![]() ![]() Hereto, we first optimized the expression vector for secreted target proteins. In this study, we investigated the potential of the plasmid-based High Five expression system for production of secreted proteins with a focus on antibody and Fc-fusion protein production. Our previous studies already demonstrated a higher protein yield of a secreted protein compared to BEVS with our plasmid-based High Five expression system 25. Without baculoviral infection, the host cells maintain their original secretory pathway integrity, normal cell growth and high viability, resulting in a higher quality of the secreted protein. Different protocols and expression vectors exist but in each case the expression vector is quite efficiently delivered by Polyethylenimine (PEI) and no baculovirus is required. Recently, the plasmid-based production in insect cells without the use of baculovirus was reported 21, 22, 23, 24, 25, 26. This bottleneck is critical for production of antibodies and therefore only few attempts have been made to produce antibodies by BEVS 17, 18, 19, 20 as the resulting yield was rather low. This impairs both yield and protein quality, in particular when the highly active but very late promoters polH or p10 are used 16. Yet, the baculovirus expression vector system (BEVS) still is not optimal for the production of secreted proteins as the virus infection negatively affects the secretory pathway of its host cells 15. Substantial optimization regarding handling, protein yield, deletion of proteases and other factors was achieved over time 12, 13, 14. Production of recombinant proteins in insect cells with baculovirus has a long history dating back to the mid 1980s 9, 10, 11. Thus, insect cells are an ideal alternative to reduce efforts and cost, as they combine ease of culture (at 27 ☌ without requirement of CO 2) with higher tolerance to osmolality of the medium, by-product concentration 6 and cheaper media 7, 8. In addition, diagnostic antibodies do not require mammalian glycosylation as they do not have to interact with the human immune system, allowing the use of alternative expression systems. ![]() For diagnostic antibodies this is not necessary thus fast transient plasmid-based production is the method of choice to produce research-scale quantities of antibodies 5. However, setting up an optimal production system with an optimized cell clone is time- and cost-intensive. Thanks to process optimization production, costs decreased from US$300/g to under US$30/g at ideal condtions 2, 3, 4. Most of these monoclonal antibodies are produced in mammalian expression systems. The global market for monoclonal antibodies used in therapy or diagnostics has grown over the past years and is estimated to reach US$132 billion dollar by 2023 1. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. To improve yields, we optimized the expression vector, media and feeding strategies. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. Yet, production remains expensive as it is mostly done in mammalian expression systems. Antibodies are essential tools for therapy and diagnostics. ![]()
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