Advances in Purification of SARS-CoV-2 Spike Ectodomain Protein Using High-Throughput Screening and Non-Affinity Methods

Abstract

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

Figure 1.. Process Flow Diagram for purification of stabilized S protein.

20MS: Clarisolve 20MS Depth Filter; F0HC: Millistak+ F0HC Depth Filter; 2XLG: Sartorius 2XLG 0.8/0.2 capsule filter; UF: ultrafiltration; DF: diafiltration; AEX: anion exchange; CEX: cation exchange.

Figure 2. Example Resin Screen Data Analysis.

Pathlength-corrected A280 (closed circles, black) and Octet binding data (open circles, blue) were plotted for increasing NaCl concentration elution fractions from 50 mM to 500 mM NaCl and, post-split, the 1000 mM NaCl strip for a selection of chromatography resins included in the pH 7 AEX resin screen. Top row: “hits” exhibited clear peaks in Octet binding < 500 mM NaCl and resolved peaks in A280 separately (either at varying NaCl concentration in the step elutions or in the 1000 mM NaCl strip), indicating successful purification. In contrast, resins not suited for capture, bottom row, showed various patterns, including gradient-like trailing with no clear peak (POROS 50 PI), overall lower Octet binding AUC (NH2–750F), or overlapping A280 peaks with no clear separation (DEAE-650M).

Figure 3. AEX Capture Step Resin Selection and Optimization.

(a) Diagram depicting experimental design. (b) Representative SDS-PAGE from POROS 50 D. Left: MES pH 6.5. Right: Sodium Phosphate pH 7.0. In each gel 1: BenchMark Protein Ladder; 2 and 9: Load FT/Chase; 3 and 10: 100 mM NaCl; 4 and 11: 200 mM NaCl; 5 and 12: 300 mM NaCl; 6 and 13: 400 mM NaCl; 7 and 14: 500 mM NaCl; 8 and 15: 1000 mM NaCl. Lanes 2 – 8 in each gel: 100 kDa UF/DF I; Lanes 9 – 15: 300 kDa UF/DF I. (c) S_dF_2P recovery by product-specific octet titer in FT/Chase, 100 mM NaCl, and 200 mM NaCl fractions for each selected top resin.

Figure 4. Cation Exchange Screen & Proof of Concept Results

(a) Example subset of resin screen graphs with A280, black line/left axis, and percent purity by GXII, blue bar/right axis. First column: example candidate flow through chromatography resins. Second column: example bind and elute candidate resins. Third column: example low separation/broad product peak resins. (b) ToyoPearl SP-650M SDS-PAGE. FT: flow through; M: BenchMark Protein Ladder; L: Load; 75: 75 mM NaCl Wash; S: Strip. (c) Nuvia HR-S Bind and Elute SDS-PAGE. L: Load; FT: Flow through; 100 through 500: mM NaCl step gradient; S: strip. (d) TEM 2D Classes of Nuvia HR-S elution.

Figure 5. 20 nm Filtration flux decay vs. mass throughput.

Flux through the 20 nm filter is plotted against Mass throughput, measured by load A280 and volume.

Figure 6. HexaPro characterization and process data.

(a) NS-EM 2D Classes of purified HexaPro protein in 10 mM Histidine, 150 mM NaCl, 5% Sucrose (w/v) pH 6.5 (b) Differential Scanning Calorimetry in duplicate (overlapping curves) shows a Tm of 59.3°C (SD 0.1°C) (c) Octet binding to three SARS-CoV-2 spike-binding antibodies (d) Final material characterization data. Abbreviations include HCP: host cell protein; UF/DF: ultrafiltration/diafiltration; SEC: size exclusion chromatography; DLS: dynamic light scattering; Rh: hydrodynamic radius; % Pd: percent polydispersity. (e) Mean HCP (ppm) value (n = 2 except UF/DF II product n = 1) across purification unit operations.