The use of FectoPRO in ExpiCHO-S and comparison with ExpiFectamine CHO

2019 Apr 8;14(4):e0215106. doi: 10.1371/journal.pone.0215106. eCollection 2019.


Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

Cupo A, Cruz Portillo VM, Gelfand P, Yasmeen A, Klasse PJ, Moore JP.

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Abstract: 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.

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