebook records.

Design Synthesis ScreeningPurification Characterization Registration

HTPP/Singleton

Purification Service DAS Separations

Med ChemD S

P C

R

S Biology

Crude reaction mixtures

are submitted

MS-Directed

Purification

1. Product Vial

2. Solubility Vial

3. NMR Plate

4. LC/MS QC Plate

Output

General Procedure for the HTPP-NMR Process

Sample collection is triggered by Mass Spec and the selected product is

dried, weighed and formatted into Sample Bank and solubility vials and

two 96-well plates are prepared in automation using a Tecan Evo robot.

4

Mass Directed PurificationUV

MS

Purification Sample Drying Sample Reformatting

Genovac Tecan EvoWaters LC-MS

2 Vials per Sample

LC-MS QC Plate

NMR Plate0.5 mmol / well

2 Plates per Library

NMR Workflow

HTPP Library and Singleton Plates

• Metadata collected from separation process is organized by

Pipeline Pilot - saved as a SD file.

• SD files are downloaded to the One-Minute NMR computer on

the Protasis flow-NMR system – file used to run the NMR.

• SD Plate map is deconstructed into single-sample sdf’ s which

are embedded in the raw NMR data tree after data acquisition.

• Structures are included in mol format within the SD file.

• Completed data is copied to a sweep-folder for automated

processing by ACD/Labs Automation Server software.

5

Flow-NMR is reliable and reproducible with minimal downtime in practice: more

than 5000 plate samples are run per year with this Protasis system.

Samples are dissolved in 25 mL of dmso-d6 (20 mM) covered and placed on LEAP.

Samples and solvent are kept dry with closed system, using dry N2 and He blanket.

Greatly reduced sample requirements - 15 mL injections used to load samples.

HTPP Flow-NMR System - using a 500 MHz Bruker AVIII

with a Robotic Sampling Station

Protasis NMR

Probe with

10 mL flow-cell

LEAP

Pump/Solvent

Conditioner

Vial or Plate

Injector

Loop

25 mL

syringe

DMSO-d6

6

HTPP-NMR and Singleton Plates

Pipeline Pilot is used to download sample metadata for direct NMR

submissions. SD file is used to collect and process the NMR data.

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HTPP-NMR and Singleton Plates

SD file from Pipeline Pilot is used to initiate the NMR data acquisition - assembles the combined metadata for each plate into a single file.

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Running HTPP/Singleton-NMR in Automation

9

NMR is Setup by Shooting a Barcode

Reads

SD File

from

Pipeline Pilot

• SD file is imported directly

into One-Minute NMR

• Generates sample queue

• Click “Queue Run” to start the

NMR for the entire plate

SD File

Click Run

One-Minute NMR

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HTPP-NMR and Singleton Plates

One-Minute NMR software reads each sample from the SD plate file “one

line at a time” and sends a single-sample sdf file to the NMR spectrometer.

• The individual sdf file is a simple formatted text file – it is saved in the

Bruker data tree in the same location where the fid is stored.

• When the data collection is complete the spectrometer software copies

the NMR file to a remote server in a sweep directory named AS_data.

• The appearance of the data file in the sweep directory triggers ACD/Labs

Automation Server to process, distribute and archive the data in a

searchable NMR database that is distributed to the NMR users.

Example of a Single-Sample sdf file – 117442-03-12.sdf

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Automation Server Output - Processed-Data Folder

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12

Processed Spectra are deposited in esp Folder

ACD/Spectrus Processor for processing and viewing spectra.

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Example Output: Plate-Database Files (nd9)- in Table View

Click on spectrum to start processor

Browse

with

scroll tab

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Plate-Database File - in Plate View

Click on a well - shows the

processed spectrum

Click on this spectrum -

starts the ACD-processor

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Example Report – four page pdf

Data summary

for notebook in

journal format

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Four Page Report – Expansions pages 2-3

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Page 4 - Contains NMR Summary

NMR reports are deposited in the chemist’s reports folder and a copy is

sent to the inbox of their Electronic Notebook.

Review of the pdf report can often be a suitable for final review without the

need for spectrum reprocessing.

