Figure 1. Membrane performance during (a) clarification and diafiltration; (b) protein passage; and (c) protein concentration.
The closed circles are the total permeate volume for that step. The open circles are the LMH (liters of permeate per square
meter of membrane per hour) rates at that time. A Purosep LT-2Q system with an Optisep 3000 holder (NCSRT, Apex, NC) was used
for all TFF experiments. The same module with a modified polysulfone membrane with an 0.8 µm pore size was used for the clarification
and diafiltration and passing the protein. A module with a regenerated cellulose membrane with a 100-kD pore size was used
for the concentrating the protein. In Figure 1a, the concentration (i) and diafiltration (ii) steps are marked. The arrow
in Figure 1b indicates the point at which the retentate flow rate increased from 3.2 to 7.2 LPM. The bar in Figure 1c indicates
the time at which diafiltration was performed during the ultrafiltration step.
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The 1.2 L of SARS-CoV spike protein was concentrated using ultrafiltration. The retentate flow rate was set at 12.5 LPM, which
corresponds to a shear rate of 6,700 sec–1 . The back pressure was maintained between 20 and 30 psi. To ensure the appropriate buffer for loading the HPLC column, a
1X diafiltration was performed using TBS.
Results and Discussion
Clarification and Protein Extraction
The membrane performance is displayed in Figure 1a for the initial clarification and diafiltration step. The initial permeate
flux was 750 mL/min. As expected in a microfiltration process, the flux slowed as a gel layer built up on the membrane. The
cell concentration was increased to 10X in 27 min., which is an average LMH (liters of permeate per square meter of membrane
per hour) of 109 L m–2 h–1 . After concentrating, diafiltration was performed on the concentrated cells to remove the medium components and place the
cells in an appropriate buffer for additional processing. The permeate flux further slowed as diafiltration was performed
on the concentrated cells, yet even at the end of the diafiltration a flux rate of 10 L m–2 h–1 was maintained. The overall average flux rate for the concentration and diafiltration step was 41 L m–2 h–1 .
The SARS-CoV spike protein was extracted from the cells using a Triton 100 extraction buffer, which was simply added to the
solution in the filtration skid tank. The desired protein passed through the membrane and was separated from the larger cellular
components (figure 1b). The protein recovery was increased by performing a 1X diafiltration, which would result in a 75% theoretical
yield of the protein. The protein passage step proceeded with an average flux rate of 10 L m–2 h–1 . The large increase seen in the LMH value (the arrow in Figure 1b) occurred when the retentate flow rate was increased as
described in the materials and methods section above. However, the permeate flux quickly decreased, returning to the previous
steady state value.
Filtering the cells after the extraction buffer was added was expected to proceed more slowly than the initial clarification
for two reasons. First, a gel layer would already have formed on the membrane during the clarification. Second, after the
extraction buffer was added, cell lysis would have increased the number of particles in the retentate. Some of these newly
formed particles would cause additional membrane fouling.
Ultrafiltration to Concentrate Protein
The ultrafiltration step to concentrate the protein with a buffer exchange diafiltration step proceeded with an average flux
rate of 35 L m–2 h–1
(Figure 1c). The permeate flux remained constant during diafiltration and then decreased as the protein was concentrated
to the final 4X concentration. In a commercial operation, performing diafiltration and concentration before loading the HPLC
column would achieve two objectives. First, the buffer could be adjusted to the optimal buffer to load the column, and second,
the volume to load the column could be minimized.
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