LGC SCIENCE & TECHNOLOGY Conceptual challenges of RMs in … · 2016-06-03 · proteomic, metabolomic measurements may be required ... predominantly “calibrants”, in support of

LGC SCIENCE & TECHNOLOGY

Conceptual challenges of RMs in support of

multiparametric biomarker measurements: A changing healthcare paradigm?

S.Pang, J.Huggett, W.Burkitt, C.Pritchard, G.O’Connor, C.Foy

& HELEN PARKES, LGC, UK

“Future of Reference Materials Science & Innovation”,

IRMM, 24 November 2010

21st Century Healthcare

• Increase in ageing population developing complex diseases such as cancer, Alzheimer’s and heart disease - burden on healthcare systems

• These complex, multifactor disorders are challenging diagnosis, treatment and monitoring

• Advances in ‘omics’ technologies are leading to identification of many novel putative disease related biomarkers /biomarker panels

• Promises significant advances in healthcare management for the future

• However, these emerging clinical diagnostics and therapeutics have a number of related measurement challenges

New Measurement Challenges:

• Increasingly, panels of biomarkers or multiparametric “fingerprints” derived from multiple data types are needed to give accurate diagnosis of complex disorders.

• Novel measurement issues to be addressed including:-

– Multiple analytical platforms - issues of comparability

– Large dynamic range of putative biomarkers in blood

– Detection of overall shifts and trends in the datasets (cf accurate quantification of individual analytes)

– Combination of genomic, transcriptomic, epigenomic, proteomic, metabolomic measurements may be required

Setting new demands for measurement traceability and design of appropriate reference standards

Normal Disease

Development of RNA (gene expression) and

protein biomarker profiling standards - A

quality metric “tool kit” for biomarker discovery and validation

Biomarker Issues

• Rate of approval of validated biomarkers is low as companies often fail to develop robust validation strategies– Assessing measurement

performance characteristics is a critical step

• Broad dynamic range of proteins in plasma – No single platform enables

detection of all proteins within plasma

• Issues span multiple technology platforms

• Overlap of results not guaranteed between platforms– Difficulties in validating

findings

Biomarker Technical Validation

(FDA)

To meet technical requirements the analytical process must be shown to be “fit for purpose”:-

• Performance evaluation of reproducibility, accuracy, sensitivity etc.

• Robustness across multiple labs/platforms/technologies

This requires:-

• Standards and control materials

• Process definition

– Analytical guidelines to use multivariate tools appropriately

• Quality metrics

– To allow objective assessment of data

Candidate protein biomarker

panel - reference material

production

• Preparation of a mixed panel of proteins with characterisation data

– 6 non-human proteins sourced from commercial suppliers (mouse CCL6, mouse lungkine, chicken caronte, mouse soggy, firefly luciferase and chicken egg lysozyme)

– Protein abundance: 50 – 10e7 pg/mL.

– Gravimetrically prepared a 10x stock of protein panel in buffer.

– Homogeneity, short and long term stability studies were performed.

Panel evaluation

• Two immunoassay platforms (Luminex and MSD) and a mass spectrometry

platform (QSTAR) were used for the study.

• Demonstrate applicability of panel for evaluating measurement

performance:

- Reproducibility, sensitivity, accuracy, dynamic range, signal to noise,

matrix effects.

- Platform performance indicator (by spiking protein panel into normal and

ovarian cancer diseased individual donor plasma samples).

Performance of the QC markers

- ovarian cancer biomarkers

Caronte

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Lysozyme

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Lungkine

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Soggy

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CCL6

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Donors 1-6: normal plasma;

Donors 7-12: ovarian cancer diseased plasma

• 3 component proteins of the QC material produced the same signal output when spiked in distinct individual donor plasma samples

- these may serve as generic indicators of the platform function

• Others all exhibit similar trends in sensitivity to matrix effects among the 12 donor samples

- these may act to indicate that sample matrix may interfere with the assay for diagnostic biomarker

•Evaluated with IL8 biomarker

Genomic Approach -• Collaboration with NIST using ERCC (External RNA Controls Consortium)

materials– set of 96 RNA control transcripts (non human/artificial) developed to assess

technical performance of gene expression assays

• Calibrator samples for gene expression multi-analyte measurements covering a range of concentrations

– Pool of RNA transcripts (8-20) of variable known abundance

– Multiple pools constructed with relative amounts of transcripts varied

• Used to monitor multi-parameter measurements – evaluate dynamic range, comparability and technical precision of assays

– Microarrays, qRT-PCR, digital PCR, Next Gen Sequencing….

• Transcripts “certified” by a range of approaches:-– International qRT-PCR inter-lab studies (CCQM - P103)

– Digital RT-PCR

• Transcripts spiked-in to a background of total RNA

International comparisons - (CCQM P103)

‘biomarker’

K-61

RNA instead of

DNA

single RNA

transcript,

matched calibrant

single RNA

transcript with

matched

calibrant in

matrix

multiple RNA

transcripts,

matched

calibrant

multiple “biomarker”

panel

RNA spike in a

complex

background of

human RNA

multiple RNAs

in a panel

RNA spikes in a

complex

background of

human RNA

- Multiplex measurement

C

CQ

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B

AW

G

Development of traceable reference

methods for structural measurements of

clinical protein biomarkers

Biomolecular structure / function:

• WHO report highlighted the increased recognition of structure function links for biologicals

• A protein’s structure, folding state and interactions with other proteins/ligands define its [immuno] activity

• Relationship between the measured “signal” and the “clinical” value, depends on many influences and effects. Variability in protein structure has a significant influence on signal measured.

