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.