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Development of methods to purify SARS CoV-2 Virus-like particles at scale
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  • Melissa A. Edeling,
  • Linda Earnest,
  • Julio Carrera Montoya,
  • Ashley Huey Yiing Yap,
  • Jamie Mumford,
  • Jason Roberts,
  • Chinn Yi Wong,
  • Dhiraj Hans,
  • Joseph Grima,
  • Nicole Bisset,
  • Jessie Bodle,
  • Steven Rockman,
  • Joseph Torresi
Melissa A. Edeling
The University of Melbourne Department of Microbiology and Immunology
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Linda Earnest
The University of Melbourne Department of Microbiology and Immunology
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Julio Carrera Montoya
The University of Melbourne Department of Microbiology and Immunology
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Ashley Huey Yiing Yap
The University of Melbourne Department of Microbiology and Immunology
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Jamie Mumford
The Peter Doherty Institute for Infection and Immunity
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Jason Roberts
The Peter Doherty Institute for Infection and Immunity
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Chinn Yi Wong
The University of Melbourne Department of Microbiology and Immunology
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Dhiraj Hans
The University of Melbourne Faculty of Medicine Dentistry and Health Sciences
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Joseph Grima
Seqirus Australia Pty Ltd
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Nicole Bisset
Seqirus Australia Pty Ltd
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Jessie Bodle
Seqirus Australia Pty Ltd
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Steven Rockman
Seqirus Australia Pty Ltd
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Joseph Torresi
The University of Melbourne Department of Microbiology and Immunology

Corresponding Author:[email protected]

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Abstract

The devastating global toll precipitated by the SARS CoV-2 outbreak and the profound impact of vaccines in stemming that outbreak has established the need for molecular platforms capable of rapidly delivering effective, safe and accessible medical interventions in pandemic preparedness. We describe a simple, efficient and adaptable process to purify SARS CoV-2 virus-like particles (VLPs) that can be readily scaled for manufacturing. A rapid but gentle method of tangential flow filtration using a 100 kDa semi-permeable membrane concentrates and buffer exchanges 0.5 L of SARS CoV-2 VLP containing supernatant into low salt and optimal pH for anion exchange chromatography. VLPs are washed, eluted under high salt, dialysed into physiological buffer, sterile filtered and aliquoted for storage at – 80 0C. Purification is completed in less than two days. A simple quality control process includes Western blot for coincident detection of Spike, Membrane and Envelope protein as a proxy for intact VLP, ELISA to detect conformationally sensitive Spike using readily available anti-Spike and/or anti-RBD antibodies, and negative stain and immunogold electron microscopy to validate particulate, Spike crowned VLPs. This process to purify SARS CoV-2 VLPs for preclinical studies serves as a roadmap for preparation of more distantly related VLPs for pandemic preparedness.