GSK srl, Via Fiorentina 1, 53100 Siena, Italy.
University of Calabria, Ponte Pietro Bucci, 87036 Arcavacata di Rende, Cosenza, Italy.
Vaccine. 2019 Jun 27;37(29):3754-3760. doi: 10.1016/j.vaccine.2018.10.082. Epub 2018 Nov 14.
Pyrogen content is one of the critical quality attributes impacting the safety of a product, and there is an increasing need for assays that can reliably measure this attribute in vaccines. The Limulus amebocyte lysate (LAL) assay and the rabbit pyrogen test (RPT) are the canonical animal-based pyrogen tests currently used to release vaccines; however, there are several drawbacks associated with these tests when applied to Bexsero, intrinsically pyrogenic product, containing a meningococcal Outer Membrane Vesicle component. While the RPT, as applied to Bexsero at its given dilution, ensures safe vaccine, it is highly variable and prone to false positive results. On the other hand, the LAL assay although quantitative, can detect only endotoxin pyrogens and is not sufficient for monitoring the safety of Bexsero, which contains both LPS and non-endotoxin pyrogens. Being aware of these limitations of the RPT and LAL when applied to Bexsero, the Monocyte Activation Test (MAT) which is sensitive to both endotoxin and non-endotoxin based pyrogens has been developed as an alternative pyrogen test. Here, the development and the validation of a MAT assay adapted from the European pharmacopoeia for Bexsero, is described. The MAT assay is then used for monitoring the safety and consistency of Bexsero vaccines at release, providing great advantages in terms of reduced variability with respect to RPT, reduction of animal use, in line with the 3Rs principle concerning the protection of animals and faster time to market. In addition the correlation of the MAT to the RPT has been demonstrated supporting the replacement of the in vivo method and the potential application of the assay to other intrinsically pyrogenic vaccines.
热原含量是影响产品安全性的关键质量属性之一，因此越来越需要能够可靠地测量疫苗中该属性的检测方法。鲎变形细胞溶解物（LAL）检测法和兔热原检测法（RPT）是目前用于放行疫苗的经典动物源热原检测法；然而，当应用于含有脑膜炎奈瑟菌外膜囊泡成分的固有发热产品 Bexsero 时，这些检测法存在几个缺点。虽然 RPT 在应用于 Bexsero 时可以确保疫苗安全，但它的变异性很大，容易出现假阳性结果。另一方面，虽然 LAL 检测法是定量的，但只能检测内毒素热原，不足以监测 Bexsero 的安全性，因为 Bexsero 既含有 LPS 又含有非内毒素热原。鉴于 RPT 和 LAL 应用于 Bexsero 时存在这些局限性，人们开发了一种替代热原检测法——单核细胞激活检测法（MAT），该检测法对内毒素和非内毒素热原均敏感。本文介绍了从欧洲药典改编而来的用于 Bexsero 的 MAT 检测法的开发和验证。然后，将 MAT 检测法用于放行时监测 Bexsero 疫苗的安全性和一致性，与 RPT 相比，该检测法具有变异性更小、动物使用量减少、符合 3R 原则（关于动物保护）和更快推向市场的优势。此外，还证明了 MAT 与 RPT 的相关性，支持替代体内方法，并且该检测法可能适用于其他固有发热疫苗。
