Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection

Agnes Hajduczki; William C. Chang; Rafael De La Barrera; James F. Wood; Wei Hung Chen; Elizabeth J. Martinez; Jaime L. Jensen; Rajeshwer S. Sankhala; Clayton Smith; Alexander Anderson; Elaine B. Morrison; Caroline E. Peterson; Phyllis A. Rees; Sandrine Soman; Caitlin Kuklis; Aslaa Ahmed; Jocelyn King; Farooq Nasar; Courtney Corbitt; Misook Choe; Paul V. Thomas; Michelle Zemil; Lindsay Wieczorek; Victoria R. Polonis; Helen M. Dooley; John R. Mascola; Natalie de Val; Gary R. Matyas; Mangala Rao; Gregory D. Gromowski; Kayvon Modjarrad; Sandhya Vasan; Jeffrey W. Froude; Nelson L. Michael; M. Gordon Joyce; Stasya Zarling

doi:10.3390/vaccines13101041

Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection

Agnes Hajduczki, William C. Chang, Rafael De La Barrera, James F. Wood, Wei Hung Chen, Elizabeth J. Martinez, Jaime L. Jensen, Rajeshwer S. Sankhala, Clayton Smith, Alexander Anderson, Elaine B. Morrison, Caroline E. Peterson, Phyllis A. Rees, Sandrine Soman, Caitlin Kuklis, Aslaa Ahmed, Jocelyn King, Farooq Nasar, Courtney Corbitt, Misook ChoePaul V. Thomas, Michelle Zemil, Lindsay Wieczorek, Victoria R. Polonis, Helen M. Dooley, John R. Mascola, Natalie de Val, Gary R. Matyas, Mangala Rao, Gregory D. Gromowski, Kayvon Modjarrad, Sandhya Vasan, Jeffrey W. Froude, Nelson L. Michael, M. Gordon Joyce^*, Stasya Zarling^*

^*Corresponding author for this work

Medicine

Research output: Contribution to journal › Article › peer-review

Abstract

Background/Objectives: In response to the COVID-19 pandemic, we developed a vaccine candidate against SARS-CoV-2. Spike Ferritin Nanoparticle (SpFN) comprises 24 identical prefusion-stabilized spike proteins anchored to a self-assembled nanoparticle. Organized along the three-fold axis of the ferritin particle, eight SARS-CoV-2 spike trimers are presented per nanoparticle. Methods: Here, we describe the CGMP processes for manufacturing SpFN using transient transfection of Expi293F cells. Results: The final yield of SpFN was ~10 mg per liter of media supernatant. The resulting protein is stable in cold storage for two years at −20 °C, as well as for a month at room temperature, and can withstand multiple freeze/thaw cycles. SpFN material produced using the CGMP protocols adjuvanted with Army Liposomal Formulation-QS-21 (ALFQ) elicited potent neutralizing antibodies against WA-1, Alpha, Beta, and Delta variants in mice as measured by a pseudovirus neutralization assay. Conclusions: This work demonstrates rapid development and scaled-up production of clinical-grade SARS-CoV-2 vaccine protein material, allowing permissive storage and transport conditions, and serves as a framework for recombinant protein production for future emergent pathogens.

Original language	English
Article number	1041
Journal	Vaccines
Volume	13
Issue number	10
DOIs	https://doi.org/10.3390/vaccines13101041
State	Published - Oct 2025

Keywords

ALFQ adjuvant
CGMP production
COVID-19
SARS-CoV-2
adjuvanted vaccine
pandemic preparedness
recombinant protein vaccine
structure-based vaccine design
transient transfection

Access to Document

10.3390/vaccines13101041

Cite this

Hajduczki, A., Chang, W. C., De La Barrera, R., Wood, J. F., Chen, W. H., Martinez, E. J., Jensen, J. L., Sankhala, R. S., Smith, C., Anderson, A., Morrison, E. B., Peterson, C. E., Rees, P. A., Soman, S., Kuklis, C., Ahmed, A., King, J., Nasar, F., Corbitt, C., ... Zarling, S. (2025). Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection. Vaccines, 13(10), Article 1041. https://doi.org/10.3390/vaccines13101041

