cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate

Abstract

We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log₁₀ . The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes.

Keywords: HIV-1 vaccine; SOSIP; affinity purification; cGMP; native-like Env trimers; recombinant vaccine development.

Figure 1

Overview of the process used to purify BG505 SOSIP.664 trimers. Schematic of the 10 steps involved in the purification of the trimer fraction from the initial gp140 protein harvest. The four dedicated viral clearance/inactivation steps are highlighted by an *. MM‐AEX, Mixed mode anion exchange

Figure 2

NS‐EM images of BG505 SOSIP.664 trimer Drug Substance products from small (50 L) and large (200 L) scale production runs. (a) 50 L Confirmation Run; (b) 200 L Demonstration Run; (c) 200 L cGMP Run. All three runs yielded trimers that were 100% native‐like

Figure 3

Properties of BG505 SOSIP.664 trimers purified from the Demonstration Run. (a) CD profile of BG505 SOSIP.664 trimer, (b) DSC profile of BG505 SOSIP.664 trimer showing a T_onset of 59.4°C and T_m of 66.6°C

Figure 4

N‐linked glycosylation analysis. (a) Total glycan profiling of glycans enzymatically released from BG505 SOSIP.664 gp140 (200 L Demonstration Run), fluorescently labelled and analyzed by HILIC‐UPLC. Oligomannose‐type and hybrid glycans (green) were identified by their sensitivity to Endo H digestion. Peaks containing complex‐type glycans are colored in pink. The pie charts summarize the quantification of the peak areas. (b) Percentage quantification of the oligomannose‐type series Man5GlcNAc2 to Man9GlcNAc2 (M5 to M9) of the glycan profiles shown in a. (c) Glycan sequencing of glycan profile shown in a. Peaks were assigned by a sequential enzymatic digestion of 2‐AA labelled glycans with a panel of exoglycosidases, followed by HILIC‐UPLC analysis. The top panel shows the undigested glycan profile of BG505 SOSIP.664 trimers produced in CHO cells. The profiles below represent digestions with the following exoglycosidases: Neuraminidase from Clostridium perfringens, α‐L‐fucosidase from bovine kidney, β1, 4‐galactosidase from Streptococcus pneumonia, β‐N‐acetylglucosaminidase from S. pneumonia and Jack bean α‐mannosidase. (d) Quantitative site‐specific N‐glycosylation of 23 of the 28 sites on BG505 Env. The glycoprotein was digested with trypsin, chymotrypsin, pronase, and GluC, and then analyzed by LC‐ESI MS. Glycans are categorized as oligomannose series (M5 to M9), hybrids (H), and fucosylated hybrids (FH), and also by the number of branching antennae (A) of complex type glycans. An, number (n) of antennae; Gn, number (n) of galactose residues; F, presence of a core fucose (Behrens et al., 2016). The bar graphs represent the means of two analytical replicates, and the quantification of oligomannose‐type (green) and complex/hybrid‐type glycans (pink) on the individual sites is summarized in the pie charts

Figure 5

SPR analysis of bNAb binding to BG505 SOSIP.664 trimers. The binding of BG505 SOSIP.664 trimer in solution to the indicated immobilized bNAbs (3BNC117, 2G12, PGT130, PG9, PGT145, PGT151, 35022, and 3BC315) was analyzed by SPR. The green curves of the sensorgram show one of two replicate profiles of the specific response for each NAb (see SOM Methods) during 300 s of association and 600 s of dissociation. Note that the y‐axis scales are adjusted according to the maximum binding to each bNAb. The red dotted lines correspond to 0 response units (RU) on the y‐axis