Bioprocessing of Avian Influenza VLP Vaccine using Baculovirus-Insect Cell Expression System

  • Matthew Chrisdianto Indonesia International Institute for Life Sciences
  • Fedric Intan Damai Indonesia International Institute for Life Sciences
  • Roselyn Mulyono Indonesia International Institute for Life Sciences
  • Jesslyn Audrey Virginia Indonesia International Institute for Life Sciences
  • Katherine K, PhD Indonesia International Institute for Life Sciences
Keywords: bioprocessing, virus-like particles, baculovirus-insect cell expression system, avian influenza, vaccine


Vaccines are widely used as a preventive measure against influenza virus infection. However, these vaccines gain concerns regarding their biosafety due to implementing the highly pathogenic avian influenza in the production process. A breakthrough that uses insect cells due to their ability to produce protein rapidly, especially viral antigens for the potential avian influenza outbreak, is being extensively researched. Insect cells infected by baculovirus (BV) are utilized to express proteins known as virus-like protein (VLP). The objective of this review is to assess the production of the avian influenza vaccine (i.e., H5N1 and H7N9 strains) made from VLP by utilizing a baculovirus-insect cell (BV-IC) expression system. A narrative review was conducted by screening international indexed journals from the last 10 years about the topic. The result shows that VLP vaccine development using BV-IC expression can be a cheaper and safer alternative to conventional vaccines while also producing a high yield. The upstream process consists of the IC infection by the BV and BV-IC cell cultivation inside the bioreactor. The downstream process consists of the purification of the VLP product until it becomes a functioning vaccine. The VLP vaccine has improved immunogenic quality, enabling a more specific immune response than other vaccines. However, studies performed on avian influenza vaccines produced by the BV-IC expression system are still lacking. Therefore, further studies are required to improve the current VLP vaccine production processes.


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Author Biographies

Matthew Chrisdianto, Indonesia International Institute for Life Sciences

Department of Biotechnology, Indonesia International Institute for Life Sciences (I3L), Jakarta, Indonesia.

Fedric Intan Damai, Indonesia International Institute for Life Sciences

Department of Biotechnology, Indonesia International Institute for Life Sciences, Jakarta, Indonesia

Roselyn Mulyono, Indonesia International Institute for Life Sciences

Department of Biotechnology, Indonesia International Institute for Life Sciences, Jakarta, Indonesia

Jesslyn Audrey Virginia, Indonesia International Institute for Life Sciences

Department of Biotechnology, Indonesia International Institute for Life Sciences, Jakarta, Indonesia

Katherine K, PhD, Indonesia International Institute for Life Sciences

Department of Biotechnology, Indonesia International Institute for Life Sciences, Jakarta, Indonesia


