Preparation of brain homogenate
Scrapie brain homogenates (20% w/v) were prepared in 0.32 M sucrose from brains of healthy mice (mock homogenate) or from
CD1 mice intracerebrally infected with RML6 (Rocky Mountain Laboratories mouse-adapted scrapie strain). Brains were cut into
pieces using a scalpel and subsequently homogenized in a ribolyzer using two bursts of 45 s at maximum speed of 6.5. Samples
were then stored in 1 mL aliquots at –80°C. The 20% homogenate was diluted 1:1 in phospate buffered saline (PBS) to produce
a 10% homogenate.
Design of experiments
Table I: Summary of three experiments. FSH is follicle stimulating hormone, NFP is normal flow parvovirus, PTA is phosphotungstic
acid, and RML is Rocky Mountain Labs.
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During the filtration study, the input and the filtrate were tested for prion infectivity. The total amount of prion infectivity
in the filtrate was then compared with the total amount of infectivity in the spiked input. In this study, three independent
scaled-down experiments were conducted to evaluate the nanofiltration step used by IBSA during the extraction of FSH from
human urine. Operative conditions were selected according to the actual manufacturing process parameters. Table I provides
a summary of the three experiments.
Experiment 1: Crude scrapie brain homogenate spiked into filtration buffer. Crude scrapie brain homogenate was used as a spike at a final concentration of 0.1% and subjected to nanofiltration. One
mL of a 10% RML6 scrapie brain homogenate (SBH) was centrifuged at 500 G for 5 min at 4°C. 800 μL of the supernatant was
spiked 1:100 into 80 mL of the filtration buffer. This step produced a final concentration of 0.1% or 10–3 dilution of homongenate. One aliquot (5 mL) was removed and frozen at –80°C (input). A 0.1% dilution in filtration buffer
of SBH was subjected to nanofiltration using an OptiScale–25 filter at a constant pressure of 2 bar.
Experiment 2: Purified prion preparation spiked into filtration buffer. Because particles significantly reduced the flow rate of the filtration in the previous spike preparation, purification of
the prion infectivity solution using phosphotungstic acid (PTA) precipitation was conducted for the second and third experiments.
This step minimized the risk of filter fouling while preserving as much infectivity (i.e., high prion titer) as possible.
This solution was subjected to nanofiltration using an Optiscale–25 filter at a constant pressure of 2 bar.
Experiment 3: Purified prion preparation spiked into filtration buffer containing FSH (purification intermediate). In a third experiment, the PTA-precipitated prions were spiked into the FSH purification intermediate (0.07 mg/mL FSH) in
filtration buffer. This solution was subjected to nanofiltration using an Optiscale–25 filter at a constant pressure of 2
bar.
Preparation of dilution series for FSH interference assay
Five μL of 10% RML6 were serially diluted 1:10 into 45 μL of 10% healthy brain homogenate. Four μL of the resulting dilutions
were then diluted 1:1000 into 4 mL of antibiotic-supplemented Opti-MEM (Invitrogen) with 10% fetal calf serum (OFCS). To
each of the 4 mL aliquots of OFCS containing the dilutions of the RML6 brain homogenate, 11 μL of FSH solution F200505/Q were
added for a concentration of 0.007 mg/mL FSH. This corresponds to the FSH concentration in the 10–1 diluted input or permeate of Experiment 3. The dilution series was then compared with the dilution series containing serial
dilutions of 10% RML6, but no FSH.
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