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

ADVERTISEMENT

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.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

AbbVie/Shire Deal Officially Off
October 20, 2014
Amgen Sues Sanofi and Regeneron over Patent for mAb Targeting PCSK9
October 20, 2014
EMA Works to Speed Up Ebola Treatment
October 20, 2014
Lilly to Close Manufacturing Facility in Puerto Rico
October 17, 2014
BioReliance Introduces New Predictive Assays
October 17, 2014
Author Guidelines
Source: BioPharm International,
Click here