| 1. |
Plant Sf9 or
High Five™* cells in shakers, Fernbachs or bioreactor at 5 x 105
cells/mL and grow for 3-5 days in HyQ SFX-Insect until the cell
density reaches 4-8 x 106 cells/mL
with 95% viability.
|
| 2. |
Remove spent
medium by aspiration from sedimented/concentrated cells or hollow
fiber and resuspend the cells to original volume in a resident vessel. |
| 3. |
Subculture the
cells from the resident vessel by planting 2 x 106
to 5 x 106 cells/mL into appropriate
production vessels using fresh HyQ SFX-Insect. Incubate the cells
for one hour for physiological and physio-chemical equilibrium. Perform
viable cell count which should be about 95%-98% viable. Infect the
cells with desired MOI. (0.5-2.5). |
| 4. |
Harvest at optimum
expression time. Upstream processing: Cell separation using cross-flow
filtration, HF or centrifugation. |
| 5. |
Concentration
of secreted rprotein(s) from spent medium. Cell lysis/disruption
to liberate intracellular rprotein(s). |
| 6. |
Preparation for
Chromatography (pH and conductivity adjustment, buffer exchange, filtration
and process testing such as SDS-PAGE, BIACOR, LAL, and Sterility). |
| 7. |
Initiate downstream
processing:
| a) |
Primary
& Polishing Chromatography: Buffer exchange, elution/gradient,
IEC, HIC, GFC, affinity.
|
| b) |
Product
into final formulation buffer followed by dialysis, diafiltration
& concentration. Testing for product recovery, sterility. |
|
| 8. |
Fill & Storage:
Store as liquid, frozen or lyophilized. QC, QA
|
*High
Five is a trademark of Invitrogen Corp.
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