As a next step, non-GLP spiking studies using PPV and the same scaled-down model were performed at Catalent Pharma Solutions
to verify the PP7 data obtained. As required by regulatory guidance, the scaled down model used during the GLP validation
run should reflect process conditions as closely as possible unless a justification to deviate from this can be provided (7,
Consequently, the volume to be filtered using the scaled-down model is derived from the required filtration volume at process-scale.
This volume was initially established by testing the virus filter without a virus spike to determine the flow rate and capacity.
It has been reported that virus spike preparations can lead to significant fouling of nanofiltration membranes in spiking
studies, and properties of the virus spike itself are an often-overlooked source of contaminants that can affect filter performance
(9). Potential contaminants in virus stocks may include serum proteins, host-cell proteins, host-cell DNA, and lipids (10,
To determine the effect of the virus spike on product filterability, nonpurified and purified spike preparations of PPV were
filtered using the same scaled-down model. The purified virus was prepared using a Q-Sepharose chromatography step.
Figure 3 outlines the filtration characteristics of the Fab product using Virosart CPV, comparing a 1% virus spike of chromatography-purified
PPV with a 1% spike of non-purified PPV. A nonspiked control is also shown. The purified PPV samples gave a better flow rate
and capacity than nonpurified virus, which demonstrated that contaminants within the viral spike were affecting filter performance.
As a result of these studies, a Q-chromatography purified PPV preparation was used for subsequent validation of the nanofiltration
step to achieve the required scaled-down volume of product (400 L/m2 ).
Figure 3: Filtration characteristics of purified versus nonpurified PPV at 1% spike. (Figure 3 courtesy of the authors.)
A series of scaled-down runs was then performed using purified PPV at different concentrations, to determine the viral spike
concentration that would give the best log reduction without affecting filter performance. Table III outlines PPV retention
data for three runs using different spike concentrations of purified PPV.
Table III: Non-GLP PPV test results for Virosart CPV.
All studies used a test pressure of 2 bar (30 psi) and filter lot number 0650773R50Z3/2. For all runs, a scaled-down filtrate
volume equivalent to 400 L/m2 was obtained to reflect the full-scale filter capacity requirements.
All three viral spike concentrations resulted in acceptable viral reduction. A range of 0.5–1% was therefore selected for
validation runs to maximize the flow rate and capacity of the filter, and to minimize the amount of virus required.
To validate the nanofiltration step, GLP runs were performed using the following panel of viruses as model viruses: PPV, murine
leukemia virus (MuLV), bovine viral diarrhea virus (BVDV), and reovirus type 3 (Reo 3) virus. The overall GLP spiking study
results are outlined in Table IV.
Table IV: Overall GLP spiking study results for Virosart CPV.
All studies used a viral spike concentration of 0.5%, a test pressure of 2 bar (30 psi), and filter lot number 0650773R50Z3/2.
For all runs, a scaled-down filtrate volume equivalent to 400 L/m2 was obtained, to reflect the full-scale filter capacity requirements.
GLP studies also included robustness studies using PPV as the model virus. The robustness studies incorporated anticipated
worst-case protein concentration based on laboratory data for the SP Sepharose process, and worst-case pressure. Results are
outlined in Table V.
Table V: Results from GLP robustness spiking study for Virosart CPV.
All studies used filter lot number 0650773R50Z3/2. For all runs, a scaled-down filtrate volume equivalent to 400 L/m2 was obtained, to reflect the full-scale filter capacity requirements.