Materials and Methods
Cell Growth Conditions
The gene encoding the S glycoprotein was cloned from a lysate of culture SARS-CoV 3200300841 (Passage #3) in Trizol LS Reagent
(Sigma, St. Louis, MO), kindly provided by Dr. Dean Erdman (CDC, Atlanta, GA). The cDNA was cloned into a baculovirus transfer
vector pSC12 (Protein Sciences Corporation, Meriden, CT) downstream from the baculovirus very late promoter of the polyhedron
gene and in frame with the chitinase secretion signal sequence at the N-terminus and 6-his at the C terminus.
To generate a recombinant baculovirus, linearized Autographa California Nuclear Polyhedrosis Virus (AcNPV) DNA, and the recombination
plasmid DNA containing the S gene were mixed and co-precipitated with calcium phosphate, and Sf9 cells were transfected. Recombinant
viruses were identified by their distinctive plaque morphology. A single recombinant virus plaque was isolated. The virus
was further amplified using Sf9 cells for P1 and ExpresSF+ cells in the absence of serum for P2 and P3. The recombinant virus
was further scaled up before added to 10 L of ExpresSF+ cell culture in a bioreactor at a multiplicity of infection of 1.0
plaque forming unit/cell. The infected culture was incubated at 27–308 C for 48 h. During this period, recombinant SARS-CoV
FL–His-6 was not secreted and was associated intracellularly.
Filtration Setup and Operation
A PUROSEP LT-2Q filtration skid equipped with an OPTISEP 3000 holder (NCSRT, Inc., Apex, NC) was used for all the TFF experiments.
An OPTISEP 3000 module containing 0.186 m2 of a modified polysulphone membrane with a pore size of 0.8 µm and channel height of 0.5 mm was used for the clarification
and protein passage. The protein concentration was performed using an OPTISEP 3000 module containing 0.093 m2 of a regenerated cellulose ultrafiltration membrane with a rating of 100 kD and channel height of 0.75 mm.
At the start of the clarification, a 2-L tank was filled with culture harvest. As permeate left the system, the tank was filled
with culture harvest to maintain a constant tank volume until the entire 10 L of culture harvest had been added. The retentate
flow rate was set at 1.6 liters per meter (LPM), which corresponds to a shear rate of 1400 sec–1 . After a 10X concentration was reached, the retentate was diafiltered with 2 L of tris-buffered saline (TBS) to remove any
small particles or proteins remaining in the medium from the concentrated cells. As the diafiltration continued, the retentate
flow rate was slowly ramped up until a shear rate of 7,000 sec–1 was reached.
The SARS-CoV spike protein was extracted from the cells by adding 150 mL of 10X extraction buffer (10% Triton 100 in TBS)
to the tank containing the 1.5-L culture harvest after diafiltration. The concentrated cells were circulated in the extraction
buffer for 30 min. with the permeate port closed to permit ample mixing and extraction time. The extracted protein was then
passed through the same membrane that was used for clarification. The retentate flow rate started at 3.2 LPM and then increased
to 7.2 LPM, which corresponds to a shear rate of 6,000 sec–1 . To increase the protein yield, a 1X diafiltration was performed.