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[ BUSINESS SOLUTION ]
1
WORKING WITH WATERS – DEVELOPING THE APPLICATIONClear goals and a clear division
of labor have characterized the
NPC’s partnership with Waters. The
relationship between the company and
several of the key founding members at
the NPC goes back to well before the
set-up of the organization in 2012. At that
time, as the idea for the NPC crystallized
and the labs were set up, establishing
the high-throughput application of
Waters UHPLC-MS systems was the
first task for the partners. Since 2013,
the researchers and experts at the NPC
have driven the program, with a set of
very specific application-based needs
in order to make the system fit for their
purpose. In turn, Waters’ know-how
and deep technical understanding of
what the instruments are capable of
has allowed the company to respond
and deliver modified features and
performance to support the application.
TECHNOLOGY: ULTRA HIGH PERFORMANCE LC-MS (WATERS ACQUITY UPLC AND XEVO TANDEM QUADRUPOLE MS)
BUILT ON THE LONDON 2012 OLYMPIC LEGACYThe MRC-NIHR National Phenome Centre (NPC) is located at Imperial College London and led by Professor Jeremy Nicholson, pioneer in metabolic profiling and molecular phenotyping. The focus of the NPC is to deliver access to a world-class capability in high precision metabolic phenotyping. In practice, the NPC offers the UK research and translational medicine community, both academic and industrial, a wide range of services including high precision untargeted profiling and bespoke quantitative assays. The Centre most commonly operates on a collaborative basis but also provides a fee-for-service offering built on the Centre’s high quality data generation platform. The experience developed within NPC has been further leveraged to nucleate an International Phenome Centre Network (IPCN) of collaborating institutions, accelerating their growth and development by providing direct access to the methodology and novel processes of the Centre.
The establishment of the NPC in 2012 took advantage of an unprecedented opportunity offered by the legacy of the London Olympics’ state-of-the-art drug testing/analytical laboratory. A significant amount of equipment and instrumentation from this lab was made available and, in addition to an initial £10m establishment grant, there were significant contributions of staff, equipment and technical support from two major instrument suppliers: Waters Corporation for UltraPerformance Liquid Chromatography® coupled to Mass Spectrometry (UPLC®-MS) instruments, and Bruker Biospin GmbH with Nuclear-Magnetic-Resonance (NMR) spectrometers.
MRC-NIHR National Phenome Centre and Waters Partnership Establishes a Powerful New Approach to Stratified Medicine
Building robust high-throughput methods moves metabolic profiling into a mainstream technique
Dr Matthew Lewis in the National Phenome Centre, London.
[ BUSINESS SOLUTION ]
2MRC-NIHR National Phenome Centre and Waters Partnership Establishes a Powerful New Approach to Stratified Medicine 2
Both companies have worked with the NPC to refine the technological platform underpinning its assays, and to establish a major training centre. Today, the NPC still enjoys very close partnerships with these companies and continues to develop and innovate technologies for phenotyping applications.
Commenting on the team at the NPC and the role of Waters in the early days of the project, Chief Operating Officer (COO), Dr Matthew Lewis explains:
“Within the NPC we have a fantastic group of talented scientists. Researchers come to us because we have deep knowledge within the related fields of metabonomics and molecular phenotyping – a real mastery of the type of science that we’re doing. Our group has a long history in these areas and related applications, so they come to us for the experience, the knowledge – the whole package. We have people working on technology development, bio-informatics and chemometrics all under the same roof as the team that are designing the methods, working with samples, and acquiring the data. It’s the quality of our team and the joined up thinking that have enabled us to achieve what we have and advance the field.”
He continues: “Collaboration with Waters has played a key role in this advancement, specifically for application of UPLC-MS technology to large scale profiling. Our partnership predates the NPC, meaning the right people were already known and at hand when we began designing the new Centre. They provided valuable input based in part on their own experiences from the 2012 Olympics lab environment, complementing our own experiences and ultimately helping us achieve our vision for a fully integrated world-class laboratory. During initial setup, installation, and commissioning, Waters staff were an integral part of the team – they were among the first people working with us in the new facility.”
THE POTENTIAL OF METABOLIC PROFILINGIt is widely recognized that people’s genetics alone do not account for all aspects of disease risk and development, and that prevention, detection and treatment can be improved by understanding the interactions between our genes, environments, microbiomes, diets and lifestyles, and their expression in diverse individuals and populations.