Proteins - 100-1000 times larger than small molecules

Heterogeneous - single AB may have 35 glycovariants

Complex biomolecular traceability:

• Key issue for biomolecular traceability, is not simply SI traceable measurement of “amount of substance”, as achieved by IDMS for small molecules - challenge is to progress beyond state of the art and develop SI traceable reference measurements and RMs linked to biomolecule “function/activity”

• Requires advances in measurement science, providing a route to traceable complex protein measurements that will impact significantly on healthcare measurement comparability

Complete digestion to constituent peptides

Add isotopically labelled peptides to protein

*

*

*

*

Complete hydrolysis to

constituent amino acids

*

*

*

*

Add isotopically labelled amino acids to peptide

WP3

WP4

Labelled Peptides (Secondary Standard)

Quantitative separation of target

protein from clinical

matrix

WP5Target Protein

H/D exchange and

IMS studies to

characterise

structure

WP6

Immunoassay

StudiesWP7

Co

mp

ari

so

n

IDMS Quantitation of

Amino Acids

Select clinically

relevant model proteins

WP2

Patient Diagnosis & Care

Labelled Amino Acids (Primary Standard)

IDMS Quantitation of

Amino Acids

Labelled Peptides (Secondary Standard)

Pure Protein Standard

Matrix Protein Standard

IDMS

Quantitation

CLINBIOTRACE - Traceability of complex biomolecules and biomarkers in diagnostics – effecting measurement comparability in clinical medicine

hGH

CRP

“Model” clinical biomarkers:

• Human Growth Hormone (hGH / rhGH / somatropin)

– Growth defect marker/ biopharmaceutical

– multimer formation, isoforms

– plasma-based heterogeneity

• C-Reactive Protein (CRP)

– Inflammmation Cardiac marker

– oliogomer formation, additives

Aim: develop methods for traceable quantification of the proteins tertiary structure and relate to protein activity(eg immunoassay)

Structural measurement

• A number of physical and immuno-affinity based separation methods are being investigated:

• HD exchange MS (HDX) enables interrogation of protein structure under physiological conditions.

– By monitoring the exchange rates of different peptides an assessment of solvent proximity and protein folding structure can be determined

– Can be performed in the presence of specified ligands enabling the determination of binding constants and the degree of activity.

• Ion mobility separates ions on the basis of collisional cross section. This enables proteins of the same mass but different folding states to be separated.

• In combination with high resolution chromatography, capable of separating protein isoforms, we aim to traceably quantity the active form

HDX: recombinant human growth hormone (somatropin)

• Issues with protein solubility

• Measured in water – pH of starting solutions unknown

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Work undertaken by Will Burkitt & Caroline Pritchard,LGC

WHO

EP

drift time

%%

drift time (bins)

m/z

m/z

EP

WHO

IMS differences observed between WHO/EP standards, analysis on SYNAPT TWIMS

Somatotropin/hGH - structure Ion mobility mass spectrometry (IMS)

Work undertaken by Caroline Pritchard,LGC

Clinbiotrace - progress• hGH

– hGH quantitation in serum

– Isoform study undertaken

– Correlating SI traceable LCMS quantified hGH, & HDX structural analysis with routine IVD methods via a commutability study

• CRP

– Structure defining immunoassay result - monomer /pentamer differences

– LC/MS approaches to quantify oligomeric states

– [IMS aggregation measurement)

– Link to commutability study results

Real metrics on how structure can influence in vitrodiagnostic method - inform commutable CRM development & MU determinations

Summary

• We have developed a number of approaches for development of protein and nucleic acid biomarker reference materials -predominantly “calibrants”, in support of biomarker discovery and valid measurement

• Issues of rapidly changing technologies - RMs should be platform independent (microarray experience…)

• Key issue - how to establish traceability? - particularly for multiparametric measurements?

• Clinbiotrace project investigations of MS (IDMS, LCMS, HDEX, IMS) approaches to protein [structure] quantification have started toaddress traceability for complex protein biomarkers and link to immunoassay performance - but long way to go!

• Resource intensive and slow process - collaboration important

Acknowledgements

• Colleagues at LGC

– Will Burkitt

– Carole Foy

– Jim Huggett

– Gavin O’Connor

– Susan Pang

– Caroline Pritchard

– Milena Quaglia

• EMRP collaborators

– Heinz Schimmel, Ingrid Zegers IRMM

– Andre Henrion PTB

Where we are heading…..

• Development of reference methods/materials for trace detection and absolute nucleic acid quantification (digital PCR) - defined copy number

• Development of reference methods for multi-parametric nucleic acid measurements

• Development of reference standards for miRNA analysis

• Developments of reference methods and materials for DNA methylation measurements

• Standardisation framework for NGS (sample and library preparation)

• Validation of alternative DNA amplification approaches for POC diagnostics (microbiology/ infectious disease focus)

• Provision of traceable standards for the standardisation of multi-parametric platforms….