The HTPP process also distributes LC/MS data generated on all HTPP-

Singleton samples – data is sent directly to each users directory on LC/MS

file server.

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LC/MS Reports Generated for Each Sample

Chromatogram with Mass Spec

Improving Open-Access NMR

• Use automated data processing features that were developed

for the HTPP/Singleton process in Open-Access NMR.

• Build new web tools to simplify the sample submission process:

- Structures can be added to sample submission to take advantage

. of the ACD processing tools.

- Auto-processing - peak-picking, scaling, integration and plotting

. all are improved when the structure is included with the data.

• NMR Visor - New sample-submittal/tracking software from Protasis

provides real time scheduling and tracking of all NMR submissions

over the network from the chemists desktop computers.

• Integrates with existing AS processing system and Bruker Iconnmr.

Building with Automation Server

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NMR Visor – a New Tool for Open-Access NMR

NMR VisorScheduling Tool

Data processing uses

existing HTPP/Singleton

workflow

Runs

NMR

Logs in Sample

with Structure

ACD/Labs

Automation ServerOpen-Access NMR Network

Chemist

Reports

to e-LN

Spectrometer

Processing Done by ACD/Labs

• Integrated NMR output

• Creates pdf reports

• Structures included

• NMR database created

• Can search by structure

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NMR Visor: Open-Access Submission and Tracking Tool

Can draw or attach structures, track projects, and more…

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NMR Visor Architecture

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NMR

Spectrometer Two

Spectrometer Three

Spectrometer N

Location Two

NMR Visor Architecture

Browser-based Web application

Attractive JavaScript Front-End

Distributed Across Enterprise

Secure Enterprise Network

Secure WebAPI Server & Oracle SQL Database

Secure, Remote Server(s)

Real-time Instrument Controller

Schedule/Monitor Experiments

NMR

RTIC

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NMR Visor Architecture – Web Site

Enterprise Web Application

– JavaScript Framework• MVC Architecture

– Rich, responsive graphics

– Browsers supported:• IE 11

– Operating Systems:• Windows

• Mac

• Linux

– Devices supported:• Desktops

• Notebooks

COC1=CC2=C(C=CN=C2C=C1)

C(C3CC4CCN3CC4C=C)O

C20H24N2O2

324.41676

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NMR Visor Architecture – Web API Server

Application DB Server

– WebAPI• REST Architecture

• Microsoft C# Controllers

– Oracle SQL Standard Edition

• Local or remote

• Consistency constraints

• Custom Fields

– Active Directory • Secure LDAP Authentication

• Windows Authentication

WebAPIC# REST

Controller

SQLsamples

LDAPdirectory

File

Serverspectra

CloudVPN

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NMR Visor Architecture - Instrument Controller

Real-time scheduler

– Custom Priorities

– Overnight Samples

– Experiment Chains

Windows Application

– Runs on Spectrometer Computer (with Topspin)

– Network Interface

– Connects to DB

– Shared Drop Folder

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Getting to the Answer – “Did I Make What I Thought?”

Submit Acquire Report

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NMR Visor Starts by Showing Spectrometer Status

Submit a sample here

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Login Windows Domain

Authenticate against

Amgen active directory

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Select NMR Spectrometer

Add Sample ID and Choose Experiment Details with Dropdown Menus

The Project field will automatically

populate with the last Project used.

The Experiment field will automatically

populate with the last experiment that you

entered if it’s on the same spectrometer.

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Add Structure with Plugin Tool

Draw freehand,

Drag ‘n Drop or

Copy/Paste

From ChemDraw or ELN

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Add More Experiments or Additional Samples (optional)

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Submit Sample

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Put Sample in Holder and Confirm when Ready

After submitting, the samples are in the ‘Pending’ state.

Walk to the instrument and place the sample in the holder

Confirm sample – after placing in auto-sampler

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Monitor the Sample Queue from Your Desk

• Sample is waiting to be run on the instrument

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Review Completed Samples

Displays samples that have completed within the last 7 days.

If a user is logged in, list is filtered by that user.

Filtered by site, spectrometer, chemist.

