The National Institute for Biological Standards and Control, Potter Bar, UK.
Haemophilia. 2020 Mar;26(2):346-353. doi: 10.1111/hae.13949. Epub 2020 Mar 6.
Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration.
In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods.
Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One-Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma-derived concentrate and recombinant FIX samples were also included for comparison in every assay.
Substantial discrepancies were observed when assaying dalcinonacog alfa using the one-stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one-stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma-derived or recombinant samples. However, incubation time influenced in the one-stage clotting assay using Dapttin.
Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.
许多重组和改良的 FIX 产品已经并将继续被开发，旨在改善乙型血友病患者的治疗效果。其中一种新产品是 dalcinonacog alfa，这是一种经过改良的 FIX，具有改善皮下给药等特性。
鉴于先前观察到改良 FIX 治疗药物的检测差异，本研究旨在评估不同检测方法之间和之内测定 dalcinonacog alfa 效价时可能存在的差异。
通过一期凝血测定法，使用 9 种不同的 APTT 试剂和 2 种市售的 FIX 显色试剂盒，分别用第 5 届国际标准（IS）FIX 浓缩物和第 4 届 ISFIX 血浆对 dalcinonacog alfa 的效价进行检测。在每个检测中还包括血浆来源的浓缩物和重组 FIX 样品进行比较。
使用一期凝血测定法，对两种标准物检测 dalcinonacog alfa 时，观察到大量差异。当使用两种显色试剂盒检测 dalcinonacog alfa 时，未获得具有统计学意义的有效结果。在两种显色试剂盒中，增加与激活试剂的孵育时间，会产生有效的检测，使效价与一期凝血测定法的效价更一致。增加显色试剂盒中孵育时间对血浆来源或重组样本的效价没有影响。然而，在使用 Dapttin 的一期凝血测定法中，孵育时间会产生影响。
在检测 dalcinonacog alfa 时，发现了方法内和方法间的差异。效价标记和临床监测的方法应给予仔细考虑。
