(265d) Protein Pegylation Using Micro-Reactors

PEGylation, which involves the conjugation of polyethylene glycol (or PEG) with a protein, is one of the main chemical modification techniques used for improving properties of biopharmaceutical proteins. Protein PEGylation is typically carried out as liquid-phase batch reactions. The mono-PEGylated protein is usually the preferred form and various strategies have been used to improve both its yield and selectivity. A large number of reactions have to be carried out to optimize such protein-polymer conjugation reactions. Scale-down or miniaturization of reaction has commonly been successfully used in such situations. This work focuses on the design and development of reactor systems suitable for studying PEGylation at a micro-scale where the protein and the PEGylating reagent are reacted in a micro-channel. Miniaturization of protein PEGylation using microfluidics would enable parallel, high-throughput study of factors likely to affect reaction yield and selectivity such as PEGylation chemistry (e.g. aldehyde reaction, NHS reaction), reactor volume, residence time, reagent concentrations, polymer to protein molar ratios, pH and quencher addition.

Industrial-scale protein PEGylation is usually carried out in stirred tank reactors in which turbulent flow predominates. However, the flow of liquid within the micro-channels is laminar. In order to make the micro-reactor system more representative of the actual macro-scale process, various active and passive mixing strategies available in the domain of microfluidics were examined. Amongst these, chaotic mixing by the using grooved-channels was found to be most promising. In the micro-reactors used in this study, reactants are efficiently mixed by the chaotic advection due to groove cycles embedded in the floor of the channel. Small volumes of the fluid are stretched and folded over the cross section of the channel and the mixing length is thereby reduced. The micro-reactor was fabricated by casting a single PDMS layer on a 3D-printed acrylic-based mold. The PDMS layer was attached to a glass slide using air plasma to create the micro-channel. The PEGylating reagent and protein solutions entered the micro-reactor through a Y-shaped terminal and the product stream leaving the reactor was quenched in-situ using glycine solution. The PEGylated protein containing product stream was analyzed for conversion and selectivity using appropriate analytical techniques.