Aucoin, M. G., Mena, J. A., & Kamen, A. A. (2010). Bioprocessing of Baculovirus Vectors: A Review. Current Gene Therapy, 10(3), 174–186.
Beas-Catena, A., Sánchez-Mirón, A., García-Camacho, F., Contreras-Gómez, A., & Molina-Grima, E. (2013). The effect of spent medium recycle on cell proliferation, metabolism and baculovirus production by the lepidopteran Se301 cell line infected at very low MOI. Journal of Microbiology and Biotechnology, 23(12), 1747–1756.
Besnard, L., Fabre, V., Fettig, M., Gousseinov, E., Kawakami, Y., Laroudie, N., … Pattnaik, P. (2016). Clarification of vaccines: An overview of filter based technology trends and best practices. Biotechnology Advances. Elsevier Inc. -
Carinhas, N., Bernal, V., Monteiro, F., Carrondo, M. J. T., Oliveira, R., & Alves, P. M. (2010). Improving baculovirus production at high cell density through manipulation of energy metabolism. Metabolic Engineering, 12(1), 39–52.
Carvalho, S. B., Fortuna, A. R., Wolff, M. W., Peixoto, C., Alves, P. M., Reichl, U., & Carrondo, M. J. T. (2018). Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers. Journal of Chemical Technology and Biotechnology, 93(7), 1988–1996.
Carvalho, S. B., Silva, R. J. S., Moleirinho, M. G., Cunha, B., Moreira, A. S., Xenopoulos, A., … Peixoto, C. (2019). Membrane-Based Approach for the Downstream Processing of Influenza Virus-Like Particles. Biotechnology Journal, 14(8).
Carvalho, S. B., Silva, R. J. S., Moreira, A. S., Cunha, B., Clemente, J. J., Alves, P. M., … Peixoto, C. (2019). Efficient filtration strategies for the clarification of influenza virus-like particles derived from insect cells. Separation and Purification Technology, 218, 81–88.
CDC. (2019). 1918 Pandemic (H1N1 virus) | Pandemic Influenza (Flu) | CDC. Retrieved 1 November 2020, from
Chen, H. B., Chen, M., Peng, H. H., Xu, Q. F., Li, X. C., & Bai, B. (2019). Relationship between the benefits of paraspinal mapping and diffusion tensor imaging and the increase of decompression levels determined by conventional magnetic resonance imaging in degenerative lumbar spinal stenosis. Journal of Orthopaedic Surgery and Research, 14(1).
Cherradi, Y., Le Merdy, S., Sim, L. J., Ito, T., Pattnaik, P., & Haas, J. (2018). Filter-based clarification of viral vaccines and vectors. BioProcess Int, 16(4).
Chisti, Y., & Moo-Young, M. (1994). Clean-in-place systems for industrial bioreactors: Design, validation and operation. Journal of Industrial Microbiology. Springer-Verlag. -
Contreras-Gómez, A., Sánchez-Mirón, A., García-Camacho, F., Molina-Grima, E., & Chisti, Y. (2014). Protein production using the baculovirus-insect cell expression system. Biotechnology Progress, 30(1), 1–18. -
Cox, M. M. J. (2012). Recombinant protein vaccines produced in insect cells. Vaccine. -
Drugmand, J. C., Schneider, Y. J., & Agathos, S. N. (2012, September). Insect cells as factories for biomanufacturing. Biotechnology Advances. -
Durous, L., Rosa-Calatrava, M., & Petiot, E. (2019). Advances in influenza virus-like particles bioprocesses. Expert Review of Vaccines. Taylor and Francis Ltd. -
Effio, C. L. (2016). Novel development tools for processing of recombinant virus-like particles. [Doctoral dissertation, Karlsruher Institut für Technologie].
Effio, C. L., & Hubbuch, J. (2015). Next generation vaccines and vectors: Designing downstream processes for recombinant protein-based virus-like particles. Biotechnology Journal. Wiley-VCH Verlag.
Eibl, R., Steiger, N., Wellnitz, S., Vicente, T., John, C., & Eibl, D. (2014). Fast Single-Use VLP Vaccine Productions Based on Insect Cells and the Baculovirus Expression Vector System: Influenza as Case Study. Advances in Biochemical Engineering/Biotechnology, 138, 99–125.
Flickinger, M. C. (2009). Encyclopedia of Industrial Biotechnology. Encyclopedia of Industrial Biotechnology. John Wiley & Sons, Inc.
Fries, L. F., Smith, G. E., & Glenn, G. M. (2013). A recombinant virus-like particle influenza A (H7N9) vaccine. New England Journal of Medicine, 369(26), 2564-2566.
