Effect of Freezing Rates and Excipients on the Infectivity of a Live Viral Vaccine during Lyophilization

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• 作者：Suling Zhai ; Raino K. Hansen ; Richard Taylor ; Jeremy N. Skepper ; Raquel Sanches ; and Nigel K. H. Slater
• 刊名：Biotechnology Progress
• 出版年：2004
• 出版时间：August 2004
• 年：2004
• 卷：20
• 期：4
• 页码：1113 - 1120
• 全文大小：269K
• 年卷期：v.20,no.4(August 2004)
• ISSN：1520-6033

文摘

Lyophilization is the most popular method for achieving improved stability of labilebiopharmaceuticals, but a significant fraction of product activity can be lost duringprocessing due to stresses that occur in both the freezing and the drying stages. Theeffect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivityin the presence of varying concentrations of cryoprotectant excipients is reported here.The freezing conditions investigated were shelf cooling (223 K), quenching into slushnitrogen (SN₂), and plunging into melting propane cooled in liquid nitrogen (LN₂).The corresponding freezing rates were measured, and the ice crystal sizes formedwithin the samples were determined using scanning electron microscopy (SEM). Theviral activity assay demonstrated the highest viral titer recovery for nitrogen coolingin the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viraltiter in the sample cooled by melting propane was consistently the highest amongthose results from the alternative cooling methods. However, this loss could beminimized by lyophilization at lower temperature and higher vacuum conditions. Wesuggest that this is due to a higher ratio of ice recrystallization for the sample cooledby melting propane during warming to the temperature at which freeze-drying wascarried out, as smaller ice crystals readily enlarge during warming. Under the samefreezing condition, a higher viral titer recovery was obtained with a formulationcontaining a higher concentration of sugar excipients. The reason was thought to betwofold. First, sugars stabilize membranes and proteins by hydrogen bonding to thepolar residues of the biomolecules, working as a water substitute. Second, theconcentrated sugar solution lowers the nucleation temperature of the water insidethe virus membrane and prevents large ice crystal formation within both the virusand the external medium.