Efficient Prion Removal from Gonadotropin Solutions by Nanofiltration Membranes - The authors explore whether a nanofiltration process can be effectively leveraged for removal of prions under conditio

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Efficient Prion Removal from Gonadotropin Solutions by Nanofiltration Membranes
The authors explore whether a nanofiltration process can be effectively leveraged for removal of prions under conditions used for the manufacture of urine-derived gonadotropins.


BioPharm International
Volume 24, Issue 12, pp. 36-49

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.

Filtration methods

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.


Figure 1: Pore distribution and size of different types of biological contaminants. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
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.

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.


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