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The baculovirus-insect cell system can produce large quantities of complex protein in a short period of time.
Successful protein production, particularly in large quantities, will depend on using the right expression system. For commercial manufacture of biopharmaceuticals, such as antibodies and vaccines, the ideal system would be one that is easy to culture and maintain, grows rapidly, and produces large amounts of protein (1). Protein expression systems range from bacteria, yeasts, and insects to plants and mammalian systems. Each system has its own strengths and challenges. When deciding on which platform to use, it is important to consider factors such as protein solubility, functionality, purification speed, and yield (2).
Over the past decades, there has been an increasing interest in the use of insect cells for heterologous protein expression, particularly following the European approval of GlaxoSmithKline’s (GSK) cervical cancer vaccine, Cervarix, in September 2007 (3), and FDA’s approval of the influenza vaccine FluBlok in January 2013 (4). The popularity of insect cells, often used in conjunction with the baculovirus expression vector (BEV) system, comes from the ability to produce relatively large quantities of eukaryotic protein with complex post-translational modifications in a relatively short period of time (5). The advantage with this system is the simplified cell growth that can be readily adapted to high-density suspension culture for large-scale expression. Moreover, scale up is easy. Because insect cells have most of the post-translational modification pathways present in mammalian systems, they can produce recombinant proteins that are more antigenically, immunogenically, and functionally similar to the native mammalian protein than if expressed in yeast or other eukaryotes (2).
Baculoviruses are highly specific to insect cells. They are versatile vectors that are easy to manipulate, able to carry large and multiple DNA inserts, and can be readily produced and
purified at high titers (6). Baculoviruses are non-pathogenic to mammals due to the fact that they have a restricted host range, which is limited to specific invertebrate species. Lepidopteran insect cell lines such as the Trichoplusia ni and Spodoptera frugiperda (subclone 9, known as Sf-9) cells are commercially used. Because insect cell lines are not transformed by pathogenic or infectious viruses, they can be cared for under minimal containment conditions (7).
Cervarix, the first vaccine produced using the BEV system in an insect cell line (Hi-5 Rix4446 derived from Trichoplusia ni), consists of two monovalent antigen bulks, containing C-terminally truncated versions of the major capsid L1 proteins of the human papillomavirus (HPV) types 16 and 18 (8). The manufacturing process involves amplification of the seed recombinant baculovirus, extraction of the recombinant baculovirus innocula, and infection of Trichoplusia ni Hi-5 production cell lines in the fermenter. L1 protein is released from the cells by osmotic shock, and subsequently purified by a series of chromatographic columns, a nanometric filtration, an ultrafiltration, and a final sterile filtration to generate the L1 virus-like particles (VLP) in purified bulks (8).
Flublok, developed by Protein Sciences, is the first trivalent influenza vaccine grown in insect cells to be approved by FDA (4). Influenza vaccines are predominantly produced using an egg-based manufacturing process, in which the chicken eggs are inoculated with the influenza virus. Production is, therefore, not flexible, because it is highly dependent on the supply of eggs. Vaccine shortages are prone to happen as a result, especially in the event of a pandemic. With Flublok, the active ingredient, hemagglutinin, is produced by infecting expresSF+ cells--a Spodoptera frugiperda insect cell line--with re-engineered baculovirus that programs the infected insect cells to generate large quantities of the desired influenza virus protein. Influenza virus or eggs are not used in the production of Flublok, which means its production does not depend on egg supply or the availability of the influenza virus (4).
The materials and methods for insect cell culture have evolved together with advances in the BEV system. Serum-supplemented media have been replaced with serum-free media, especially in large-scale production because of increasing emphasis on eliminating animal-source components from cell culture media. Serum-free media eliminate the need for costly fetal bovine and other animal sera supplements and offer better lot-to-lot consistency. The newer optimized formulations of serum-free media have been reported to support faster population doubling times and higher saturation cell densities compared with traditional media, giving higher titers and yields of recombinant protein expression (7).
1. Sigma-Aldrich, “Protein Expression Systems,” accessed Nov. 3, 2016.
2. Thermo Fischer Scientific, Protein Expression Handbook, accessed Nov. 3, 2016.
3. GSK, “Cervarix, GSK’s Cervical Cancer Vaccine, Approved in Europe,” Press Release, Sept. 24, 2007.
4. FDA, “FDA Approves New Seasonal Influenza Vaccine Made Using Novel Technology,” Press Release, Jan. 16, 2013.
5. GTP, “Insect Cells,” accessed Nov. 3, 2016.
6. K. Airenne et al., Molecular Therapy 21 (4) 739-749 (2013).
7. Invitrogen, “Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques,” accessed Nov. 3, 2016.
8. EMA, Cervarix: EPAR-Scientific Discussion, accessed Nov. 3, 2016.
Vol. 29, No. 12
When referring to this article, please cite as A. Siew, “Large-Scale Protein Expression in Baculovirus-Infected Insect Cells," BioPharm International 29 (12) 2016.