• Journal of Microbiology
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• v.39 no.1
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• pp.67-73
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• 2001

A validation study was conducted to evaluate the efficacy and mechanism of the cryo-precipitation, monoclonal anti-FVIIIc antibody (mAb) chromatography, Q-Sepharose chromatography, and lyophilization steps involved in the manufacture of high purity factor VIII (GreenMono) from human plasma, in the removal and/or inactivation of 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 the high purity factor VIII concentrate. Samples were collected at each step and immediately titrated using a 50% tissue culture infectious dose (TCID$\_$50/) and then the virus reduction factors were evaluated. HAV was effectively partitioned from factor VⅢ during cryo-precipitation with the log reduction factor of 3.2. The mAb chromatography was the most effective step far removal of HAV with the log reduction factor of $\geq$4.3. HAV infectivity was not detected in the fraction of factor VⅢ, while most of HAV infectivity was recovered in the fractions of flow through and wash during mAb chromatography. Q-Sepharose chromatography showed the lowest efficacy for partitioning HAV with the log reduction factor of 0.7. Lyophilization was an effective step in inactivating HAV with the log reduction factor of 2.3. The cumulative lag reduction factor, $\geq$10.5, achieved for tile entire manufacturing process was several magnitudes greater than the potential HAV load of current plasma pools.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.1
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• pp.65-68
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• 2004

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60$^{\circ}C$ heat treatment for 10 h), involved in the manufacture of albumin from human plasma, in 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 the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID$\_$50/). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID$\_$50/ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was $\geq$4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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• pp.340-346
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• 2002

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 (TCID$\_$50/), 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 Viresolve NFP filtration with a log reduction factor of $\geq$ 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 log$\_$10/ TCID$\_$50/ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was $\geq$ 4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was $\geq$ 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.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.242-247
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• 2003

Murine encephalomyocarditis virus (EMCV) has been used as a surrogate for hepatitis A virus (HAV) for the validation of virus removal and/or inactivation during the manufacturing process of biopharmaceuticals. Recently international regulation for the validation of HAV safety has been reinforced because of the reported cases of HAV transmission to hemophiliac patients who had received ntihemophilic factors prepared from human plasma. The purpose of the present study was to compare the resistance of HAV and EMCV to various viral inactivation processes and then to standardize the HAV validation method. HAV was more resistant than EMCV to pasteurization (60oC heat treatment for 10 hr), low pH incubation (pH 3.9 at 25oC for 14 days), 0.1 M NaOH treatment, and lyophilization. EMCV was completely inactivated to undetectable levels within 2 hr of pasteurization, however, HAV was completely inactivated to undetectable levels after 5 hr treatment. EMCV was completely inactivated to undetectable levels within 15 min of 0.1 M NaOH treatment, however, residual infectivity of HAV still remained even after 120 min of treatment. The log reduction factors achieved during low pH incubation were 1.63 for HAV and 3.84 for EMCV. Also the log reduction factors achieved during a lyophilization process of antihemophilic factor VIII were 1.21 for HAV and 4.57 for EMCV. These results indicate that HAV rather than EMCV should be used for the virus validation study and the validation results obtained using EMCV should be precisely reviewed.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.377-382
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• 2009

Viral safety is an important prerequisite for clinical preparations of all biopharmaceuticals derived from plasma, cell lines, or tissues of human or animal origin. To ensure the safety, implementation of multiple viral clearance (inactivation and/or removal) steps has been highly recommended for manufacturing of biopharmaceuticals. Of the possible viral clearance strategies, Ultraviolet-C (UVC) irradiation has been known as an effective viral inactivating method. However it has been dismissed by biopharmaceutical industry as a result of the potential for protein damage and the difficulty in delivering uniform doses. Recently a continuous flow UVC reactor (UVivatec) was developed to provide highly efficient mixing and maximize virus exposure to the UV light. In order to investigate the effectiveness of UVivatec to inactivate viruses without causing significant protein damage, the feasibility of the UVC irradiation process was studied with a commercial therapeutic protein. Recovery yield in the optimized condition of $3,000\;J/m^2$ irradiation was more than 98%. The efficacy and robustness of the UVC reactor was evaluated with regard to the inactivation of human immunodeficiency virus (HIV), hepatitis A virus (HAV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), bovine parvovirus (BPV), minute virus of mice (MVM), reovirus type 3 (REO), and bovine parainfluenza virus type 3 (BPIV). Non enveloped viruses (HAV, PPV, BPV, MVM, and REO) were completely inactivated to undetectable levels by $3,000\;J/m^2$ irradiation. Enveloped viruses such as HIV, BVDV, and BPIV were completely inactivated to undetectable levels. However BHV was incompletely inactivated with slight residual infectivity remaining even after $3,000\;J/m^2$ irradiation. The log reduction factors achieved by UVC irradiation were ${\geq}3.89$ for HIV, ${\geq}5.27$ for HAV, 5.29 for BHV, ${\geq}5.96$ for BVDV, ${\geq}4.37$ for PPV, ${\geq}3.55$ for BPV, ${\geq}3.51$ for MVM, ${\geq}4.20$ for REO, and ${\geq}4.15$ for BPIV. These results indicate that UVC irradiation using UVivatec was very effective and robust in inactivating all the viruses tested.