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

RESULTS

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.


Table II: Summary of experimental results.
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).

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.


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).
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.

DISCUSSION

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.


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