The manufacturing process of MAb CB.Hep-1 is based primarily on Protein A–Sepharose affinity chromatography. In this work,
the affinity chromatography's effectiveness in removing enveloped and nonenveloped viruses was revalidated after a scale-up
of the purification process (scale-up factor = 6). Two earlier viral clearance validation studies of the MAb CB.Hep-1 purification
process demonstrated that the unique step showing high removal capacity of model viruses was Protein A–Sepharose affinity
chromatography.15,16 For this reason, we only performed a revalidation study of this purification step.
Scale-down of a chromatographic process can generate different removal factors even when the same virus and resin are used.7 Several factors can be responsible for these differences; notably the buffer system, linear flowrate, bead height-to-diameter
ratio, protein content, and temperature.17 We have reason to believe that the residence time is the most important parameter to scale-down affinity chromatography.
The availability of small-scale commercial affinity chromatography columns facilitate this task. In this study, the scale-down
process was specified so as to keep the residence time and the protein load-per-mL of matrix constant.
A major concern in validation clearance studies is determining which viruses should be used. Preference should be given to
viruses with different shapes, genomes, sizes, envelopes, and resistance to physical and chemical agents. We selected our
model viruses on the basis of recommendations from the literature and also because we wanted to use the same viruses used
in the previous validation study. Detailed information about the selection appears in reference 15.
Figure 1. Flowsheet of monoclonal antibody CB.Hep-1 purification process. The study starts at the red arrow.
The number of viruses is another aspect that requires special attention. The number will depend on the level of characterization
of the starting material and the production process. To demonstrate the viral removal factor of the MAb CB.Hep-1 scale-up
purification process, HSV-1, HIV-1, HPV-2, and CPV were individually added to one tenth of the desalted material before the
affinity chromatography step (Figure 1). To ensure the validity of this revalidation work, the model viruses used were the
same viruses used during the first validation study (Table 1).18
Table 1. General characteristics of the model viruses used in the validation studies.
MATERIALS AND METHODS
We used the same MAb that was used in production. CB.Hep-1 MAb is an IgG-2b secreted by the hybridoma cell line 48/1/5/4.
This hybridoma is a mouse–mouse hybridoma cell generated by the fusion of the HBsAg immunized BALB/c mouse lymphocytes and
the heterohybridoma SP2/0-Ag14.12