Dr Lewis elaborates: “Research tells us that an individual’s phenome is dynamic, changing in response to varying external influences. Lifestyle and environment, therefore, play an important role in determining a person’s measurable phenome.”
As more is learned about these interrelationships through metabolomic profiling, knowledge of physical characteristics and disease states looks set to be transformed, permitting significant advances in medical treatments, and enabling governments and medical authorities to address global public health in ways not previously foreseen. World-leading scientists, science-driven companies and governments alike recognize the potential revolutionary impact of this approach.
A single technology platform or assay cannot provide the comprehensive coverage of the metabolome needed to provide the insight into this complex human biochemistry.
In response, the NPC has developed and implemented a complementary mix of two analytical technologies working in concert to maximize metabolome coverage and make the entire platform as robust and efficient as possible.
WORKING WITH WATERS – TESTING AND TRAININGThis way of working first delivered
results for the hardware, columns
and chemistry, and helped the NPC
reach the goal of robust, reliable
methods that were able to run large
batches of samples in a highly
automated way. Subsequently, the
same iterative process between
the NPC and Waters software
R&D teams has facilitated the new
data processing elements of the
lab set-up. Today, the partnership
continues to work together to refine
its methodology and practices
which are supported by extensive
documentation and testing as
well as a training program and
ultimate dissemination to the IPCN
laboratories (see below).
Dr Lewis sums up : “We have
enjoyed and benefit ted greatly
from direct access to some very
bright people deep in the Waters
organization. This kind of unbarred
integration is unique, in my
experience. It has been an open and
trusting partnership with a clear
division of labor – and together
we have achieved powerful results
which speak for themselves .”
Dr Matthew Lewis, Chief Operating Officer (COO) MRC-NIHR National Phenome Centre
[ BUSINESS SOLUTION ]
3MRC-NIHR National Phenome Centre and Waters Partnership Establishes a Powerful New Approach to Stratified Medicine
The combination of NMR spectroscopy and UPLC-MS gives unparalleled INSIGHT into the molecular composition of samples.
NMR provides valuable information on sample quality and constitutes a first line profiling analysis for the most abundant metabolites, including those not readily measured by UPLC-MS. Subsequent UPLC-MS profiling assays provide unprecedented metabolic coverage, comprising quantification or relative quantification of thousands of individual metabolites and complex lipids.
INITIAL STEPS IN UPLC-MS AND A ‘BIG IDEA’UPLC-MS provides multidimensional high-resolution separations and sensitive detection across a broad range of chemical species. However, despite it being a ‘mature technology’, instrumentation is generally designed for research applications or small batch analysis. Early work that pushed the technique with larger batches and large patient cohorts found batch-to-batch or study-to-study variation, unacceptably high in-run CVs (coefficients of variance), run order effects, and lack of reproducibility. This is due in part to the complexity of the hyphenated system: involving the distinct processes of high-pressure liquid separation, followed by analyte ionization, and finally mass spectrometric separation and detection.
In addition, scientists in a research environment most often think about extracting the highest density of data and the ultimate in sensitivity from a technique. This approach is often not compatible with the reproducible collection of very large datasets.
However, the allure of epidemiological-scale metabolic data sets has continued, and it was this goal that the NPC initially set out to achieve. Dr Lewis explains how the need for population-scale analysis capability drove the thinking that led to their ‘big idea’ – perhaps best described as the industrialization of metabolic profiling:
“We set out to ensure that the systems would be capable of 24/7 operation with minimal intervention, and without running out of samples. We pulled back from pursuing the ultimate in per-sample analytical performance and sensitivity, and put the focus firmly on making reproducible, robust methodology.
Bespoke chromatographic methods were developed to specific time constraints, providing regular cycle of sample preparation and analysis as well as excellent analyte retention and peak separation performance.”
“Working with Waters experts, we optimized the instrument configuration and developed automatic adjustment capability allowing for continuous analysis of large sample sets, ultimately delivering unprecedented precision at such a scale.”
