MATERIAL AND METHODS
The risk of prion contamination is a possibility in all biological products of human origin. Evaluation of a step process
can be achieved by spiking a significant amount of prions to the material to be nanofiltered, scaling down the original manufacturing
step, and determining prion removal.
Description of virus removal filters
Viresolve NFP filters are designed for the removal of small viruses (i.e., 18 nm diameter and largeer) from highly purified
proteins. These protein products are produced from recombinant culture, transgenic animals, tissue or body-fluid extractions,
and Cohn plasma fractions. The primary mechanism of removal is size exclusion using a composite polyvinylidene fluoride (PVDF)
membrane. The structure of the membrane allows proteins as large as 160 kDa to pass, while retaining small particles and viruses
such as parvovirus. A small virus model of φX174, a 28 nm nonenveloped bacteriophage, has been validated for the release of
Viresolve NFP membrane and filters.
The Viresolve NFP filter has been demonstrated to clear parvovirus in excess of 4 logs in the presence of various protein
solutions. The membranes are used in normal flow filtration (NFF) mode. Filtration can be performed under constant flow, by
using a peristaltic pumping system, or under constant pressure.
The operation is similar to that based on the 0.22 μm filters widely used in many laboratories. These filters provide robust
clearance of viruses that is relatively independent of operating pressure and protein concentration. The membrane has a composite
structure wherein a thin ultrafiltration (UF) layer is cast on top of a microporous substrate. The thin UF skin retains viruses
effectively without excessively limiting fluid permeability. Various filter formats are available for optimization trials
or for pilot- and industrial-scale production, all of which incorporate the same membrane type.
For the small-scale filterability experiments, the constant pressure V-max model was used (23). V-max is defined as the maximum
product solution volume (L) that can be filtered by 1 m2 of membrane before complete plugging. It is a method for predicting the throughput of filters (capacity = L/m2) based on the gradual pore-plugging model. Gradual pore plugging occurs when colloids or suspended matter collects on the
sides of filter pores to gradually block them off, until a state of total occlusion is eventually reached. This gradual blocking
of the pores occurs in a distinct geometric pattern.
Membrane pore-size distribution in a virus filter device is represented in Figure 1. The largest pores overlap in size with
the smallest viruses. The separation challenge calls for carefully characterizing large pores; the smallest (or functional)
pores provide robust virus clearance, while large pores lead to virus leakage.
Figure 1: Pore distribution and size of different types of biological contaminants. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
When gradual pore plugging occurs, the smallest pores are the first to be plugged. For example, when more than 75% of a membrane's
pores are plugged, the probability of relatively large molecules or viruses passing through the largest pores of the membrane
increases. Hence, it is crucial to define the part of the distribution pore area in which the membrane is working. EMD Millipore
recommends that the Viresolve NFP be sized using the V75 approach, which is the capacity reached when the flow rate has declined
to 25% of the initial flow rate (i.e., 75% of the membrane is plugged).
Selection of the prion strain
For this study, the Rocky Mountain Laboratories (RML6) mouse-adapted scrapie strain was used. Rodent-adapted prion agents
have several advantages for this type of study, including a high-titer source of prion infectivity, a high concentration of
pathogenic PrPSc, and, in the case of RML, a cell-culture-based assay to titrate prion infectivity (24). A host animal (i.e., tga20 mice)
can be used to assay for infectivity with a relatively short incubation period. Mouse prions represent a low biohazard risk
because of the lack of scrapie pathogenicity for humans.