Optimizing Protein-Polymer Conjugation through Course Grain Simulation and Cell-Free Protein Synthesis: Toward Better 2nd Generation Biologics

Most 2nd generation protein therapeutics are simply PEGylated versions of a prior biologic. Conjugating PEG significantly improves pharmacokinetics and protein stability, however this often occurs at the cost of activity. Indeed there are FDA approved drugs that lose over 90% of their specific activity but are still over 3 times more effective due to the advantages of PEGylation. Here we report our work to integrate course grain simulation results with rapid cell-free protein synthesis screening to determine the optimal locations to attach PEG to proteins that retain activity and further improve protein stability. We also report that traditionally held design heuristics for PEGylation (including solvent accessible surface area and secondary structure) often lead to sub-optimal location choices for PEGylation. However, using the computationally inexpensive approach of course grain simulation is an excellent tool to determine optimal locations for PEGylation and cell-free protein synthesis can be used as a rapid screen to verify computationally determined targets.