Ge, J., An, Q., Gao, D., Liu, Y., & Ping, W. (2016). Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity. Scientific Reports, 6.
GE Healthcare. (2011). WAVE Bioreactor™ 2/10 and 20/50 systems. Retrieved 1 November 2020, from
Hahn, T., Courbron, D., Hamer, M., Masoud, M., Wong, J., Taylor, K., … Smith, G. (2013). Rapid Manufacture and Release of a GMP Batch of Avian Influenza A(H7N9) Virus-Like Particle Vaccine Made Using Recombinant Baculovirus-Sf9 Insect Cell Culture Technology. BioProcessing Journal, 12(2), 4–17.
Hattori, T., Nakanishi, K., Mori, T., Tomita, M., & Tsumoto, K. (2016). The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins. Bioscience, Biotechnology and Biochemistry, 80(3), 445–451.
Hebel, D. (2014). Making glass bioreactors CIP and SIP capable: Infors HT develops an approach designed to increase system throughput. Genetic Engineering and Biotechnology News, 34(16), 34–35.
Hillebrandt, N., Vormittag, P., Bluthardt, N., Dietrich, A., & Hubbuch, J. (2020). Integrated Process for Capture and Purification of Virus-Like Particles: Enhancing Process Performance by Cross-Flow Filtration. Frontiers in Bioengineering and Biotechnology, 8.
Hutchins, B., Sajjadi, N., Seaver, S., Shepherd, A., Bauer, S. R., Simek, S., … Aguilar-Cordova, E. (2000). Working toward an adenoviral vector testing standard. Molecular Therapy.
Kirkland, B. (2015). Cleaning in Place: A Guide to Cleaning Technology in the Food Processing Industry : Handbook. (T. P. P. Systems, Ed.), Chemical Engineer (London) (p. 40).
Kis, Z., Shattock, R., Shah, N., & Kontoravdi, C. (2019). Emerging Technologies for Low-Cost, Rapid Vaccine Manufacture. Biotechnology Journal. Wiley-VCH Verlag.
Koller, K. (2020). CIP Processes. Retrieved 1 November 2020, from
Koho, T., Koivunen, M. R. L., Oikarinen, S., Kummola, L., Mäkinen, S., Mähönen, A. J., … Laitinen, O. H. (2014). Coxsackievirus B3 VLPs purified by ion exchange chromatography elicit strong immune responses in mice. Antiviral Research, 104(1), 93–101.
Koho, T., Mäntylä, T., Laurinmäki, P., Huhti, L., Butcher, S. J., Vesikari, T., … Hytönen, V. P. (2012). Purification of norovirus-like particles (VLPs) by ion exchange chromatography. Journal of Virological Methods, 181(1), 6–11.
Ladd Effio, C., Wenger, L., Ötes, O., Oelmeier, S. A., Kneusel, R., & Hubbuch, J. (2015). Downstream processing of virus-like particles: Single-stage and multi-stage aqueous two-phase extraction. Journal of Chromatography A, 1383, 35–46.
Lai, C. C., Cheng, Y. C., Chen, P. W., Lin, T. H., Tzeng, T. T., Lu, C. C., … Hu, A. Y. C. (2019). Process development for pandemic influenza VLP vaccine production using a baculovirus expression system. Journal of Biological Engineering, 13(1).
Le Merdy, S. (2015). Selection of Clarification Methods for Improved Downstream Performance and Economics. BioProcessing Journal, 14(3).
Li, Z., Wei, J., Yang, Y., Ma, X., Hou, B., An, W., … Su, Z. (2018). Strong hydrophobicity enables efficient purification of HBc VLPs displaying various antigen epitopes through hydrophobic interaction chromatography. Biochemical Engineering Journal, 140, 157–167.
López-Vidal, J., Gómez-Sebastián, S., Bárcena, J., Del Carmen Nuñez, M., Martínez-Alonso, D., Dudognon, B., … Escribano, J. M. (2015). Improved production efficiency of virus-like particles by the baculovirus expression vector system. PLoS ONE, 10(10).
Merck Millipore. (2016). Generic Process of Virus-Like Particle (VLP) Based Vaccine Manufacturing (Application Note No. TN1246EN00). Retrieved at
Meyer, H. P., & Schmidhalter, D. (2014). Industrial scale suspension culture of living cells. John Wiley & Sons.
Michalsky, R., Passarelli, A. L., Pfromm, P. H., & Czermak, P. (2010). Concentration of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by ultrafiltration. Desalination, 250(3), 1125-1127.
Michl, J., Park, K. C., & Swietach, P. (2019). Evidence-based guidelines for controlling pH in mammalian live-cell culture systems. Communications biology, 2(1), 1-12.
Moleirinho, M. S. G. (2015). Exploring novel downstream strategies for the purification of enveloped VLPs [Unpublished Bachelor’s Thesis]. Instituto Superior Técnico.
