Experiments were assessed using a scale-down version equivalent to 1% (1:100) of the MAb CB.Hep-1 manufacturing purification
scale, which showed no significant differences in MAb yield (p = 0.0545) or specific activity (p = 0.0861). The recovery (p
= 0.0299) and purity (p = 0.0327) showed significant differences with the Reference "22" validation study but all recovery
and purity values were over the pre-determined specification limits (Figure 2). Therefore, the scale-down process performed
within the limits established for the manufacturing purification scale and for the validation study.
Figure 2. Four graphs show that the yield, recovery, purity, and specific activity of the Mab CB.Hep-1 purification process
are consistent in manufacturing scale and validation (laboratory) scale. Bars represent the average of the results of each
parameter and the confidence limit. All experiments were performed in triplicate.
The model viruses used in this study, which are listed in Table 1, cover a wide range of physical–chemical and structural
characteristics of viruses.18 Sources for the four viruses are listed in Table 2. Cell lines employed in the virus titration were: African green monkey
kidney cell line (Vero), guinea pig fibroblast cell line (LFBC) and the HIV negative human T cell line (MT4). The source of
these cell lines is listed in Table 2. The log TCID50/mL virus titers in the starting stocks were: 8.4 for HSV-1; 5.8 for
HIV-1; 6.5 for HPV-2; and 7.5 for CPV. All the model viruses were purified by an ultracentrifugation method and then filtered
under sterile conditions in separate laboratories and at different times to ensure that each virus preparation was pure.
Table 2. Source of the cell line used for titration and detection of model viruses in both validation and revalidation studies
Protein A–Sepharose affinity chromatography viral removal
This revalidation study was carried out following the same principles as the viral validation study.15 It was performed in a separate laboratory using the same affinity-chromatography residence time and protein concentration
in the column's applied material as in production. The purification process flow is illustrated in Figure 1. The starting
material was individually loaded with each model virus and spiked into the Protein A–Sepharose affinity column (three independent
replicas for each virus). The IgG adsorption buffer was PBS (pH 8.0) and 0.1 M citric acid (pH 3.0) was used as the elution
buffer. All experiments were conducted at 4 °C. The affinity column used was an Amersham-Bioscences XK26/30 loaded with 54.2
mL of matrix and operated at 100 cm/h of linear flowrate. The protein absorbance was registered using a 280 nm filter and
the applied IgG per run was 90% of the dynamic IgG binding capacity of the matrix.
Samples for virus detection were taken from each elution fraction (not from the pool of elution fractions). These fractions
were then dialyzed with PBS to raise the pH. Because viruses were detected in the elution fractions, we believe viral clearance
occurred primarily as a result of virus removal, but inactivation could not be completely avoided, because, as we have demonstrated,
enveloped viruses are easily inactivated at low pH.