– the intergovernmental organization through which Member States act together
     on matters related to measurement science and measurement standards.
Search facility:


| Site map | News | Contact us
Large organic molecules

    A model for core competency key comparisons for large molecule primary calibrators

    The BIPM, together with the CCQM-PAWG and the PAWG Focus Group I, has developed a model for organizing comparisons on peptide purity in terms of their relative molecular mass, the amount of cross-linking, and the extent of any modifications (see Figure 1, Table 1)[1]. The degree of difficulty for the purity characterization of peptide/protein calibrators is expected to increase from quadrant A to G because of the increasing lengths of the molecules, incorporation of peptide/protein cross-links (disulfide bonds), and modifications (e.g. glycation). Synthetic (e.g. proteotypic) pure calibrator peptides/proteins will typically fit in the lower space (quadrants A-C), while more complex calibrators that are commonly produced by recombinant techniques will usually be part of the upper space (quadrants D-G).

      Peptide AAs kDa Sss
      Glycated Hexapeptide of HbA1c (glcVHLTPE) 6 ˜0.8 0
      Tryptic Peptide Solution (tP) ˜10 ˜1 0
      Oxytocin (OXT) 9 1 1
      Antiotensin I (ANG I) 10 1.2 0
      Human Hepcidin-25 (hHEP-25) 25 2.8 4
      Human C-Peptide (hCP) 31 3 0
      Calcitonin Salmon (CALC1) 32 3.4 1
      Brain Natriuretic Peptide (BNP) 32 3.5 1
      Amyloid-Beta-42 (Abeta-42) 42 4.5 0
      Human Insulin (hINS) 51 5.8 3
      Insulin-like Growth Factor 1 (IGF-1) 70 7.6 3
      Parathyroid Hormone (PTH) 84 9.4 0

    The CCQM-K115/P55.2 core competency comparison series for large molecule primary calibrators

    The CCQM-K115/P55.2 comparison for human C-peptide (hCP) and other peptides of current interest identified by NMIs are marked in Figure 1. The CCQM-K115/P55.2 comparison covers the space of quadrant A for short (1 kDa to 5 kDa), non-cross-linked, and non-modified synthetic peptides. This comparison has been proposed after validation work was carried out by the BIPM and the NIST on angiotensin I (ANG I) which also fits in the space of quadrant A. Future comparisons will address the other six quadrants of the model. As part of the CCQM comparison strategy the BIPM in collaboration with the National Institute for Metrology of China (NIM) has finalized in 2016/2017 the first peptide purity key comparison, on human C-peptide (diabetes differential diagnosis marker and forensic relevance):

    The C-peptide comparison (CCQM-K115/P55.2) is the first comparison addressing capabilities for the value assignment of large organic molecule primary calibrators, with hCP representing a model straight-chain peptide with atomic weight of less than 5 kDa, in addition to being an important marker in diabetes diagnosis for which NMIs have developed CRMs and reference measurement methods. Nine NMIs have submitted results for the key comparison. The BIPM and NMI activities in peptide purity reference measurements are of interest not only to the diagnostics community, but also in the area of reference standards for peptide therapeutic products.

    CCQM-K115/P55.2 follow-up studies are proposed on oxytocin (OXT) for 2018 by BIPM/NIM and on HbA1c hexapeptide (GE) and HbA0 hexapeptide (VE) for 2019 by HSA/NIM/BIPM to cover the quadrants B (small, cross-linked and non-modified) and C (small, non-cross-linked, and modified), respectively.



    1. Josephs R.D., Stoppacher N., Westwood S., Wielgosz R.I., Li M., Quaglia M., Melanson J., Martos G., Prevoo D., Wu L., Scapin S., Öztug Senal M., Wong L., Jeong J.-S., Chan K.W.Y., Arsene C.G., Park S.-R., Concept Paper on SI Value Assignment of Purity - Model for the Classification of Peptide/Protein Purity Determinations, J. Chem. Metr., 2017; in print.

