Key comparison study on peptide purity—synthetic human C-peptide

Abstract

Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Eight Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of human C-peptide (hCP) present as the main component in the comparison sample for CCQM-K115. The comparison samples were prepared from synthetic human hCP purchased from a commercial supplier and used as provided without further treatment or purification. hCP was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of short (up to 5 kDa), non-cross-linked synthetic peptides/proteins. 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 a peptide impurity corrected amino acid analysis (PICAA) approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the hCP 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 hCP purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the hCP 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 hCP mass fraction assignment. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most exhaustive and elaborate set of results is taken for the calculation of the KCRVPepImp. The KCRVPepImp for the peptide related impurity mass fractions of the material was 83.3 mg/g with a combined standard uncertainty of 1.5 mg/g. 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 only a very small number of impurities have been identified and quantified resulting in an underestimation of the peptide related impurity mass fractions. The approach to obtain a KCRVhCP for the mass fraction of hCP is based on a mass balance calculation that takes into account the most exhaustive and elaborate set of results for the peptide related impurities KCRVPepImp, the TFA mass fraction value, water and other minor counter ions obtained by the coordinating laboratories. Differences in the quality of the results obtained for both peptides related impurity mass fractions and hCP mass fractions are better weighted and reflected in smaller uncertainties. The KCRVhCP for CCQM-K115 is 801.8 mg/g with a corresponding combined standard uncertainty of 3.1 mg/g. In general, mass balance approaches show smaller uncertainties than PICAA approaches and the majority of results obtained by the PICAA approach are in agreement because of larger corresponding uncertainties. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. 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).

Pilot study on peptide purity—synthetic human C-peptide

“…The PAWG has decided in April 2016 to use the TFA mass fraction and corresponding combined uncertainty of 57.1 ± 1.9 mg/g obtained by the NIM by ion chromatography for the calculation of the key comparison reference value for the mass fraction of hCP. The TFA mass fraction value obtained by the NIM in CCQM-K115 has been confirmed by the NRC through qNMR in CCQM-P55.2 [20].…”

“…Sorption balance measurements indicated that weighings of the CCQM-K115/P55.2 comparison material need to be performed under controlled conditions of temperature and relative humidity (RH) as the water content of the comparison material changes reversibly as a function of the RH. A full summary of the results for hCP mass fraction and of the methods used to investigate, assign and confirm the composition of the CCQM-K115/P55.2 candidate material and to demonstrate the fitness for purpose of the homogeneity, stability and reversible water adsorption/desorption of the material are given in detail in the CCQM-K115 Draft B Report [20].…”

“…In addition, the BIPM and NIM, China who employed a mass balance (summation of impurities) procedure to determine the hCP content were required to report the combined mass fraction assignment and corresponding uncertainty for the sub-classes of total related peptide impurities, water, total residual organic solvent / volatile organic compounds (VOCs) and total non-volatile organics & inorganics. Details are provided in the CCQM-K115 Draft B Report [20].…”

“…The major shortcoming is the peptide related impurities (PepImp) identification and quantification as described in detail in the CCQM-K115 Draft B Report [20]. In many cases, only a very small number of impurities have been identified/quantified resulting in an underestimation of the peptide related impurity mass fractions and consequently in an overestimation of the mass fraction value for hCP.…”

Metrologically traceable quantification of trifluoroacetic acid content in peptide reference materials by19F solid-state NMR

“…Despite the high performance of this method, it requires a relatively large amount of peptide sample per analysis (typically 1-10 mg) [7], which generally cannot be recycled once in deuterated solvent containing the calibrant. This sample requirement is particularly detrimental when a large number of related peptide impurities are acquired that need purity assigned prior to use as quantitative standards for measuring these impurities by mass spectrometry in a peptide reference material [9][10][11]. The cost of these custom synthesized peptides can be very high, especially in the case of isotopically labelled peptides, even for quantities such as 10 mg.…”