@article{fc7cc07339424773884bb7b8a6a620d0,

title = "Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection",

abstract = "Background/Objectives: In response to the COVID-19 pandemic, we developed a vaccine candidate against SARS-CoV-2. Spike Ferritin Nanoparticle (SpFN) comprises 24 identical prefusion-stabilized spike proteins anchored to a self-assembled nanoparticle. Organized along the three-fold axis of the ferritin particle, eight SARS-CoV-2 spike trimers are presented per nanoparticle. Methods: Here, we describe the CGMP processes for manufacturing SpFN using transient transfection of Expi293F cells. Results: The final yield of SpFN was ~10 mg per liter of media supernatant. The resulting protein is stable in cold storage for two years at −20 °C, as well as for a month at room temperature, and can withstand multiple freeze/thaw cycles. SpFN material produced using the CGMP protocols adjuvanted with Army Liposomal Formulation-QS-21 (ALFQ) elicited potent neutralizing antibodies against WA-1, Alpha, Beta, and Delta variants in mice as measured by a pseudovirus neutralization assay. Conclusions: This work demonstrates rapid development and scaled-up production of clinical-grade SARS-CoV-2 vaccine protein material, allowing permissive storage and transport conditions, and serves as a framework for recombinant protein production for future emergent pathogens.",

keywords = "ALFQ adjuvant, CGMP production, COVID-19, SARS-CoV-2, adjuvanted vaccine, pandemic preparedness, recombinant protein vaccine, structure-based vaccine design, transient transfection",

author = "Agnes Hajduczki and Chang, {William C.} and {De La Barrera}, Rafael and Wood, {James F.} and Chen, {Wei Hung} and Martinez, {Elizabeth J.} and Jensen, {Jaime L.} and Sankhala, {Rajeshwer S.} and Clayton Smith and Alexander Anderson and Morrison, {Elaine B.} and Peterson, {Caroline E.} and Rees, {Phyllis A.} and Sandrine Soman and Caitlin Kuklis and Aslaa Ahmed and Jocelyn King and Farooq Nasar and Courtney Corbitt and Misook Choe and Thomas, {Paul V.} and Michelle Zemil and Lindsay Wieczorek and Polonis, {Victoria R.} and Dooley, {Helen M.} and Mascola, {John R.} and {de Val}, Natalie and Matyas, {Gary R.} and Mangala Rao and Gromowski, {Gregory D.} and Kayvon Modjarrad and Sandhya Vasan and Froude, {Jeffrey W.} and Michael, {Nelson L.} and Joyce, {M. Gordon} and Stasya Zarling",

note = "Publisher Copyright: {\textcopyright} 2025 by the authors.",

year = "2025",

month = oct,

doi = "10.3390/vaccines13101041",

language = "English",

volume = "13",

journal = "Vaccines",

issn = "2076-393X",

number = "10",

}

Hajduczki, A, Chang, WC, De La Barrera, R, Wood, JF, Chen, WH, Martinez, EJ, Jensen, JL, Sankhala, RS, Smith, C, Anderson, A, Morrison, EB, Peterson, CE, Rees, PA, Soman, S, Kuklis, C, Ahmed, A, King, J, Nasar, F, Corbitt, C, Choe, M, Thomas, PV, Zemil, M, Wieczorek, L, Polonis, VR, Dooley, HM, Mascola, JR, de Val, N, Matyas, GR, Rao, M, Gromowski, GD, Modjarrad, K, Vasan, S, Froude, JW, Michael, NL, Joyce, MG & Zarling, S 2025, 'Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection', Vaccines, vol. 13, no. 10, 1041. https://doi.org/10.3390/vaccines13101041

TY - JOUR

T1 - Production of Clinical-Grade SARS-CoV-2 Spike Ferritin Nanoparticle Protein Immunogen by Transient Transfection

AU - Hajduczki, Agnes

AU - Chang, William C.

AU - De La Barrera, Rafael

AU - Wood, James F.

AU - Chen, Wei Hung

AU - Martinez, Elizabeth J.

AU - Jensen, Jaime L.

