BIOTECHNOLOGY
Fig. 1. Expression levels of VHHs in yeast strains from Phenotypeca’s diverse library were assessed using fluorescence, allowing the best strains to be picked for further development
can select for high-producing clones sooner and eliminate future issues during manufacturing scale-up, such as those encountered with other microbes or mammalian cells. With this in mind, Isogenica tasked Phenotypeca with investigating whether high-quality, functional VHH proteins could be produced, secreted and displayed on the surface of a S. cerevisiae yeast cell.
APPROACH Te first task involved verifying that Phenotypeca’s unique collection of strains
Fig. 3. VHHs were expressed with fluorescent tags, and surface-display was determined by assessing whether the fluorescent signal was retained on solid culture media after the cells were washed away. The fluorescent patches in the middle of the second plate show where cells grew that were secreting product, whereas no fluorescence is retained where the VHHs were displayed on the surface of the cells (top and bottom). This was also confirmed by flow cytometry and ELISA-based studies showing antibody binding to the surface displayed VHHs
could be modified to successfully produce a range of VHH proteins. Expression of these proteins was confirmed in several different yeast strains (Fig.1), showing a very clean homogeneous product even in un-purified culture supernatant (Fig. 2). Te absence of significant host-derived protein in these samples means that the product can be purified without the need for immunogenic tags or expensive affinity resins, further reducing cost of goods. In addition, it showed that Phenotypeca’s strains could also produce multi-target (bi-specific) VHH antibodies, a particular challenge for E. coli systems. Phenotypeca then sought
Fig.4. ELISA showing equivalent target binding of anti- PD-L1 VHHs, produced using E. coli and Phenotypeca’s Saccharomyces system, including a bi-specific variant
improving secretion levels from 10-20- fold for a variety of antibody fragments. In addition, Isogenica has been able to confirm that VHH molecules produced by Saccharomyces at Phenotypeca are fully active and able to bind to target antigens, with affinities comparable to antibody fragments produced in E. coli (Fig. 4).
to improve the production and secretion of these VHHs in various yeast strains, as well as developing a method to successfully display these molecules on the surface of the yeast cell (Fig. 3).
Fig. 2. VHHs were secreted by yeast into culture media, which has been visualised here using SDS-PAGE, showing high levels of purity even before purification
RESULTS Phenotypeca successfully produced several different VHHs, both secreted and displayed, in a range of engineered yeast strains,
CONCLUSION Phenotypeca’s platform has paved the way for the creation of a multi-functional toolbox for VHH discovery and production. Tis will lead to a reduction in time and cost of producing high-quality, functional VHHs when compared with production in E. coli and mammalian systems, and improved safety both for manufacturing and for patients compared with production in Pichia.
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