This makes available a significant amount of expressed protein with which to perform bioanalytical assays even at this small scale. Typically, we perform a two-tiered assay scheme. Sample preparation is automated, with harvest, sonication, and separation of soluble and insoluble fractions performed on a single robotic platform.
The first round of analysis is designed to provide information on the titer of active Fab expressed in the soluble fraction, using high-throughput, automated biolayer interferometry. Binding of assembled, active Fab to its antigen can be used to rank the expression strains, rapidly identifying the top yielding 1-5% of the expression strains evaluated. Alternately, binding to protein L or protein A can be used to quantitate soluble Fab.
The scatter plot depicted in Figure 1 is an example of results from the first-tier analysis of a typical 1,000-strain screen. Both host strain and expression plasmid can influence the overall yield of active Fab.
In this example, strains carrying monocistronic expression plasmids are shown in black and those carrying bicistronic expression plasmids are shown in red. The top yielding strains typically produce 100 to 500 mg/L of active Fab at the 96-well scale.
The second tier of assays is performed on selected samples and can include affinity enrichment of active Fab, SDS-CGE, Western blot, and even LC-MS to further assess the quality of the expressed Fab. The ability to perform intact mass analyses at this scale can give us information on the fidelity of signal peptidase cleavage of the secretion leaders on both the heavy and light chains.
All of these steps, from receipt of optimized gene fragments through completion of the analyses and selection of strains to evaluate in fermentation, take approximately five weeks to complete. In the case that the goal is the isolation of research amounts of protein, sufficient amounts of protein can be isolated from shake flask cultures due to the large amount of biomass (i.e., 30–50 A600 units) that can be recovered from such cultures.
When larger quantities of material and/or an optimized production strain is the end goal, up to 10 high-yielding strains, representing a diversity of expression strategies and host strain combinations are selected for fermentation “range-finding” experiments to identify the most robust strain for further development.