Date Published: April 8, 2019
Publisher: Public Library of Science
Author(s): Albert Cupo, Victor M. Cruz Portillo, Paul Gelfand, Anila Yasmeen, P. J. Klasse, John P. Moore, Nicholas J Mantis.
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.
Native-like, recombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are widely used as a vaccine development platform in programs aimed at inducing neutralizing antibodies (NAbs) [1, 2]. The prototype of this immunogen class, the BG505 SOSIP.664 trimer, has been produced under current Good Manufacturing Practice (cGMP) conditions for clinical trials and others are being made in this way for the same reason [3–5]. The most common method to produce SOSIP trimers in cGMP programs, and often also in the research laboratory setting, involves stably transfected Chinese hamster ovary (CHO) cells followed by affinity purification of the secreted trimers, initially via an anti-trimer monoclonal antibody (MAb)-based column [3, 6]. However, transient transfection of various cell types, including 293F and CHO, is commonly used in pre-clinical research, again followed by MAb affinity-column purification [1, 5, 7, 8]. One human MAb that is particularly useful for this purpose, PGT145, recognizes a trimer-specific (quaternary) epitope at the trimer apex [8–11]. PGT145 columns can therefore be used as a one-step method to purify native-like trimers from culture supernatants, at least in the pre-clinical environment [12–14].
Here, we show that the otherwise considerable benefits of the ExpiCHO-S cell transient transfection method for producing multi-milligram amounts of PGT145-purifiable SOSIP trimers are compromised by the presence of an inhibitor in the Enhancer component of the manufacturer-recommended culture system. This inhibitor impedes the binding of SOSIP trimers to the PGT145 affinity column. The most likely culprit is the polyanionic anti-clumping agent dextran sulfate, which is present in the Enhancer component and which we showed does act as an inhibitor of PGT145 binding to SOSIP trimers in immunoassays. The mechanism is most probably a charge-based interaction between the polyanion and the cationic apex of the trimer where the PGT145 epitope is located. A similar PGT145 epitope-occluding effect was previously seen with CpG ODNs, a DNA-based polyanionic adjuvant .