(194c) Integrated Processes for High Yield Protein Pegylation

Considering the production cost of therapeutic proteins, post translational modifications, as PEGylation, have to be highly efficient and optimized, not to waste the valuable and highly purified protein to be modified. Because of the multiple conjugation sites often present at the protein surface, PEGylation suffers from limited yield when it comes to selectively target one single degree of PEGylation. Regarding this limitation, the integration of a chromatographic step in the process can bring highly competitive advantages. Ion-exchange chromatography is used as fractionation technique in order to isolate and recycle the unreacted protein from the PEGylated products. This allows operating the reactor with short reaction times so as to minimize the production of multi-PEGylated proteins. That is, the coupling reaction is stopped before the desired product (i.e. the mono-PEGylated protein) can further react, thus leading to limited conversion but high yield. The recycling of the unreacted protein was then considered to drive the protein overall conversion to completion.

In a second part of this work, the integration is pushed at is paroxysm by investigating an on-column PEGylation process. Integrated processes, efficiently combining reaction and separation in a single step are very attractive to perform protein PEGylation at high yield and high conversion. By adsorbing the protein on an ion-exchange media before contacting it with the functionalized PEG solution, this approach has the potential to inhibit the formation of multi-PEGylated species by restricting the coupling reaction to the few accessible sites. After developing the batch on-column PEGylation process, a multicolumn counter-current chromatography set-up was considered to turn the batch on-column PEGylation process to a continuous one. Using the parameters previously estimated in batch mode, the reactive multicolumn countercurrent solvent gradient purification (rMCSGP) process was simulated and the performance of the process assessed. The rMCSGP can potentially improve the throughput, purity and yield for the production of mono-PEGylated protein.