CCF, HCCF, recombinant proteins, and other materials
CHO CCF and recombinant proteins were produced at WuXi Biologics using
standard cell culture procedures. The bioreactors used for cell cultures
varied from a lab scale to at-scale manufacturing. The cell cultures
were harvested either using centrifugation followed by depth filtration
or depth filtration only. The molecules studied in this article are
produced by CHO cell. The information of the molecule types is listed in
the table1. Nicotinamide adenine dinucleotide phosphate (NADPH) was from
EDM Chemicals; 2,6-Dichlorophenolindophenol (DCPIP) from Sigma-Aldrich;
Thioredoxin reductase (TrxR) from
Sigma-Aldrich.
0.22µm filter from Millipore and D0HC depth filter from Millipore.
Low-speed centrifugation supernatant and cell lysate: CCF was
centrifuged in a
Sorvall
ST 16R rotor centrifuge (Thermo) at 800 x g for 10min at room
temperature. The 800 x g for 10min is considered as a moderate
centrifugation condition and would not cause cell lysis. The obtained
up-liquid is the low-speed centrifugation supernatant; i.e., the
supernatant of CCF. The cell pellets were re-suspended into original
volume using 50 mM HAc-NaAc buffer. The cell lysis of the suspension was
realized either by centrifugation in a Sorvall ST 16R rotor centrifuge
at 10000 x g for 30min or by freeze-thaw at -70oC/room
temperature followed by a centrifugation at 10000 x g for 30 min. The
up-liquid is the cell lysate.
Reduction simulation study: 1-2 mL of samples were transferred into 10ml
glass bottles filled with nitrogen and sealed with a rubber plug. The
bottles with the samples were incubated at intended temperature for up
to 12 hours. The samples were taken at pre-determined time-points with
syringe and immediately kept at <-70℃until analysis.
Harvest depth filtration processing: CCF was clarified with lab-scale
depth filters (23cm2 D0HC). The filters were rinsed
with purified water at a flow rate of 600LMH (L/m2/h),
equilibrated with the equilibration buffer (50mM Tris, HAc 50mM NaCl,
pH7.4) at a flow rate of 300LMH for more than 20L/m2before use. The speed of the peristaltic pump was set at 50-60LMH for
CCF filtrations. The delta pressure of depth filter was monitored with
pressure sensors (PENDOTECH). After the CCF filtration, the filters were
flushed with the equilibration buffer.