“The lab layout and infrastructure itself was designed for efficiency and industrial-scale data generation, simultaneously addressing challenges in hardware accessibility to maximize the ease and safety of daily work as well as efficient handling of the noise and heat produced. We managed to fit a good deal of instrumentation in a relatively small space using simple but elegant designs which have been replicated in a number of labs since including Waters’ own Wilmslow headquarters. We are proud of the fact that our collaboration has been one characterized by mutual benefit, in terms of both practical and technical exchange of information and experience.”
He concludes: “Today, there are 16 Waters systems in daily use at the NPC – six Xevo® G2-S tandem quadrupole mass spectrometers and ten Xevo G2-S QToF mass spectrometers, each equipped with an ACQUITY UPLC System for reliable performance. The platform is managed in such a way that each instrument is dedicated to a specific analysis type on a specific biofluid.”
“We’ve designed a world-leading research
facility and really made the whole
process fit for the purpose of large scale
molecular profiling. That was a huge
effort on both ours and Waters behalf,
and an achievement in its own right. ”
DR MATTHEW LEWISCOO, MRC-NIHR National Phenome Centre
[ BUSINESS SOLUTION ]
4MRC-NIHR National Phenome Centre and Waters Partnership Establishes a Powerful New Approach to Stratified Medicine 4
They run in parallel, which means that when a sample set comes into the Centre, all assays can be performed simultaneously for fast turnaround of data.
However, the configuration is flexible and adaptive to the needs and timelines of our collaborators. Having an analytical platform composed of many instances of the same technology running in parallel ensures that the lab always has capacity even if one of the instruments requires attention – or a service intervention. On the whole, troubleshooting, firefighting and instrument downtime are absolutely minimized.”
And what about the results? Dr Lewis again:
“We’ve actually now been able to achieve continuous analysis on thousands of samples, which is very efficient for the lab operations, but also producing very high quality raw data. We’re doing metabolic profiling, but achieving a higher standard of data generation more commonly associated with a bioananlysis approach. We see percent relative standard deviation (%RSD) values in the single digits and low teens – very high precision by any measure, and across large scale datasets. We’re getting that routinely now on all of our studies.”
The optimization process has extended to data processing and quality review too. Collecting very large data sets creates a bottleneck in processing if traditional practices of serial data generation followed by data review and dataset processing are followed. Waiting for all data to be collected before further analysis simply wastes too much time, and doesn’t allow for adjustments or corrections to be made if and when they are needed.
Working with Waters’ software team, a new software approach has been developed to enable processing alongside data generation, allowing the user to monitor instrument performance and data collected in near real time. A remarkable improvement, and one that perfectly supports the industrialization of the methodology.
ACCREDITED LABS AROUND THE WORLDLaunched in 2016 by the MRC-NIHR
National Phenome Centre at Imperial
College London in the UK and its
corporate partners – Waters and
Bruker – and announced at The World
Innovation Summit for Health in Doha
(29-30 November 2016, Qatar National
Convention Centre), the International
Phenome Centre Network (IPCN) is
a research consortium working to
transform health care globally and
improve disease prevention, detection
and treatment.
The IPCN already includes a number
of international partners with regional,
multi-institutional hubs in: Australia,
Canada, Japan, Singapore, Taiwan, the
United States and the United Kingdom,
all working to understand the dynamic
interactions between our genes,
environments, microbiomes, diets and
lifestyles and their expression in diverse
individuals and populations.
The IPCN applies the NPC’s cutting-
edge analytical and mathematical
approaches to study large, global data
sets of rich biological information to
unravel the molecular underpinnings
of disease risk in individuals and
populations. In addition to broadening
public awareness of metabolic profiling
for future healthcare, the scientific goals
of the IPCN include:
■ The elimination of erroneous
and inconsistent laboratory
practices by employing a
harmonized approach across
multiple institutions worldwide
■ The establishment of a framework
for quality, consistency, and
accuracy in data generation –
from acquisition and processing
to interpretation and visualization
■ The education of both scientific
and clinical communities on
the importance/relevance of
metabolic phenotyping.
NPC has developed a fit-for-purpose UPLC-MS platform, method set and sample analysis workflow, capable of sustained analysis on an industrial scale and allowing batch-free operation for large studies.1
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5MRC-NIHR National Phenome Centre and Waters Partnership Establishes a Powerful New Approach to Stratified Medicine
HIGH THROUGHPUT AND HIGH PERFORMANCE: TODAY’S OPTIMIZED WORKFLOWA recently published paper1 provides comprehensive details of the preparative, analytical, and data processing procedures as well as the experimental workflow developed by the NPC for the metabolic phenotyping of human urine by UPLC-MS.
