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Application of capillary electrophoresis for the standardisation of measurements of protein

biomarkers for In Vitro Diagnostics.

Guy.Auclair@ec.europa.eu

Joint Research Centre IRMM

Outline

• Introduction – EC-JRC-IRMM

• Metrological traceability chain

• Role of CE for primary calibrator characterisation

• Purity assessment

• Oligomerisation state

• Conclusions

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Directorates-General

European Commission

Directorates/ Institutes

(Scientific) Units

The European Commission’s Joint Research Centre

JRC

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JRC-IRMM's mission

Confidence in Measurements®

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to support industrial competitiveness,

quality of life, safety and security in the

EU

by developing advanced measurement

standards and providing state-of-the-

art scientific advice

in the area of measurements and

standards for EU policies.

Health: Standardisation and traceability for healthcare measurements

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• Scientific policy advice

• Co-operation with IFCC, IVD industry, hospitalclinicians

• Certified reference materials for complex biomarkers: enzymes,

proteins (including markers for cancer, infectious and cardio vascular diseases),

for genetic testing & leukaemia monitoring

• On-going standardisation: clinical biomarkers for chronic diseases

(autoimmune and Alzheimer diagnostics) and cancer

Routine sample result

e.g. AAA, dry mass determination, UV spectrophotometry, total nitrogen,

refractive index

Primary calibrator

Secondary calibrator

reference procedure, immunoassay ring trial

value transfer 1: LC-MS, immunoassay, referenceprocedure

value transfer 2: immunoassay, reference procedure

Immunoassay

Definition of the SI unit

Manufacturer’sworking calibrator

Manufacturer’sproduct calibrator

Traceability chain Uncertainty

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Primary calibrator

Solution of "pure" protein usually at a high concentration compared to classical routine samples.

Salt concentration adapted to preserve the integrity of the protein (stability, isoform profile, oligomerisation)

Commercially available materials often not pure enough. Need for further purify the proteins with chromatographic techniques (immuno affinity, size exclusion, ion exchange).

Solutions of a higher purity will be characterised with lower uncertainties.

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Which purity matters ?

Anything that can have an impact on value assignment.

- Dry mass determination - Amino acid analysis - UV spectrophotometry…

Most of the relevant impurities can be looked at with CE based measurements (GCE, cIEF, CZE)

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Impurity (MW around 145.8 kDa)

(3.8 area %)

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Purity assessment by GCE – Non reduced

GCEcapillary 30.2 cmElectrokinetic injection15.0 kV

Standard MW (10 kDa)

Human Serum Albumin calibrant

Theoretical mass 66.5 kDaMeasured mass 61.6 kDa

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Similar measurements are performed with cIEF method

Most of the time, we do get a single peak apart from the markers

If no other peak present, then purity uncertainty estimated as the uncertainty of the LOD of the method

CE is not used to perform the value assignment itself

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For the SDS-MW, methods are usually optimized for a specific range of MW

May lead to difficulty in using them for smaller proteins

* no separation between the 10 kD marker and

the protein of interest

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Purity assessment by CZE

Can be of interest for proteins in the lower range of MW

Normal polarityCapillary 30.2 cmHydrodynamic injectionBGE 100mM HCl – 70 mM TRIS pH 1.815 kV

Beta-2-microglobulin calibrant (11.7 kDa)

Problem: Definition of measurand & unit

““““Straightforward” for:

total amount of defined molecules

C-reactive protein

whole molecule?

part(s) of the molecule?

activity / reactivityLess evident:

amount of 'active' molecules

Traceability: Identity of the measurand

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C-reactive protein

Clinical analysers: most measure the formation of aggregates between target protein and antibodies

→→→→ Relevance of aggregation state of protein

aggregateAb trimer+

Ab aggregate+

Ab monomer+

Processing of calibrant may cause changes in oligomeric state

→→→→ Breaks in traceability chain depending on technique

No signal!

Variable signalnormal signal

Oligomerisation

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C-Reactive Proteinonly in the monomeric form (23 kDa)

No discrimination between the two forms

SDS-GCE

Purity assessment by GCE

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

AU

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

PDA - 220nm

CRP pentamerCRP pentamerCRP pentamerCRP pentamer0.115 mg/mL

1723 March 2015

Normal polarityCapillary 60.2 cmHydrodynamic injection

Oligomeric state confirmed

Purity assessment by CZE

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

AU

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

PDA - 220nm

CRP pentamerCRP pentamerCRP pentamerCRP pentamer0.115 mg/mL

1823 March 2015

Normal polarityCapillary 60.2 cmHydrodynamic injection

Oligomeric state confirmed

Purity assessment by CZE

See Poster P-117

1923 March 2015

Conclusions

CE is a versatile technique which can play a role in the purity assessment of primary calibrants for protein measurements

Sensitivity allows to reduce the uncertainty contribution resulting from purity testing

Different CE principles can allow a better screening of possible relevant sources of impurities.

Thanks!Virginie TrégoatAmalia Muñoz