    2. Josephs R.D., Li M., Song D., Westwood S., Stoppacher N., Daireaux A., Choteau T., Wielgosz R.I., Xiao P., Liu Y., Gao X., Zhang C., Zhang T., Mi W., Quan C., Huang T., Li H., Flatschart R., Borges Oliveira R., Melanson J.E., Ohlendorf R., Henrion A., Kinumi T., Wong L., Liu Q., Öztug Senal M., Vatansever B., Ün I., Gören A.C., Akgöz M., Quaglia M., Warren J., Final Report on key comparison CCQM-K115: Peptide Purity - Synthetic Human C-Peptide, Metrologia 2017 54 Tech. Suppl. 08007

    3. Josephs R.D., Li M., Song D, Daireaux A, Choteau T, Stoppacher N, Westwood S, Wielgosz RI, Xiao P, Liu Y, Gao X, Zhang C., Zhang T., Mi W., Quan C., Huang T., Li H., Melanson J.E., Ün I., Gören A.C., Quaglia M., Warren J., Final Report on pilot study CCQM-P55.2: Peptide Purity - Synthetic Human C-Peptide, Metrologia 2017 54 Tech. Suppl. 08011
    FINAL REPORT: Study of Measurement Service and Comparison Needs for an
    International Measurement Infrastructure for the Biosciences and Biotechnology:
    Input for the BIPM Work Programme

    This study was commissioned by the BIPM and delivered by LGC under contract in response to the call for tender number AO/BIPM/CHIM/2009/015. The key aim of the study is to provide a report on:

    1. Measurement services required to establish an International Measurement Infrastructure for the Biosciences that:
      • are expected to be delivered and/or developed in the next 3-5 years and 5-10 years by NMIs, or other organizations developing measurement standards, or methods for the biosciences
      • are required by industry and other identified stakeholders over the next 3-5 and 5-10 years

    2. International comparisons that are required to demonstrate the degree of equivalence of the measurement services that are, or will be developed and delivered

    3. R&D activities necessary for the development of higher metrological order measurement standards and methods for the biosciences

    The Study focuses on protein and nucleic acid measurements for healthcare, which were identified as fields of high interest by the BIPM at the start of the Study.

    The report provides input for BIPM proposals on future requirements for BIPM laboratory activities in Metrology for Biotechnology and the Biosciences and is intended to be a useful reference for National Measurement Institutes developing programmes in these fields.

    The BIPM has developed and cross-validated different approaches for the purity value assignment of peptide/protein primary calibrators in collaboration with different NMIs in support of the CCQM Protein Analysis Working Group's (PAWG) model on pure peptides/proteins. Approaches and analytical methods are further developed at the BIPM to address increasingly demanding peptides/proteins.


    Angiotensin I (ANGI) and human insulin (INS)

    The BIPM has developed and cross-validated different approaches for the purity value assignment of angiotensin I (hypertension biomarker) in collaboration with the National Institute of Standards and Technology of the United States of America (NIST) and for the more complex human insulin (diabetes biomarker). Good agreement of results was found for the application of the full mass balance, PICAA, PICCHN and PICqNMR approaches for both model compounds. Liquid chromatography coupled to (high-resolution) mass spectrometry was found to be a key analytical technique for the characterization of impurities[1, 2] and indispensable for correcting results for other peptide-related impurities. The study results have been submitted published[3].

    ANG I covers the space of quadrant A for short (1 kDa to 5 kDa), non-cross-linked, non-modified synthetic peptides. INS is a larger model peptide/protein (5k Da to 10 kDa range) with multiple cross-links representing peptides/proteins of quadrant E. In addition, the BIPM has value-assigned five AA materials (F, I, L, P and V) for purity by use of the mass balance approach in order to achieve traceable results for the application of the PICAA approach based on LC-IDMS.


