Pilot study on peptide purity—synthetic oxytocin
Authors: Josephs R D , Li M , Daireaux A , Choteau T , Martos G , Westwood S , Wielgosz R I , Li H , Wang S , Feng L , Huang T , Pan M , Zhang T , Gonzalez-Rojano N , Balderas-Escamilla M , Perez-Castorena A , Perez-Urquiza M , Ün, I , Bilsel M 
Published: August 2020

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.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) 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 oxytocin (OXT) present as the main component in the comparison sample for CCQM-P55.2.b. The comparison samples were prepared from synthetic OXT purchased from a commercial supplier and used as provided without further treatment or purification.
OXT 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, with one cross-link and up to 5 kDa. 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 quantitative nuclear magnetic resonance (PICqNMR) spectroscopy with a correction for structurally-related peptide impurities. Other participants provided results obtained by peptide impurity corrected amino acid analysis (PICAA) or Inductively coupled plasma mass spectrometry (PICICP). It was decided to assign reference values based on the KCRVs of CCQM-K115.b for both the OXT 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 OXT purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the OXT 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 and the hydrolysis efficiency 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 OXT mass fraction assignment.
The assessment of the mass fraction of peptide impurities is based on the assumption that only the Largest Consistent Subset (LCS) of results is taken for the calculation of the KCRVPepImp by use of the weighted mean. The KCRVPepImp of 31.6 mg/g is associated with a small corresponding expanded uncertainty of ±1.4 mg/g (k = 2) providing a more realistic basis of evaluation for the capabilities of the participants to identify/quantify peptide related impurities. Inspection of the degree of equivalence plots for the mass fraction of peptide impurities and additional information obtained from the peptide related impurity profile indicates that in many cases the major related peptide impurities have been identified and quantified.
The approach selected to obtain a KCRVOXT for the mass fraction of OXT is based on random-effects meta-analysis (DerSimonian-Laird (DSL) variance-weighted mean). The DSLmean takes into account the uncertainties of the results while introducing sufficient excess variance to allow for their observed dispersion resulting in a larger expanded uncertainty U(KCRVOXT). The KCRVOXT for CCQM-K115.b is 787.2 mg/g with a corresponding expanded uncertainty of the KCRVOXT of ±12.9 mg/g. It should be pointed out that the mass balance approaches show smaller uncertainties than PICAA, PICqNMR or PICICP approaches. Inspection of the degree of equivalence plots for CCQM-P55.2.b for the mass fraction of OXT shows that all results, except the UME qNMR result, agree with the reference value.

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The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).