Severe Acute Respiratory Syndrome (SARS), a coronavirus (SARS CoV) is a respiratory disease, the main symptoms of which include
fever, cough, shortness of breath, and pneumonia. Because SARS has the potential to reappear as a new, naturally acquired
outbreak or by accidental or intentional release, an effective vaccine is sought. One of the most promising candidates for
a SARS vaccine is the S glycoprotein protein. This glycoprotein forms large spikes in the viral envelope and mediates the
binding of SARS CoV to the host cell through the host cell receptor, Angiotensin Converting Enzyme II (ACE2).1 A promising platform for the production of this SARS vaccine is insect cell cultures. Insect cells have the ability to produce
proteins at high concentrations with translational modifications that permit proper folding and function. Additional advantages
of insect cells over mammalian cells include the ease of culture, high molarity tolerance, and high expression levels.
This increased demand for insect cell products leads, in turn, to greater need for effective harvesting methods for large-scale
cultures. Techniques have been established for harvesting insect cells using batch centrifugation,2 continuous centrifugation,3 and tangential flow filtration using either hollow fibers4 or flat sheets.5 Batch centrifugation is the simplest method for small-scale cultures, but it is very difficult to scale up to larger sizes
because each batch usually is limited to only a few liters. Continuous centrifugation permits larger scale operation, but
requires considerable initial investment. Also, the concentrate contains many particles that are 1 Ám or larger, which means
an additional filtration step is necessary before further processing. Finally, the product that remains in the sediment-containing
phase is not recovered.6
An alternative to centrifugation is tangential flow filtration (TFF). TFF has the advantage of being linearly scalable from
the bench top to large scale production.7 A major drawback to TFF is the tendency of microfiltration membranes to foul with particles in the media, which lowers membrane
performance and makes cleaning difficult.8,9
Another complication of using TFF is the shear sensitivity of infected insect cells.5 If TFF is operated at too high a flow rate, the cells will break apart as a result of the shear. These cell parts further
foul the membrane.
This study tests the performance of a modified TFF system, "SmartFlow TFF" (SFTFF, NCSRT, Apex, NC), in obtaining a high yield
of a target protein from insect cell culture. In SFTFF, ribs create uniform retentate channels with uniform flow patterns
over the entire functional surface of the membrane module. These uniform flow patterns maximize membrane efficiency and minimize
fouling of the membrane module. The uniform flow also makes it possible to scale the system linearly from the laboratory bench
to commercial production. In installed systems for other applications, the system has been scaled up directly from 5-m2 laboratory studies to commercial operations that process process 1.4 million L/d.
In this work, a single tank, single-module process was developed using SFTFF to harvest the SARS-CoV spike protein, which
was expressed intracellularly from an insect cell culture. One microfiltration module was used for the following steps: (i)
clarification; (ii) diafiltration to replace the medium with buffer; (iii) lysis of cells and release of the desired protein
by the addition of extraction buffer; and (iv) passage of the released protein through the membrane. Then, an ultrafiltration
membrane was used to concentrate the protein and perform a buffer exchange before loading the protein onto an HPLC column.
No lysis was observed, which means that SFTFF did minimal harm to the cells during clarification. The one tank–one module
method simplifies the harvest in comparison to multiple centrifugation steps. The implications of these results for both small-scale
and large-scale harvesting of insect cells are discussed below.