Abstract：
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.
摘要：
欧洲药典（Ph。欧尔.)以确定总蛋白质含量。然而,治疗性免疫球蛋白中蛋白质含量的测定方法。欧尔.专著是Kjeldahl方法。Kjeldahl方法耗时,需要使用大量的危险试剂,这也导致了大量危险化学废物的产生。这项工作的目的是验证一种不需要危险试剂并节省时间的替代色谱方法，使用Ph.中指定的相同仪器条件。欧尔.用于人免疫球蛋白大小排阻高效液相色谱（SEC-HPLC）分子大小分布测定。用TSKgelG3000SW(600×7.5mm，10µm)柱，使用等度洗脱，在280nm波长下检测。流动相由0.03M磷酸氢二钠的水溶液组成。0.01M磷酸二氢钠一水合物，0.2M氯化钠和1mM叠氮化钠。参照具有已知蛋白质浓度的标准品(即人免疫球蛋白(分子大小)生物参比制剂)测定测试样品的蛋白质含量。根据ICHQ2指南对该方法进行了验证，评估了特性的精确性和真实性，并评估了信号响应的线性拟合优度(仅供参考)。此外,方法的等效性通过两个单侧t检验（TOST）分析与Kjeldahl方法进行评估。欧尔.关于治疗性免疫球蛋白的专著，并用Bland-Altman分析SEC-HPLC和制造商的数据(凯氏定氮和双缩脲方法)。还计算了测量的不确定度，以评估结果的准确性和质量，从而促进可靠的合规/不合规决策。根据结果,该方法被认为是测定人免疫球蛋白中蛋白质含量的合适且方便的替代方法。
