Validation and application of a SEC-HPLC method for the determination of total protein in therapeutic immunoglobulins as an alternative to the European Pharmacopoeia methods.

Several analytical procedures are described in the European Pharmacopoeia (Ph. Eur.) to determine total protein content. However, the method for the determination of protein content in therapeutic immunoglobulins prescribed in the Ph. Eur. monographs is the Kjeldahl method. The Kjeldahl method is time-consuming and requires the use of large amounts of hazardous reagents, which also results in the production of a large amount of hazardous chemical waste. The purpose of this work was to validate an alternative chromatographic method that requires no hazardous reagents and saves time, using the same instrumental conditions specified in the Ph. Eur. for the human immunoglobulin size-exclusion high-performance liquid chromatography (SEC-HPLC) molecular-size distribution assay. The chromatographic separation was achieved with a TSKgel G3000SW (600 × 7.5 mm, 10 µm) column, using an isocratic elution, with detection at 280 nm wavelength. The mobile phase consisted of an aqueous solution of 0.03 M disodium hydrogen phosphate dehydrate, 0.01 M sodium dihydrogen phosphate monohydrate, 0.2 M sodium chloride and 1 mM sodium azide. The protein content of the test samples was determined referring to a standard with a known protein concentration (i.e. Human immunoglobulin (molecular size) Biological Reference Preparation). The method was validated evaluating the characteristics precision and trueness according to the ICH Q2 guideline, and the goodness of linear fit for the signal response was assessed (given for information only). In addition, the equivalence of methods was evaluated with two one-sided t-tests (TOST) analysis with the Kjeldahl method mentioned in Ph. Eur. monographs on therapeutic immunoglobulins, and with Bland-Altman analysis of SEC-HPLC and manufacturers' data (Kjeldahl and biuret methods). The uncertainty of measurement was also calculated in order to evaluate the accuracy and quality of the results, thus facilitating a reliable compliance/non-compliance decision. Based on the outcome, the method is proposed as a suitable and convenient alternative for the determination of protein content in human immunoglobulins.