Monteiro, F., Bernal, V., Chaillet, M., Berger, I., & Alves, P. M. (2016). Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system. Journal of Biotechnology, 233, 34-41.
Morenweiser, R. (2005). Downstream processing of viral vectors and vaccines. Gene Therapy, 12(1), S103-S110.
Negrete, A., Pai, A., & Shiloach, J. (2014). Use of hollow fiber tangential flow filtration for the recovery and concentration of HIV virus-like particles produced in insect cells. Journal of virological methods, 195, 240-246.
Palomares, L. A., Realpe, M., & Ramírez, O. T. (2015). An overview of cell culture engineering for the insect cell-baculovirus expression vector system (BEVS). In Animal Cell Culture (pp. 501-519). Springer, Cham.
Pidre, M. L., Ferrelli, M. L., Haase, S., & Romanowski, V. (2013). Baculovirus display: a novel tool for vaccination. Current Issues in Molecular Virology-Viral Genetics and Biotechnological Applications, 137-164. InTech.
Pushko, P., Tretyakova, I., Hidajat, R., Sun, X., Belser, J. A., & Tumpey, T. M. (2018). Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone. Vaccine, 36(29), 4346-4353.
Saxena, A., Byram, P. K., Singh, S. K., Chakraborty, J., Murhammer, D., & Giri, L. (2018). A structured review of baculovirus infection process: integration of mathematical models and biomolecular information on cell–virus interaction. Journal of General Virology, 99(9), 1151-1171. Microbiology Society.
Schmidt, S. R., & Wieschalka, S. (2017). Single-Use Depth Filters: Application in Clarifying Industrial Cell Cultures. BioProcess International.
Sequeira, D. P., Correia, R., Carrondo, M. J. T., Roldão, A., Teixeira, A. P., & Alves, P. M. (2018). Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production. Vaccine, 36(22), 3112–3123.
Shuler, M., Kargi, F., & DeLisa, M. (2017). Bioprocess Engineering: Basic Concepts (3rd ed.). Prentice Hall.
Smith, G. E., Flyer, D. C., Raghunandan, R., Liu, Y., Wei, Z., Wu, Y., … Glenn, G. M. (2013). Development of influenza H7N9 virus like particle (VLP) vaccine: Homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus. Vaccine, 31(40), 4305–4313., F., Ghorbanpour, S. M., Palmberger, D., & Striedner, G. (2020). Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells. Scientific Reports, 10(1).
Thompson, C. M., Petiot, E., Mullick, A., Aucoin, M. G., Henry, O., & Kamen, A. A. (2015). Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems. BMC Biotechnology, 15(1).
Unger, T., & Peleg, Y. (2012). Recombinant protein expression in the baculovirus-infected insect cell system. Methods in Molecular Biology, 800, 187–199.
Vedvick, T. S., Steadman, B., Richardson, C., Foubert, T. R., & Petrie, C. R. (2013). U.S. Patent No. 8,481,693. Washington, DC: U.S. Patent and Trademark Office.
Vicente, T., Roldão, A., Peixoto, C., Carrondo, M. J. T., & Alves, P. M. (2011). Large-scale production and purification of VLP-based vaccines. Journal of Invertebrate Pathology.
Whitford, W. (2015). Single-use perfusion bioreactors support continuous biomanufacturing. Pharmaceutical Bioprocessing, 3(1), 75-93.
Wickramasinghe, S. R., Stump, E. D., Grzenia, D. L., Husson, S. M., & Pellegrino, J. (2010). Understanding virus filtration membrane performance. Journal of Membrane Science, 365(1–2), 160–169.
World Health Organization. Module 2: Types of vaccine and adverse reactions. Available online:
Zepeda-Cervantes, J., Ramírez-Jarquín, J. O., & Vaca, L. (2020). Interaction Between Virus-Like Particles (VLPs) and Pattern Recognition Receptors (PRRs) From Dendritic Cells (DCs): Toward Better Engineering of VLPs. Frontiers in Immunology. Frontiers Media S.A.
Zhou, Y., McNamara, R. P., & Dittmer, D. P. (2020). Purification methods and the presence of RNA in virus particles and extracellular vesicles. Viruses, 12(9), 917. MDPI AG.
How to Cite
Chrisdianto, M., Damai, F., Mulyono, R., Virginia, J., & K, K. (2022). Bioprocessing of Avian Influenza VLP Vaccine using Baculovirus-Insect Cell Expression System. Indonesian Journal of Life Sciences, 4(1), 26-47.
Indonesian Journal of Life Sciences