Pilot study on peptide purity - glycated hexapeptide of HbA1c

Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P55.2.c, was coordinated by the Bureau International des Poids et Mesures (BIPM), the Health Sciences Authority (HSA) of Singapore and the Chinese National Institute of Metrology (NIM). Three Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of glycated hexapeptide of HbA1c (GE) present as the main component in the comparison sample for CCQM-P55.2.c. The comparison samples were prepared by HSA/BIPM from synthetic GE purchased from a commercial supplier and used as provided without further treatment or purification. GE was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of chemically synthesized peptides of known sequence, without cross-links, up to 5 kDa and modification (mono-glycation). It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used amino acid analysis (PICAA) or quantitative nuclear magnetic resonance (PICqNMR) spectroscopy with a correction for structurally-related peptide impurities. It was decided to assign reference values (RVs) based on the KCRVs of CCQM-K115.c for both the GE mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the GE purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the GE mass fraction. In particular, it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. More detailed studies on the identification/quantification of peptide related impurities revealed that the integrity of the impurity profile of the related peptide impurities obtained by the participant is crucial for the impact on accuracy of the GE mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that all results are directly taken for the calculation of the RV PepImp by use of random-effects meta-analysis (DerSimonian-Laird (DSL) variance-weighted mean). The RV PepImp of 45.4 mg/g is associated with a c