Novo Nordisk Pharmatech - Enhance VLP production

Upon expression, the structural matrix polyprotein 'Gag' from the human immunodeficiency virus (HIV) has shown to accumulate beneath the lipidic membrane. After a sufficient number of the Gag polyprotein is recruited, the assembly process is finished and the virus-like particle (VLP) buds out of the cell. The Gag polyprotein has been demonstrated to accommodate a diversity of protein antigens, underlining their potential as a multivalent vaccine. Several biological systems have been used to produce these nanoparticles, but animal cell lines are the preferred option. Specially, the insect cell/ baculovirus expression system (BES) has been proved to work properly for the expression of complex proteins, achieving high protein yields with adequate posttranslational modifications.

"Unlike mammalian cell lines, insect cells provide an easy means for complex protein expression since they can reach high cell densities, do not contain human pathogens and entail less culture requirements."

Unlike mammalian cell lines, insect cells provide an easy means for complex protein expression since they can reach high cell densities, do not contain human pathogens and entail less culture requirements. Among them, the Sf9 insect cell line from Spodoptera frugiperda is the most extensively used platform. The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the typical baculovirus employed for recombinant protein production in this cell line.

Upon infection, the baculovirus arrests the host cell machinery to replicate itself and spread the infection throughout the cell culture. Using standard cloning procedures, the gene of interest is placed after a strong promoter, normally the polyhedrin promoter, to drive the expression of high levels of the recombinant protein of interest. This system consumes an important part of the media nutrients and strategies to increase the production yields of VLPs are then of upmost importance.

Cell-doubling time reduction

Recently, a chemically defined medium for Sf9 cells was developed and there is an opportunity to enhance cell growth and VLP production through specific supplementation. In this study, the effect of recombinant insulin (r-insulin) addition in ExpiSf9 cells using the chemically defined ExpiSF CD medium (Thermo Fisher Scientific) was evaluated. An initial phase of adaptation to different r-insulin concentrations was performed prior to experimentation (1mg/L, 3mg/L and 5mg/L).

The first part of the study focused on the effect of r-insulin addition on cell growth and viability maintenance. A 1.1-fold reduction in cell-doubling time (21±0.5 vs 23.8±2.4 hours) was achieved using 1mg/L r-insulin supplementation. This way, the maximum cell growth was shortened by 24 hours. Also, a 1.2-fold improvement in maximal viable cell concentration was obtained. In the second part, r-insulin was investigated as an enhancer for HIV-1 Gag VLP production using the BES. Again, 1mg/L r-insulin supplementation turned out to be the best condition to increase VLP yield. In these conditions, a 1.2-fold increase in VLP production was attained (5.6×1,010 VLP/mL), all of them with a diameter comprised in the 100-200nm range.

Overall, this work highlights the benefits of 1mg/L r-insulin supplementation in insect cell culture to accelerate cell growth, as well as increasing the maximum VLP titre.

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