Trimers were transiently expressed in ExpiCHO-S cells using FectoPRO (Polyplus-transfection SA) transfection reagent. ExpiCHO-S cells were cultured with ExpiCHO Expression Medium (Thermo Fisher Scientific) in a shaker incubator set at 135 rpm, 37˚C and 8.0% CO2. The day before transfection, ExpiCHO-S cells were seeded at 3 x 10e6 per ml in 500 ml of ExpiCHO Expression Medium. Following the manufacturer’s recommended protocol, 320 μg of Env plasmid, 80 μg of Furin plasmid, and 800 μl of FectoPRO reagent were mixed in 50 ml of cold Opti-PRO SFM. A 250 μl quantity of FectoPRO booster was immediately added to the FectoPRO-transfected cultures. Culture supernatants were harvested when cell viability dropped below 60% (day 6 to 14),

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.