Quantitation of impurities in biological products

摘要：本项目的目标是开发和验证生物制品中可能影响产品安全或疗效的化学杂质和残留制造剂的方法学。这些包括：1)疫苗中用作灭活剂或结合剂的残留戊二醛，2)花粉变应原提取物和致敏源材料中的残留四氯乙烯，3)变应原粗提物和变应原斑贴试验材料中的非活性成分，以及4)免疫球蛋白产品中的抗病毒药物inactin。 目前正在进行的工作或过去一年开展的工作包括： 1)开发和验证四氯乙烯的测定方法，用于加工过敏原提取物的花粉来源材料。建立了一种吹扫捕集气相色谱测定致敏原提取物中四氯乙烯的方法，并对其进行了验证，获得了具有代表性的最终产品采样数据。用气相色谱/质谱仪测定致敏源材料中的这种化合物正在验证过程中，已经收集了具有代表性的花粉来源材料样本的中期数据。 2)建立了高效液相色谱测定戊二醛的方法，包括柱前衍生化和紫外检测，验证研究正在进行中。 3)对高效液相色谱-柱后衍生化/荧光法测定免疫球蛋白中去乙酰胆碱的方法进行了改进。进行了漫长的尝试以使制造商的程序合格，该程序需要进行广泛的修改，然后才能成功地检测到所需的十亿分之一的极限。 4)最近获得的离子陷阱高效液相色谱/质谱仪将与气相色谱/质谱仪一起使用，以开发一种通用程序，用于筛选各种产品中的杂质，以及表征过敏原提取物和过敏原斑贴测试产品中的低分子化合物。

Summary: The objectives of this project are to develop and validate methodologies for chemical impurities and residual manufacturing agents in biological products whose presence may affect product safety or efficacy. These include: 1) residual glutaraldehyde used as inactivating or conjugating agent in vaccines, 2) residual tetrachloroethylene in pollen allergenic extracts and allergenic source materials, 3) inactive components of crude allergenic extracts and allergen patch test materials and 4) an antiviral agent, inactine, in immune globulin products. Current work in progress, or that performed in the past year includes: 1) The development and validation of methods for the determination of tetrachloroethylene, used in the processing of pollen source materials for allergenic extracts. A purge-and-trap gas chromatographic method for tetrachloroethylene in allergenic extracts has been developed and validated, data has been obtained for a representative sampling of final products. The determination of this same compound in allergenic source materials by gas chromatography/mass spectrometry is in the process of validation, interim data on a representative sampling of pollen source materials have been collected. 2) An HPLC method for the determination of glutaraldehyde, involving pre-column derivatization with p-nitrobenzyl-hydroxylamine and UV detection has been developed, validatation studies are in progress. 3) An HPLC ion-exchange and post-column derivatization/fluourescence procedure for the determination of inactine in immune globulins was transfered and modified. A lengthy attempt was made to qualify a manufacturer's procedure, which required extensive modification before successful detection at the required limit of 1 part-per-billion was accomplished. 4) A recently acquired ion-trap HPLC/mass spectrometer will be used along with gas chromatography/mass spectrometry to develop a general procedure for impurities screening in a variety of products as well as the characterization of low-molecular weight compounds in allergenic extracts and allergen patch test products.