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Cancel a Sample in the Queue

Shows samples in the ‘Pending’ and ‘Waiting’ states.

Buttons:

Moves samples/experiments to the ‘Cancelled’

state. Cancelled samples/experiments are

shown on the “Resubmit Failed” tab.

Permanently deletes the samples/experiments.

Role restrictions:

• Users with the NMR Visor ‘Admin’ role will see everyone samples listed here.

• Users with the NMR Visor ‘Submitter’ role will see only their own samples here.

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Archive Sample Context

Spectra saved on spectrometer

Sample record stored in secure database (remote or local)

Molecule structure saved in multiple formats (.sk2, .mol, .cdx, .smi)

Spectra transferred to ACD AS for Processing

– Bruker AU script support

Quinine 01080910482D 25 28 0 0 0 0 999 V2000 -0.7145 -0.8250 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 -1.4289 -0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 -1.4289 0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 -0.7145 0.8250 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 0.0000 0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 0.0000 -0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 0.7145 -0.8250 0.0000 N 0 0 0 0 0 0 0 0 0 0 0 0 0.7145 0.8250 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 1.4289 0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 1.4289 -0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 1.4289 2.8875 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 2.8578 2.0626 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 2.8578 2.8876 0.0000 C 0 0 2 0 0 0 0 0 0 0 0 0 2.5559 2.5857 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 3.5723 3.3001 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 2.1434 3.3001 0.0000 C 0 0 1 0 0 0 0 0 0 0 0 0 2.1434 1.6501 0.0000 N 0 0 2 0 0 0 0 0 0 0 0 0 2.5560 0.9357 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 4.2868 2.8876 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 1.4289 2.0625 0.0000 C 0 0 2 0 0 0 0 0 0 0 0 0 0.7144 1.6500 0.0000 C 0 0 2 0 0 0 0 0 0 0 0 0 0.0000 2.0625 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0 -2.1435 0.8250 0.0000 O 0 0 0 0 0 0 0 0 0 0 0 0 1.4289 1.2375 0.0000 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.8579 0.4125 0.0000 C 0 0 0 0 0 0 0 0 0 0 0 0 4 3 1 0 0 0 0 3 23 1 0 0 0 0 3 2 2 0 0 0 0 1 2 1 0 0 0 0 5 4 2 0 0 0 0 6 5 1 0 0 0 0 1 6 2 0 0 0 0 7 10 2 0 0 0 0 6 7 1 0 0 0 0 8 5 1 0 0 0 0 21 8 1 0 0 0 0 10 9 1 0 0 0 0 8 9 2 0 0 0 0 20 21 1 0 0 0 0 11 20 1 0 0 0 0 20 17 1 0 0 0 0 16 11 1 0 0 0 0 12 13 1 0 0 0 0 16 13 1 0 0 0 0 13 15 1 1 0 0 0 16 14 1 6 0 0 0 14 18 1 0 0 0 0 15 19 2 0 0 0 0 17 18 1 6 0 0 0 21 22 1 1 0 0 0 20 24 1 1 0 0 0 17 12 1 0 0 0 0 23 25 1 0 0 0 0M END

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Conclusions

Flow and tube NMR offer complementary advantages. A common

processing and reporting approach allows each sample format to be

used to full advantage.

Structures are captured during sample submission so they are available

for downstream processing and reporting.

Structural graphics add visual information to improve the final report,

helping chemists to confirm and interpret spectra and prevent errors.

ACD/Labs software has proven to be a versatile and reliable tool for

processing NMR spectra in automation – using both flow and

tube-based NMR techniques.

SQLsamples

An integrated NMR sample history database is

fully integrated into the sample management

system to improve automation and provide NMR

use statistics.

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I Made What I Thought I Made!

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Acknowledgements

Wolfgang Goetzinger, Amgen

Ashley Nichols, Amgen

Paul Krolikowski, Amgen

Grace Bi, Amgen

Larry Miller, Amgen

Peter Grandsard, Amgen

Craig Milling, Protasis

Igor Teslya, ACD/Labs

Dave Adams, ACD/Labs

41