    Primary sequence of ANG I:

    DRVYIHPFHL

    Primary sequence of INS:

    Related link:




    1. Stoppacher N., Josephs R.D., Daireaux A., Westwood S., Wielgosz R.I., Impurity identification and determination for the peptide hormone angiotensin I by liquid chromatography - high resolution tandem mass spectrometry and the metrological impact on value assignments by amino acid analysis, Anal. Bioanal. Chem., 2013, 405, 8039-8051

    2. Stoppacher N., Josephs R.D., Daireaux A., Choteau T., Westwood S.W., Wielgosz R.I., Accurate quantification of impurities in pure peptide material – angiotensin I: Comparison of calibration requirements and method performance characteristics of liquid chromatography coupled to hybrid tandem mass spectrometry and linear ion trap-high resolution mass spectrometry, Rapid Comm. Mass Spectrom., 2015, 29, 1651-1660

    3. Josephs R.D., Stoppacher N., Daireaux A., Choteau T., Lippa K.A., Phinney K., Westwood S., Wielgosz R.I., State-of-the-art and Trends for the SI Traceable Value Assignment of the Purity of Peptides Using the Model Compound Angiotensin I, Trends Anal. Chem., 2017, submitted.

    Human C-peptide (hCP)

    Human C-peptide (hCP), the connecting peptide (between the A- and B-chains of insulin), is an important diabetes differentiation biomarker in laboratory medicine and plays a role in forensic medicine. Liquid chromatography/high-resolution mass spectrometry methods were developed and validated for the identification and quantification of related structure impurities in an hCP material[1]. The methods have been applied in the CCQM-K115/P55.2 studies on hCP purity[2, 3] coordinated by the BIPM in collaboration with the NIM (China). hCP covers the space of quadrant A for short (1 kDa to 5 kDa), non-cross-linked, non-modified synthetic peptides in the model for the classification of peptide/protein purity determinations.

    Primary sequence of hCP:

    EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ

    Secondment:

    • Dr Ming Li from NIM, China (2 years)

    Related link:




    1. Li M., Josephs R.D., Stoppacher N., Wielgosz R.I., Li H., Impurity determination for human C-peptide by liquid chromatography–high resolution tandem mass spectrometry, 2017, in preparation.

    2. Josephs R.D., Li M., Song D., Westwood S., Stoppacher N., Daireaux A., Choteau T., Wielgosz R.I., Xiao P., Liu Y., Gao X., Zhang C., Zhang T., Mi W., Quan C., Huang T., Li H., Flatschart R., Borges Oliveira R., Melanson J.E., Ohlendorf R., Henrion A., Kinumi T., Wong L., Liu Q., Öztug Senal M., Vatansever B., Ün I., Gören A.C., Akgöz M., Quaglia M., Warren J., Final Report on key comparison CCQM-K115: Peptide Purity - Synthetic Human C-Peptide. Metrologia, 2017, 54 Tech. Suppl., 08007

    3. Josephs R.D., Li M., Song D, Daireaux A, Choteau T, Stoppacher N, Westwood S, Wielgosz RI, Xiao P, Liu Y, Gao X, Zhang C., Zhang T., Mi W., Quan C., Huang T., Li H., Melanson J.E., Ün I., Gören A.C., Quaglia M., Warren J., Final Report on pilot study CCQM-P55.2: Peptide Purity - Synthetic Human C-Peptide, Metrologia, 2017, Tech. Suppl., 08011

    Hepcidin (hHEP-25)

    Hepcidin (hHEP-25) is a liver-specific antimicrobial peptide and iron-regulatory hormone. It could be potential recombinant candidate representing short (1 kDa to 5 kDa range) and multi-crosslinked (4 cross-links) peptides/proteins of quadrant B. hHEP has been investigated by the BIPM in collaboration with the Laboratoire national de métrologie et d'essais of France (LNE)[1]. It has not only the difficulty of multiple cross-links but also occurs in several isobaric higher order structures that would lead to a kind of 'conformational purity'. An LC-hrMS method has been developed and validated in-house to identify and quantify related structure impurities in hHEP-25 materials. However, identification, separation and quantification of the isobaric higher order structures require additional technologies that are still immature and rarely available within NMIs.

    Structure of hHEP-25:

    Secondment:

    • Dr Pauline Bros from LNE, France

    Related links:




    1. P. Bros, R.D. Josephs, N. Stoppacher, G. Cazals, S. Lehmann, C. Hirtz, R.I. Wielgosz, V. Delatour: Impurity Determination for Hepcidin by Liquid Chromatography - High Resolution and Ion Mobility Mass Spectrometry for the Value Assignment of Candidate Primary Calibrators, Anal. Bioanal. Chem. (2017). (doi:10.1007/s00216-017-0202-4) [Epub ahead of print].

