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


Cell Viability Following TFF


Figure 2. (a) Microscopic analysis of insect cells producing the SARV-CoV Spike protein. The cell viability was 79%. (b) Insect cells producing the SARV-CoV Spike protein after concentration to 7X using SFTFF. The cell viabilty was 69%. No cell lysis was observed.
One common problem encountered when harvesting insect cells by both centrifugation and TFF is the lysis and injury of the cells as a result of shear forces. To demonstrate that the cells were minimally damaged under the operating conditions for the clarification, viability measurements were taken using a CEDEX and pictures of the cells were taken using a light microscope (Figure 2). The viability of the culture after harvest was 79%. After a 7X concentration was achieved, the viability of the cells was 69%. These results indicate that 87% of the cells that were viable before clarification remained viable after the clarification. When the cells were examined microscopically, no additional cell debris or broken cells were observed after concentrating to 7X (Figure 2). The dearth of broken cells was expected because this TFF design does not use a retentate screen and the operating conditions were set to minimize the shear forces on the cells.



Protein Analysis


Figure 3. Analysis of retentate and permeate samples by (a) SDS-PAGE and (b) Western blot analysis. The arrow indicates the position of the desired protein. The lanes contain (1) marker protein; (2) harvested cell sample; (3) retentate at 10X concentration; (4) cell retentate after addition of the elutration buffer; (5) permeate before addition of elution buffer; (6) permeate after addition of extraction buffer; (7) permeate from RC 100 kD module; (9) flow-through from loading the nickel column; (10) elution fraction 1 from the nickel column; (11) elution fraction 2 from the nickel column; (12) elution fraction 3 from the nickel colum; and (13) elution fraction 4 from the nickel column.
The passage of the targeted protein for each step (clarification, protein extraction, and concentration) was checked using an SDS-PAGE gel followed by a Western blot on the retentate and supernatant samples (see Figure 3, in which the arrow indicates the desired protein). No protein passage was found before the protein extraction (lane 5) or during the concentration step (lane 8). Therefore, no protein was lost through the membrane during these steps. Good passage of the desired protein was observed after the elution buffer was added (lane 6). When comparing the column flow-through (lane 9) to the fraction eluted from the column (lanes 10 through 13, in order of elution), the eluted fraction contained the desired protein purified. Therefore, the desired protein bound and eluted from the nickel column. Thus, the buffer ex-change performed in the TFF resulted in a buffer that was suitable for loading the column, making any additional processing steps unnecessary.

Increased Speed and Lower Cost

In this study, it was shown that 10 L of culture broth could be clarified and that 75% of the protein could be extracted in 2.5 h. However, this time could be reduced by decreasing the starting volume-to-membrane-area ratio. For example, increasing the membrane area to 0.3 m2 would decrease the process time to less than 2 h. In this work, the protein yield was not optimized. By increasing the number of system diafiltrations to four during the extraction step, the protein recovery could be increased to more than 99%. Based on a continued flux rate of 5 L m–2 h–1 , the additional time needed to achieve a 99% yield would be 2 h. This time could be decreased by increasing the membrane area to 0.3 m2 such that a 99% yield could be obtained in 3.1 h.


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