Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods
Nicole L. Cibelli,G. Arias,McKenzie L. Figur,Shireen Khayat,Kristin M. Leach,Ivan Loukinov,William R. Shadrick,Watchalee Chuenchor,Yaroslav Tsybovsky,Aakash Patel,Misa Mai,Xin Wang,Karen Vickery,Tina Khin,Renata Skubutyte,Farah Vejzagic,Niutish Bastani,A.A. Vinitsky,Q. Paula Lei,Krishana C. Gulla,Daniel B. Gowetski +20 more
Reads0
Chats0
TLDR
In this paper , the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44, was presented.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. read more
Citations
More filters
Posted ContentDOI
Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices
TL;DR: In this paper , the authors present a review of literature that reported the adoption and utilisation of liquid handling devices available in the market and presents examples of their practical use in both conventional and nonconventional applications.
Journal ArticleDOI
Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices
TL;DR: In this article , the authors present a review of literature that reported the adoption and utilisation of liquid handling devices available in the market and presents examples of their practical use in both conventional and nonconventional applications.
Journal ArticleDOI
A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein
TL;DR: In this article , a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein was proposed. But, this method requires very low flow rates to be used during binding and elution, and this slows down the purification process.
References
More filters
Journal ArticleDOI
Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
Daniel Wrapp,Nianshuang Wang,Kizzmekia S. Corbett,Jory A. Goldsmith,Ching-Lin Hsieh,Olubukola M. Abiona,Barney S. Graham,Jason S. McLellan +7 more
TL;DR: The authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor, and test several published SARS-CoV RBD-specific monoclonal antibodies found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs.
Journal ArticleDOI
RELION: implementation of a Bayesian approach to cryo-EM structure determination.
TL;DR: Developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined are described.
Journal ArticleDOI
Automated electron microscope tomography using robust prediction of specimen movements.
TL;DR: A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction, using changes in specimen position at previous tilt angles to predict the position at the current tilt angle.
Journal ArticleDOI
A serological assay to detect SARS-CoV-2 seroconversion in humans.
Fatima Amanat,Daniel Stadlbauer,Shirin Strohmeier,Shirin Strohmeier,Thi H. O. Nguyen,Veronika Chromikova,Meagan McMahon,Kaijun Jiang,Guha Asthagiri Arunkumar,Denise Jurczyszak,Jose Polanco,Maria Bermudez-Gonzalez,Giulio Kleiner,Teresa Aydillo,Lisa Miorin,Daniel S. Fierer,Luz Amarilis Lugo,Erna Milunka Kojic,Jonathan Stoever,Sean T. H. Liu,Charlotte Cunningham-Rundles,Philip L. Felgner,Thomas M. Moran,Adolfo García-Sastre,Daniel Caplivski,Allen C. Cheng,Katherine Kedzierska,Olli Vapalahti,Jussi Hepojoki,Jussi Hepojoki,Viviana Simon,Florian Krammer +31 more
TL;DR: A serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters and can be adjusted to detect different antibody types in serum and plasma.
Journal ArticleDOI
Site-specific glycan analysis of the SARS-CoV-2 spike.
Yasunori Watanabe,Yasunori Watanabe,Joel D. Allen,Daniel Wrapp,Jason S. McLellan,Max Crispin +5 more
TL;DR: It is shown how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design, and enables mapping of the glycan-processing states across the trimeric viral spike.
Related Papers (5)
Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods
Nicole L. Cibelli,G. Arias,McKenzie L. Figur,Shireen Khayat,Kristin M. Leach,Ivan Loukinov,William R. Shadrick,Watchalee Chuenchor,Yaroslav Tsybovsky,Aakash Patel,Misa Mai,Xin Wang,Karen Vickery,Tina Khin,Renata Skubutyte,Farah Vejzagic,Niutish Bastani,A.A. Vinitsky,Q. Paula Lei,Krishana C. Gulla,Daniel B. Gowetski +20 more