Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

Cibelli, Nicole; Arias, G.; Figur, McKenzie; Khayat, Shireen; Leach, Kristin; Loukinov, Ivan; Shadrick, William R.; Chuenchor, W.; Tsybovsky, Yaroslav; Patel, Aakash; Mai, Misa; Wang, Xin; Vickery, Karen; Khin, Tina; Skubutyte, Renata; Vejzagic, Farah; Bastani, Niutish; Vinitsky, Alison; Lei, Q. Paula; Gulla, Krishana; Gowetski, Daniel B.

doi:10.1038/s41598-022-07485-w

Cited by 8 publications

(3 citation statements)

References 38 publications

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“…We demonstrated that, with a simple tag-free single multimodal chromatography, the particles produced from a stirred-tank bioreactor could be purified with an efficiently scalable procedure. However, a multi-column design may help to improve the purity profile, such as incorporating a weak anion exchange resin as a capture step in a neutral pH buffer [ 61 ]. Affinity mode can also be featured since the spike protein is heavily glycosylated.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool

Puarattana-aroonkorn,

Tharakaraman,

Suriyawipada

et al. 2024

Vaccines

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At times of pandemics, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the situation demands rapid development and production timelines of safe and effective vaccines for delivering life-saving medications quickly to patients. Typical biologics production relies on using the lengthy and arduous approach of stable single-cell clones. Here, we used an alternative approach, a stable cell pool that takes only weeks to generate compared to a stable single-cell clone that needs several months to complete. We employed the membrane, envelope, and highly immunogenic spike proteins of SARS-CoV-2 to produce virus-like particles (VLPs) using the HEK293-F cell line as a host system with an economical transfection reagent. The cell pool showed the stability of protein expression for more than one month. We demonstrated that the production of SARS-CoV-2 VLPs using this cell pool was scalable up to a stirred-tank 2 L bioreactor in fed-batch mode. The purified VLPs were properly assembled, and their size was consistent with the authentic virus. Our particles were functional as they specifically entered the cell that naturally expresses ACE-2. Notably, this work reports a practical and cost-effective manufacturing platform for scalable SARS-CoV-2 VLPs production and chromatographic purification.

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Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool

Puarattana-aroonkorn,

Tharakaraman,

Suriyawipada

et al. 2024

Vaccines

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“…SARS-CoV-2 S-2P protein (S2P7471) was produced as previously described. 78 Briefly, S2P7471 was expressed and purified from the CHO-DG44 cell line. S2P7471 was clarified through centrifugation and Satopore XLG 0.8/0.2 filters.…”

Section: Methodsmentioning

confidence: 99%

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates

Moliva,

Andrew,

Flynn

et al. 2023

Preprint

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SummarySARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there’s a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

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“…Purifying the recombinant spike protein at scales suitable for large-scale manufacturing remains a major challenge [9] , [11] , [12] , [13] , [14] , [15] , [16] . Due to its large size, the purification of this protein using conventional resin-based chromatography is both difficult and inefficient.…”

Section: Introductionmentioning

confidence: 99%

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein

Butani

Pan

et al. 2023

Journal of Chromatography B

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Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

Cited by 8 publications

References 38 publications

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool

Rapid and Scalable Production of Functional SARS-CoV-2 Virus-like Particles (VLPs) by a Stable HEK293 Cell Pool

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein

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