Development of a Novel Platform TFF System for Insect Cell Culture Harvest - Development of a Novel Platform TFF System for Insect Cell Culture Harvest A single-membrane system was used to clarify ins

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Development of a Novel Platform TFF System for Insect Cell Culture Harvest
Development of a Novel Platform TFF System for Insect Cell Culture Harvest A single-membrane system was used to clarify insect cells, diafilter the cell concentrates, mix with an elution buffer to release the target protein, and collect the released target protein in the filtrate. The one tank–one module method simplifies and improves the harvest in comparison to multiple centrifugation and filtration steps.


BioPharm International



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 m 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.
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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