AU - Sankhala, Rajeshwer S.

AU - Smith, Clayton

AU - Anderson, Alexander

AU - Morrison, Elaine B.

AU - Peterson, Caroline E.

AU - Rees, Phyllis A.

AU - Soman, Sandrine

AU - Kuklis, Caitlin

AU - Ahmed, Aslaa

AU - King, Jocelyn

AU - Nasar, Farooq

AU - Corbitt, Courtney

AU - Choe, Misook

AU - Thomas, Paul V.

AU - Zemil, Michelle

AU - Wieczorek, Lindsay

AU - Polonis, Victoria R.

AU - Dooley, Helen M.

AU - Mascola, John R.

AU - de Val, Natalie

AU - Matyas, Gary R.

AU - Rao, Mangala

AU - Gromowski, Gregory D.

AU - Modjarrad, Kayvon

AU - Vasan, Sandhya

AU - Froude, Jeffrey W.

AU - Michael, Nelson L.

AU - Joyce, M. Gordon

AU - Zarling, Stasya

PY - 2025/10

Y1 - 2025/10

N2 - Background/Objectives: In response to the COVID-19 pandemic, we developed a vaccine candidate against SARS-CoV-2. Spike Ferritin Nanoparticle (SpFN) comprises 24 identical prefusion-stabilized spike proteins anchored to a self-assembled nanoparticle. Organized along the three-fold axis of the ferritin particle, eight SARS-CoV-2 spike trimers are presented per nanoparticle. Methods: Here, we describe the CGMP processes for manufacturing SpFN using transient transfection of Expi293F cells. Results: The final yield of SpFN was ~10 mg per liter of media supernatant. The resulting protein is stable in cold storage for two years at −20 °C, as well as for a month at room temperature, and can withstand multiple freeze/thaw cycles. SpFN material produced using the CGMP protocols adjuvanted with Army Liposomal Formulation-QS-21 (ALFQ) elicited potent neutralizing antibodies against WA-1, Alpha, Beta, and Delta variants in mice as measured by a pseudovirus neutralization assay. Conclusions: This work demonstrates rapid development and scaled-up production of clinical-grade SARS-CoV-2 vaccine protein material, allowing permissive storage and transport conditions, and serves as a framework for recombinant protein production for future emergent pathogens.

AB - Background/Objectives: In response to the COVID-19 pandemic, we developed a vaccine candidate against SARS-CoV-2. Spike Ferritin Nanoparticle (SpFN) comprises 24 identical prefusion-stabilized spike proteins anchored to a self-assembled nanoparticle. Organized along the three-fold axis of the ferritin particle, eight SARS-CoV-2 spike trimers are presented per nanoparticle. Methods: Here, we describe the CGMP processes for manufacturing SpFN using transient transfection of Expi293F cells. Results: The final yield of SpFN was ~10 mg per liter of media supernatant. The resulting protein is stable in cold storage for two years at −20 °C, as well as for a month at room temperature, and can withstand multiple freeze/thaw cycles. SpFN material produced using the CGMP protocols adjuvanted with Army Liposomal Formulation-QS-21 (ALFQ) elicited potent neutralizing antibodies against WA-1, Alpha, Beta, and Delta variants in mice as measured by a pseudovirus neutralization assay. Conclusions: This work demonstrates rapid development and scaled-up production of clinical-grade SARS-CoV-2 vaccine protein material, allowing permissive storage and transport conditions, and serves as a framework for recombinant protein production for future emergent pathogens.

KW - ALFQ adjuvant

KW - CGMP production

KW - COVID-19

KW - SARS-CoV-2

KW - adjuvanted vaccine

KW - pandemic preparedness

KW - recombinant protein vaccine

KW - structure-based vaccine design

KW - transient transfection

UR - http://www.scopus.com/inward/record.url?scp=105020169184&partnerID=8YFLogxK

U2 - 10.3390/vaccines13101041

DO - 10.3390/vaccines13101041

M3 - Article

AN - SCOPUS:105020169184

SN - 2076-393X

VL - 13

JO - Vaccines

JF - Vaccines

IS - 10

M1 - 1041

ER -