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Updated: Dec 5, 2025
Expression and Purification of Virus-like Particles for Vaccination
Published on: June 2, 2016
Development of a High Yielding Bioprocess for a Pre-fusion RSV Subunit Vaccine
Peifeng Chen1, Mingzhong Chen1, Amritha Menon1
1Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9 West Watkins Mill Rd., Gaithersburg, MD, 20878, USA.
Insights
Developing a stabilized pre-fusion RSV fusion (F) protein vaccine candidate, DS-Cav1, achieved high yields and maintained its crucial pre-fusion conformation for potential clinical use in infants and the elderly.
Area of Science:
- Vaccinology
- Protein Engineering
- Bioprocess Development
Background:
- Respiratory syncytial virus (RSV) poses a significant threat to infants and the elderly.
- The RSV fusion (F) protein's pre-fusion conformation is a key target for neutralizing antibodies.
- Stabilizing the metastable pre-fusion F conformation is critical for vaccine development.
Purpose of the Study:
- To develop a scalable production process for the stabilized pre-fusion RSV F glycoprotein (DS-Cav1).
- To ensure the DS-Cav1 vaccine candidate maintains its pre-fusion conformation and high immunogenicity.
- To optimize protein expression for clinical manufacturing.
Main Methods:
- Designed a stabilized pre-fusion F glycoprotein (DS-Cav1).
- Optimized expression using co-transfection with individual plasmids for DS-Cav1 and furin.
- Enhanced protein yield through seeding density optimization and biphasic hypothermia.
- Scaled up the production process to 50 L.
Main Results:
- Achieved high protein expression levels of approximately 1,500 mg/L.
- Successfully maintained the pre-fusion conformation of the DS-Cav1 antigen.
- Demonstrated reproducible production at a 50 L scale.
- Preliminary Phase I data suggest promising efficacy.
Conclusions:
- The developed bioprocess enables high-yield, stable production of the DS-Cav1 pre-fusion RSV F vaccine candidate.
- The stabilized DS-Cav1 antigen is suitable for clinical manufacturing and testing.
- This advancement holds promise for a new RSV vaccine to protect vulnerable populations.
Abstract:
Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).
