Buchheit K H, Daas A, Jönsson K H
EDQM, Council of Europe, B.P. 907, F-67029 Strasbourg, France.
Pharmeuropa Spec Issue Biol. 2002 Jun;2002(1):7-27.
For interferon-alfa-2 (IFN-alfa-2) finished products a simple potency assay for batch consistency control or market surveillance studies is lacking. The European Pharmacopoeia monograph "Interferon alfa-2 concentrated solution" applies to bulk product and requires a cell culture potency assay which is neither straightforward nor are its precision and interlaboratory variability satisfying. Thus a project was initiated within the Biological Standardisation Programme to establish an HPLC assay for batch potency testing of recombinant IFN-alfa-2 finished products. In the collaborative study the suitability and transferability of an HPLC method developed in the project leaders' laboratory was checked. Twelve laboratories participated in the study. Based on a standardised method, calibration curves were established using the European Pharmacopoeia reference substances for IFN-alfa-2a and IFN-alfa-2b. The resolution between IFN-alfa and the oxidised forms of IFN-alfa was evaluated. Three marketed IFN-alfa preparations (one containing albumin) with known IFN-alfa content were used for the quantitative assay. The reproducibility of the method was not satisfactory but appears improvable with a slight modification of the method. Most laboratories succeeded in separating oxidised IFN from the main IFN peak. Five laboratories achieved very good linearity of the calibration curves while poor linearity was observed in three, mainly due to flattening of the curve at the lowest concentrations. The intercepts of the calibration curves were negative in all cases which might be due to a substantial degree of adsorption of IFN on glass and plastic surfaces. The robustness of the method concerning the choice of chromatographic column seems to be low. Using a corresponding column from a different manufacturer instead of the one proposed resulted in a loss of resolution between oxidised IFN and the main peak. Even the use of different batches of the prescribed column seemed to influence the result. The repeatability for the four consecutive injections of the three commercial IFN products does not indicate a systematic methodological error. The reproducibility (variability between laboratories) of the originally proposed method was large. Thus, the originally proposed method appears less suitable. However, the values obtained with a modified technique were less variable; in addition, an improvement of peak symmetry was obtained. Based on the results obtained with the original and the adapted method, a follow-up study with a modified technique (e.g. changes in liquid phase, application of system suitability criteria) is proposed.
对于干扰素-α-2（IFN-α-2）成品，缺乏用于批次一致性控制或市场监督研究的简单效价测定方法。欧洲药典专论“干扰素α-2浓缩溶液”适用于原料药，要求进行细胞培养效价测定，该方法既不直接，其精密度和实验室间变异性也不尽人意。因此，在生物标准化计划内启动了一个项目，以建立一种用于重组IFN-α-2成品批次效价检测的高效液相色谱（HPLC）测定法。在协作研究中，对项目负责人实验室开发的HPLC方法的适用性和可转移性进行了检查。十二个实验室参与了该研究。基于标准化方法，使用欧洲药典中IFN-α-2a和IFN-α-2b的参考物质建立校准曲线。评估了IFN-α与IFN-α氧化形式之间的分离度。使用三种已知IFN-α含量的市售IFN-α制剂进行定量测定。该方法的重现性不令人满意，但对方法稍作修改似乎可以改进。大多数实验室成功地将氧化型IFN与主要IFN峰分离。五个实验室的校准曲线线性非常好，而三个实验室观察到线性较差，主要是由于曲线在最低浓度处变平。校准曲线的截距在所有情况下均为负，这可能是由于IFN在玻璃和塑料表面上有大量吸附。该方法在色谱柱选择方面的耐用性似乎较低。使用不同制造商的相应色谱柱代替建议的色谱柱会导致氧化型IFN与主峰之间的分离度降低。即使使用不同批次的规定色谱柱似乎也会影响结果。对三种商业IFN产品连续四次进样的重复性未表明存在系统性方法误差。最初提出的方法的重现性（实验室间变异性）很大。因此，最初提出的方法似乎不太适用。但是，采用改进技术获得的值变异性较小；此外，峰对称性也得到了改善。基于原始方法和改进方法获得的结果，建议采用改进技术（例如，改变液相、应用系统适用性标准）进行后续研究。
