Fully Traceable Absolute Protein Quantification of Somatropin That Allows Independent Comparison of Somatropin Standards

Abstract

Abstract Background: Measurement traceability in clinical chemistry is required to standardize clinical results irrespective of the measurement procedure and laboratory. The traceability of many protein substances is maintained by reference to the first standard produced, which may no longer exist, with values assigned by consensus. Independent methods that provide traceability to the Système d’Unité International for all relevant properties of a protein standard could remove reliance on the original standard preparations. Methods: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present. Results: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 ± 0.21) mg/vial, European Pharmacopeia 2.06 ± 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification. Conclusions: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.