Downstream Processing: A Revalidation Study of Viral Clearance in the Purification of Monoclonal Antibody CB.Hep-1 - - BioPharm International

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Downstream Processing: A Revalidation Study of Viral Clearance in the Purification of Monoclonal Antibody CB.Hep-1

TITRATION

Human herpes simplex type 1 and human poliovirus type 2 titration

The HSV-1 and HVP-2 titers were determined by the inoculation of test solutions into Vero cell cultures (Table 2) and calculated using the Reed Müench method.21 Briefly, the assay consisted of inoculating 50 μL of viral sample in 150 μL of Gibco minimum essential medium (MEM) supplemented with 2% of Hyclone FCS containing Vero confluent cells. Ten serial dilutions were performed across the plates. Plates were maintained at 37 °C under 5–6% CO2 atmosphere. On the fifth day, cultures were carefully observed. End points were taken as the last dilution given cytopathic effect, with virus titer expressed as log10TCID50 mL-1. The same virus preparation was used as control of the experiment, and it was storage-aliquoted at –70 °C until thawing immediately prior to the titration assay. The virus titration was considered satisfactory when the difference between the expected and the true titer of the HVS-1 or HPV-2 used as control was less than 1 Log. Each sample was titrated in triplicate with eight determinations per each serial dilution.

Human immunodeficiency virus type 1 titration

The HIV-1 titers were determined by the inoculation of samples into MT4 cell culture (Table 2) and calculated using the Reed Müench method.20 Fifty μL of viral samples were inoculated in 150 μL of Gibco RPMI 1640 supplemented with 2% of Hyclone FCS containing MT4 confluent cells. Ten serial dilutions were performed across the plates. Plates were maintained at 37 °C under 5–6% CO2 atmosphere. On the seventh day the cultures were carefully observed and the virus titer was expressed as log10TCID50 mL-1 of the last dilution with cytopathic effect. The same virus preparation was used as control of the titration assay. The virus titration was considered satisfactory when the difference between the expected and the true titer of the HIV-1 used as control was less than 1 Log. Each sample was titrated in triplicate with eight determinations per each dilution.

Canine parvovirus titration

The titers of CPV were determined according to the Reed Müench method by inoculating of test solutions into LFBC cell cultures (Table 2).20 The viral samples were added to 150 μL of MEM supplemented with 2% of FCS containing LFBC confluent cells and maintained at 37 °C under 5–6% CO2 atmosphere. On the fifth day, the culture was carefully observed. The last dilution with virus cytopathic effect was taken to estimate the virus titer. The same CPV preparation was used as a control of the experiment and the titration assay also considered satisfactory when the difference between the expected and the true titer of the CPV used as control was <1 Log. Each sample was also titrated in triplicate with eight determinations per dilution.

MATHEMATICAL ANALYSIS

The Kruscall-Wallis test was executed to compare the yield, recovery, purity, and specific activity results between the scale-down and the manufacturing scale of the MAb CB.Hep-1 purification process. The Mann-Whitney (Wilcoxon) W test was used to compare medians of two samples, sorting the data from smallest to largest, and comparing the average ranks of two samples in the combine data. The program used was the STATGRAPHICS plus 5.0, 1994–2000 (Statistical Group Graphic Corporation).

Calculation of removal or inactivation factor (RF)

Each virus removal factor was calculated individually by White-Grun-Sur-Sito method as in Equation [1], below:21











RF is the log of Equation [1].


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