Biotherapeutics Division, National Institute for Biological Standards and Control, South Mimms, UK.
Vox Sang. 2021 Jan;116(1):99-105. doi: 10.1111/vox.12980. Epub 2020 Sep 28.
Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed.
A simplified one-step method has been developed to replace the existing Ph Eur two-step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one-stage method is simplified by the use of a dedicated online app.
The one-stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one-stage method. Improved methodology using true initial rate determinations and use of acid-treated prekallikrein substrate (PKS) helped understand and reduce artefactual results.
The one-stage method and associated app streamline real-time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.
激肽原酶激活剂（PKA）是一种在治疗性白蛋白和免疫球蛋白产品中发现的污染酶。通常使用欧洲药典（Ph Eur）定义的方法来测量其水平，该方法可追溯至 PKA 的世界卫生组织国际标准。这种方法通常效果很好，但有时也会出现问题。
已开发出一种简化的一步法来替代现有的 Ph Eur 两步法，该两步法包括激肽释放酶的生成，然后使用显色底物测量激肽释放酶。通过使用专用的在线应用程序，简化了对一阶段方法数据的分析。
使用白蛋白和免疫球蛋白的批次对一步法进行了验证，针对当前 Ph Eur 方法。使用一步法对免疫球蛋白的问题批次进行了研究。通过使用真实初始速率测定和酸处理激肽原底物（PKS），改进了方法学，有助于理解和减少人为结果。
一步法和相关应用程序简化了 PKA 的实时测定，促进了酶测定的良好原则，以限制底物耗竭，同时还节省了昂贵的 PKS。空白步骤和重现性得到简化。
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