Production of therapeutic proteins with baculovirus expression system in insect cell

Institute of Biotechnology, Wonkwang University, Iksan, Korea
Entomological Research 11/2008; 38(s1):S71 - S78. DOI: 10.1111/j.1748-5967.2008.00177.x

ABSTRACT Recombinant DNA technology has a major advantage in that it is capable of producing specific therapeutic proteins on demand in a heterologous expression system. The extent of this notion can be understood when one considers how crucial such proteins are, and how problematic the economical and safe production of such proteins are. Therapeutic recombinant protein production is a fundamental aspect of 21st century biotechnology industries. The improved therapeutic recombinant protein expression systems that use prokaryotic and eukaryotic cells have enabled the development of a multi-billion dollar industry. Among the variety of available heterologous expression systems, the baculovirus-based insect cell expression system has been utilized frequently for the high-level production of therapeutic recombinant proteins. Thus, the baculovirus expression system has been recognized as one of the most powerful expression technologies for production, by virtue of the achievable amount and purity, and the ease of the eukaryotic production process. The majority of therapeutic proteins are glycoproteins originating from humans. The insect-based expression system harbors glycosylation processing pathways, which constitute an advantage over other prokaryotic systems that lack glycosylation. However, there are several drawbacks which must be circumvented in order to establish an efficient system for the production of recombinant proteins. This review presents a brief overview of the perspective, particularly the glycosylation aspect, of the production of therapeutic recombinant proteins via a baculovirus-based insect cell expression system.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recombinant baculoviruses (rBac) are used for many different applications, ranging from bio-insecticides to the production of heterologous proteins, high-throughput screening of gene functions, drug delivery, in vitro assembly studies, design of antiviral drugs, bio-weapons, building blocks for electronics, biosensors and chemistry, and recently as a delivery system in gene therapy. Independent of the application, the quality, quantity and purity of rBac-based products are pre-requisites demanded by regulatory authorities for product licensing. To guarantee maximization utility, it is necessary to delineate optimized production schemes either using trial-and-error experimental setups ("brute force" approach) or rational design of experiments by aid of in silico mathematical models (Systems Biology approach). For that, one must define all of the main steps in the overall process, identify the main bioengineering issues affecting each individual step and implement, if required, accurate analytical methods for product characterization. In this review, current challenges for quality control (QC) technologies for up- and down-stream processing of rBac-based products are addressed. In addition, a collection of QC methods for monitoring/control of the production of rBac derived products are presented as well as innovative technologies for faster process optimization and more detailed product characterization.
    Journal of Invertebrate Pathology 07/2011; 107 Suppl:S94-105. · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 μL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 μL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.
    Hybridoma (2005) 10/2011; 30(5):419-26. · 0.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37 °C. We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34 °C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.
    BMC Biotechnology 11/2011; 11:114. · 2.17 Impact Factor


Available from
May 19, 2014