    2. P. Bros: Développement de méthodes de référence pour spectrométrie de masse pour le dosage de biomarqueurs de la maladie d'Alzheimer, PhD thesis, Université de Montpellier, May 2016. (https://tel.archives-ouvertes.fr/tel-01468265/document).

    Calcitonin (CALC1)

    Calcitonin salmon (CALC1) is a 32 amino acid peptide and it is commonly used for clinical purposes in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy. CALC1 has been investigated as a candidate peptide that could represent the space of quadrant B for short (1 kDa to 5 kDa), cross-linked (one crosslink) and non-modified synthetic peptides for a CCQM-K115/P55.2 follow-up study coordinated by the BIPM in collaboration with NIM (China). An LC-hrMS method has been developed and validated in-house to identify and quantify of related structure impurities in a CALC1 material in collaboration with the Health Sciences Authority of Singapore (HSA).

    Structure of CALC1:

    Secondments:

    • Dr Lingkai Wong from HSA, Singapore
    • Dr Ming Li from NIM, China

    Related link:

    Oxytocin (OXT)

    The next CCQM-K115/P55.2 follow-up study on oxytocin (OXT) accepted by PAWG for 2018/2019 and coordinated by the BIPM in collaboration with the National Institute of Metrology of China (NIM) will cover the space of quadrant B for short (1 kDa to 5 kDa), cross-linked and non-modified synthetic peptides.

    OXT is a nine amino acid residue cyclic peptide. It is a hormone, neuropeptide and active pharmaceutical compound (API) used to induce labor or to enhance uterine contractions during labor. OXT is one of the peptide hormones that is increasingly frequent prepared synthetically to avoid possible contamination with vasopressin (ADH) and other small polypeptides with biologic activity.

    The presence of cross-links denotes an additional challenge for a LC-hrMS analysis. The BIPM is currently developing LC-hrMS and LC-MS/MS methods to identify and quantify of related structure impurities in OXT materials.

    Structure of OXT:

    Secondment:

    • Dr Ming Li from NIM, China

    Related link:

    Brain natriuretic peptide (BNP)

    Brain natriuretic peptide (BNP) is produced in myoendocrine cells of the heart ventricles from which it is released into the circulatory system. It is involved in blood pressure control and cardiovascular homeostasis and has emerged as an important biomarker with an established role in the diagnosis of congestive heart failure. BNP is a biologically active 32-amino acid polypeptide and potential model peptide representing the space of quadrant B for short (1 kDa to 5 kDa) and cross-linked (one crosslink) peptides for future CCQM-K115/P55.2 follow-up studies on pure peptides.

    Existing LC-hrMS methods are further developed to identify and where possible assign the mass fraction values of individual impurity components in pure materials of BNP in collaboration with the LGC of the United Kingdom.

    Structure of BNP:

    Secondment:

    • Dr Frank A. Torma from LGC, UK

    Related link:

    HbA1c hexapeptide (GE) and HbA0 hexapeptide (VE)

    Haemoglobin A1c (HbA1c) is an important biomarker for the diagnosis of diabetes mellitus and for monitoring the long-term blood glucose level in diabetic patients to ensure proper treatment and management. Alternatively to using classical reference methods modern SI-traceable methods based on isotope dilution mass spectrometric (IDMS) can be employed to perform a separate quantification of both HbA0 (non-glycated form) and HbA1c (glycated form) using corresponding signature hexapeptides as the calibration standards. Purity assessments of the signature hexapeptides are crucial for standardization and harmonization. The non-glycated HbA0 hexapeptide (VE) represents the space of quadrant A for short (1 kDa to 5 kDa), non-cross-linked and non-modified synthetic peptides. The glycated HbA1c hexapeptide (GE) is a model peptide representing the space of quadrant C for short (1 kDa to 5 kDa), non-cross-linked and modified synthetic peptides for future CCQM-K115/P55.2 follow-up studies on pure peptides proposed as a collaboration of the BIPM, Health Sciences Authority of Singapore (HSA) and National Institute of Metrology of China (NIM).

    Structure of VE:

    VHLTPE

    Structure of GE:

    gluVHLTPE

    Related link:


    Related link: