Removal and inactivation of hepatitis A virus during manufacture of urokinase from human urine

The purpose of the present study was to examine the efficacy and mechanism of the PAB (para-amino benzamidine) affinity column chromatography, Viresolve NFP virus filtration, pasteurization (60°C heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine as regards the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing HAV with a log reduction factor of 3.24. HAV infectivity was rarely detected in the urokinase fraction, while most of the HAV infectivity was recovered in the unbound and wash fractions. HAV was completely removed during the vire solve NFP filtration with a log reduction factor of ≥4.60. Pasteurization was also found to be an effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.18 log₁₀ TCID₅₀ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was ≥4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was ≥14.08. Accordingly, these results indicate that the production process for urokinase exhibited a sufficient HAV reducing capacity to achieve a high margin of virus safety.