In experiment 1, a premature and strong plugging effect was noted (see Figure 2). No prion infectivity was detected in the
permeate (see Figure 3). This result corresponded to a ≥ 5.8 log reduction in infectivity compared wiht the input. The result
was also in agreement with the Western blot data, which suggested a PrPSc reduction of at least 3.2 logs (see Figure 4).
Figure 2: Observation of a strong and premature plugging effect.
Figure 3: Scrapie cell endpoint assay results. A: Infectivity titer in log infective tissue culture (TCI) units/mL for input
sample (red) and permeate (blue); B: reduction in infectivity titer achieved by nanofiltration for the first experiment.
Figure 4: Western blot after ultracentrifugation of serial dilutions of input compared with the permeate after nanofiltration.
No PrPSc was detected in the permeate. This corresponds to a ≥ 3.2 log fold reduction in PrPSc.
In Experiment 2, using a pretreatment approach of this preparation in filtration buffer, the authors were able to filter a
larger volume (i.e., 80 mL, see Table II). Again, no prion infectivity was detected in the permeate by SCEPA, which corresponded
to a reduction of at least 5.7 logs in TCI (see Figure 5).
Table II: Summary of experimental results.
In the third experiment, where the PTA-precipitated prions were spiked into the FSH purification intermediate, 80 mL of the
spiked solution was nanofiltered. No prion infectivity was detected in the permeate, which corresponded to a reduction of
at least 5.0 logs in TCI (see Figure 6).
Figure 6: Scrapie cell endpoint assay results. A: Infectivity titer in log infective tissue culture (TCI) units/mL for input
sample (red) and permeate (blue). B: Reduction in infectivity titer achieved by nanofiltration containing follicle stimulating
hormone at a concentration of 0.07 mg/mL in the filtration buffer.
Figure 5: Scrapie cell endpoint assay results. A: Infectivity titer in log infective tissue culture (TCI) units/mL for input
sample (red) and permeate (blue). B: Reduction in infectivity titer achieved by nanofiltration for the second experiment.
FSH interference assay
To assess whether the presence of FSH would interfere with the test system for prion infectivity (i.e., SCEPA) an interference
assay was performed. For this assay, serial dilutions of the RML6 standard inoculum in cell-culture medium were prepared.
The inoculum for each dilution contained the same amount of FSH as is present in a 10–1 dilution of the input or permeate of experiment 3 in the cell culture medium. This result was compared with the same RML6
dilution series without FSH in the inoculum.
The prion titers for the SCEPA in the presence of FSH were similar to the ones in the absence of FSH (see Figure 7). This
result indicates that the FSH did not have an influence on the performance of the SCEPA.
Figure 7: Scrapie cell endpoint assay results, follicle-stimulating hormone (FSH) interference assay. Infectivity titer in
log infective tissue culture (TCI) units/g RML6 brain in the absence (red) or presence of FSH (blue).
In this study, the authors investigated the capacity of Viresolve NFP filters to remove the scrapie prion protein, PrPSc, under the actual conditions used for the manufacture of urine-derived gonadotropins. Spiked preparations were designed to
present a serious challenge to the filters.
Western blot assays were used to monitor the partitioning of PrPSc during the first nanofiltration trial. These assays are semi-quantitative indicate the relative levels of PrPSc present in different samples. However, the sensitivity of available assays are limited and they provide only an indirect
measure of infectivity.
Results demonstrate that the Viresolve NFP membrane reduced prion infectivity in a given sample by more than 5.0 logs, both
in buffer and in gonadothropin solution. Viresolve NFP filtration consistently reduced the level of PrPSc to below the limits of detection of the SCEPA infectivity assay, suggesting that this process step is effective for the
removal of prions. Retention of prion protein seems to occur at all the membrane loading level, even when the membrane is
more than 90% plugged.
To examine the influence of protein on PrPSc removal, buffer alone was tested. In this situation, the removal of PrPSc by Viresolve NFP filters showed no significant differences. Protein (FSH) solution did not interfere in the infectivity
reduction calculation, as demonstrated in the FSH interference assay.
Filtration removal mechanisms are mainly related to size exclusion or adsorption. In a typical biopharmaceutical manufacturing
processes, the active compound should be efficiently separated from the pathogen agents without affecting its biological activity
or modifying its molecular characteristics during the filtration process. Size exclusion is the mechanism of choice for such
a purpose. This separation can be efficiently performed if the active compound and the specific pathogen agent are significantly
different in size or molecular weight.