聚乙二醇化蛋白作为人类疾病的治疗性干预的临床产品的开发正在增加。此类产品的定量依赖于适当校准的传统方法,包括反相高效液相色谱(RP-HPLC)。然而,目前可用的药典校准剂,例如,化学参考物质(CRS),是已知浓度的高度纯化的非聚乙二醇化蛋白质。这些不确定是否适合用于校准目的,其中对聚乙二醇化蛋白质的质量进行精确定量,通常与聚乙二醇(PEG)链大小不同,结构,附着位点和异构体的数量和比例,是必需的。在这项研究中,开发了定制的RP-HPLC方法,并验证了用于分析具有线性20kDaPEG链的聚乙二醇化IFN-α2a产物(PEG20-IFN-α2a;ReiferonRetard(®))。由于PEG20部分在210nm的检测波长下没有产生信号,可以测定PEG-IFN-α2a中基础IFN-α2a分子的浓度。通过计算各自色谱图的峰面积在210nm处的紫外吸光度,PEG20-IFN-α2a浓度与IFN-α2a的CRS浓度的高度相关性(r(2)≥0.995),或表征良好的PEG20-IFN-α2a内部参考物质(IRS)被发现。这一发现证实了该CRS作为通过定制的RP-HPLC方法进行PEG20-IFN-α2a质量测定的主要校准物的适用性。将该方法应用于10批ReiferonRetard(®)的定量分析,获得了准确和一致的结果,表明其用于当前和未来ReiferonRetard(®)批次的质量测定。
There is increasing development of pegylated proteins as clinical products for therapeutic interventions in human disease. Quantification of such products relies on appropriately calibrated traditional methods, including reverse phase-high performance liquid chromatography (RP-HPLC). However, currently available pharmacopoeia calibrants, e.g., chemical reference substances (CRS), are highly purified non-pegylated proteins of known concentration. These are uncertain to be suitable for calibration purposes where the precise quantification of the mass of pegylated proteins, often heterogeneous with respect to polyethylene glycol (PEG) chain size, structure, attachment sites and isomer numbers and proportions, is required. In this study, a customised RP-HPLC method was developed and validated for the analysis of a pegylated IFN-α2a product having a linear 20kDa PEG chain (PEG20-IFN-α2a; Reiferon Retard(®)). Since the PEG20 moiety generated no signal at the detection wavelength of 210nm, the concentration of the base IFN-α2a molecules in PEG-IFN-α2a could be determined. By calculating the UV absorbance at 210nm of peak areas in their respective chromatographic profiles, a high correlation (r(2) ≥ 0.995) of PEG20-IFN-α2a concentrations with equal concentrations of the CRS of IFN-α2a, or of a well-characterised PEG20-IFN-α2a internal reference substance (IRS) was found. This finding confirmed the suitability of this CRS as a primary calibrant for mass determinations of PEG20-IFN-α2a by the customised RP-HPLC method. Application of this method to the quantitative analysis of 10 batches of Reiferon Retard(®) yielded accurate and consistent results, indicating its utility for mass determinations of current and future Reiferon Retard(®) batches.