The results demonstrate the feasibility of collecting UPLC-MS data suitable for the elucidation of subtle metabolic effects within epidemiological studies. The key is continuous analysis, facilitated by use of standard 96-well plates and a 15 minute cycle, providing a regular period for preparation and analysis.
Today, this methodology is in routine use across the parallel set-up of 10 Waters profiling instruments, which are working 24 hours/day, constantly being fed with sample plates and generating data at an unprecedented scale.
The UPLC-MS systems are made up of the following three modules:
■ The sample handling component is a Waters 2777C sample
manager equipped with a 25 μL Hamilton syringe, a 2 μL loop
used for full-loop injections of prepared sample, and a 3-drawer
sample chamber thermo-stated at 4 °C with a constant flow of
dry nitrogen gas to prevent the build-up of condensation
■ The LC system is an ACQUITY UPLC composed of a binary
solvent manager and column heater/cooler module
■ The MS instrument is a Xevo G2-S QTof coupled to the UPLC
via a ZSpray electrospray ionization (ESI) source.
“I believe our achievements so far
are considerable. Standardized
high-throughput methodology with
unprecedented reproducibility at scale
has generated some very large datasets,
which have already produced new
insights into what were considered to
be well-characterized diseases. But
we are still just at the beginning, and I
see our work and the partnership with
Waters adding value long into the future.
Moving from data to clinically actionable
information remains the ultimate goal, and
a new focus on real-time data processing
as well as harmonization across studies
– and between the growing international
network of phenome centres – will,
I believe, rapidly accelerate the
advance and value of metabolic
profiling in healthcare.”
DR MATTHEW LEWISCOO, MRC-NIHR National Phenome Centre
[ BUSINESS SOLUTION ]
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, The Science of What’s Possible, ACQUITY, UPLC, UltraPerformance LC, and Xevo are registeredtrademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2017 Waters Corporation. Produced in the U.S.A. November 2017 720006128EN NPC-LM-PDF
WHERE NEXT?NPC protocols are now being disseminated to IPCN member labs, with on-site training provided in collaboration with Waters to ensure efficient adoption and international harmonization. Moreover, standards and proficiency tests are being developed with the same goal in mind.
One of the biggest remaining road blocks to deliver insight rather than just data is feature adaptation and metabolite identification. The team at the NPC is focused on solving those challenges. For example, work is in progress on automated assignment of metabolites based on in-house databases, and structure elucidation of total unknowns. Dr Lewis is seeing progress: “We’re actually innovating those things faster than I thought we would be. We’re doing quite a good job with that, but it remains a huge opportunity. We will be able to go to our current collaborators and say, “Actually, the data that we’ve given you, we can now annotate more of it.” Then in six months, we can annotate even more of it. That’s really what we’re trying to drive at in order to extract the maximum amount of actionable knowledge from the data.”
References1. Matthew R. Lewis, Jake T. M. Pearce, Konstantina Spagou,
Martin Green, Anthony C. Dona, Ada H. Y. Yuen, Mark David, David J. Berry, Katie Chappell, Verena Horneffer-van der Sluis, Rachel Shaw, Simon Lovestone, Paul Elliott, John Shockcor, John C. Lindon, Olivier Cloarec, Zoltan Takats, Elaine Holmes, and Jeremy K. Nicholson, ‘Development and Application of Ultra-Performance Liquid Chromatography-TOF MS for Precision Large Scale Urinary Metabolic Phenotyping’ Analytical Chemistry 2016 88 (18), 9004-9013
To find out more about solutions for metabolomics and metabolic profiling, visit www.waters.com/metabolomics
“We’ve produced biomarkers that
have been taken all the way through
our advanced elucidation pipeline in
order to characterize them. These are
things that aren’t well characterized in
the literature. Many of these aren’t in
databases. Yet, our approach is finding
them. We’re characterizing them. We’re
then going into our other studies and
looking for the same biomarkers there
and we’re finding them. Pulling it all
together across such an abundance
of data – it’s very powerful stuff.”
DR MATTHEW LEWISCOO, MRC-NIHR National Phenome Centre