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Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelop glycoprotein

Wednesday, September 12, 2018 — Poster Session I

12:00 p.m. – 1:30 p.m.
FAES Terrace
VRC
RSCHSUPP-18

Authors

  • NA Schneck
  • AL Vinitsky
  • VB Ivleva
  • FJ Arnold
  • JW Cooper
  • QP Lei

Abstract

Vaccine design efforts for HIV-1 have focused on developing a recombinant HIV-1 trimeric envelop glycoprotein as an immunogen to induce broadly neutralizing antibodies. One of the native-like BG505.SOSIP.664 trimer constructs is currently under development as a vaccine candidate. The trimeric structure comprises of three gp140 heterodimer proteins, and each heterodimer contains a gp120 and gp41ECTO subunit which are linked together by six arginine residues (uncleaved) and a disulfide bond (cleaved/uncleaved). Furin proteolytic cleavage happens at the gp120-41ECTO junction (hexa-arginine site), leading to a potent biostructure with a native-like trimeric conformation. To monitor the efficiency of the cleavage in the product, SDS-PAGE has been first applied; however, a newly developed RPLC-UV method has demonstrated to be more reliable due to its better resolution, sensitivity, precision and accuracy. Under reducing conditions, cleaved gp140 converted to its gp120 and gp41ECTO subunits while any remaining gp140 was indicative of uncleaved gp140. During development, the RPLC separation was optimized, as well as the reduction sample treatment step to ensure full reduction of cleaved gp140 which is held together (gp120-gp41) by an intermolecular disulfide bond. Peak identification for uncleaved gp140 under reducing/non-reducing conditions was also achieved via RPLC-UV analysis by spiking furin into the purified trimer material to induce additional (uncleaved) gp140 cleavage. Mass spectrometry was further used to confirm all RPLC-UV peaks. The method is robust and has been qualified to be specific, precise and accurate for monitoring uncleaved gp140. Finally, this novel method has been successfully applied to support product development.

Category: Research Support Services