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

Quantification of MAb CB.Hep-1 by ELISA

A Costar polystyrene microplate (Corning Life Sciences) was coated with 10 μg per well of rHBsAg in 0.1 M NaHCO3 buffer for 20 min at 50 °C. After this step, samples were added to the plate in 0.05% Tween 20 in PBS and incubated for 1 h at 37 °C. Several washes with 0.05% Tween 20/PBS were done. Then the plate was incubated for 1 h at 37 °C with a horseradish peroxidase conjugate (Sigma Chemical). The reaction was then revealed using 100 μL/well of 0.05% O-phenylenediamine and 0.015% H2O2 in citrate buffer (pH 5.0), and stopped with 50 μL/well of 1.25M H2SO4. The absorbance was measured in a Labsystems Multiskan enzyme-linked immunosorbent assay (ELISA) reader using a 492-nm filter. The range of the calibration curve was from 3.13 to 50 ng/mL. The inner standard MAb used was IgG2B070305, supplied by the Quality Control Department of Center for Genetic Engineering and Biotechnology, Havana, Cuba.

Determination of MAb CB.Hep-1 purity by SDS-PAGE

Samples were analyzed by electrophoresis on 12.5% sodium dodecyl sulphate poliacrylamide gel electrophoresis (SDS-PAGE) gels as described by Laemmli.19 Separated proteins were stained with Coomassie blue R-250 and then analyzed by densitometry. Percentage of purity was measured by Bio-RAD Version 1.4.1 Build 446 Molecular Analyst software. All samples (20 μg) were applied to the gel under reduction conditions.

Virus inactivation during affinity matrix sanitization with 70% ethanol

Nine mL of the Protein A–Sepharose matrix were previously washed and equilibrated with a fivefold column volume of 70% ethanol/30% purified water. The model viruses (HSV-1, HIV-1, HPV-2, and CPV) were individually diluted (1:10 v/v) and added to the matrix. The working temperature was 4 °C and supernatants were collected after each exposure time (0 min and 12 h) to be dialyzed against PBS (pH 7.2) to allow the virus titration. The control was a similar amount of virus added to the Protein A–Sepharose matrix previously equilibrated with PBS (pH 7.2) and kept at 4 °C during the whole experiment.

Nonenveloped virus inactivation during affinity matrix sanitization with 0.1 N HCl

The Protein A–Sepharose matrix was previously washed and equilibrated with a fivefold column volume of 0.1 N HCl (pH 1.0). Nonenveloped model viruses (HPV-2 and CPV) were diluted 1:10 (v/v) and added to the matrix. The working temperature was 4 °C and samples were collected after the following exposure times: 0 min, 3, 4, 5, 8, and 10 h to be dialyzed against PBS (pH 7.2) to allow the virus titration. The control was a similar amount of virus added to the affinity matrix previously equilibrated with PBS (pH 7.2) and kept at 4 °C during the whole experiment.

Virus inactivation during affinity matrix storage with 20% ethanol

Nine mL of the Protein A–Sepharose matrix previously washed and equilibrated with a fivefold column volume of 20% ethanol/80% purified water. The model viruses (HSV-1, HPV-2, HIV-1, CPV) were individually diluted (1:10) were added to the matrix. The working temperature was again 4 °C and samples were collected after each exposure time. For HSV-1 and HIV-1: 0 min, 15 min, 1 h, and 2 h; for HPV-2 and CPV: 0, 15, and 30 min, and 1, 24 h, 48 h, and 72 h. All samples were dialyzed against PBS (pH 7.2) to allow the virus titration. The control was a similar amount of virus added to the affinity matrix previously equilibrated with PBS (pH 7.2) at 4 °C.


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