Proteins of relatively small sizes can be PEGylated to improve pharmacokinetic profiles for therapeutic purposes. PEGylated proteins present a few challenges during the downstream processing. Ion exchange chromatography is widely used for purification of PEGylated proteins. However, the dynamic binding capacity of PEGylated proteins is significantly reduced compared with the native protein. Potential causes for this include shielding of the proteins charge by conjugated PEG polymer chains and reduced diffusivity in resin beads due to the large PEGylated protein sizes. In this study, we found that protein concentration can impact the size and structure of the PEGylated protein and thus its binding behavior in ion exchange chromatography. Another challenge from the PEGylation reaction is the catalyst used in the reaction has a UV absorbance that can interfere with the chromatogram and protein concentration determination. We utilized tangential flow filtration to remove the catalyst and other impurities, and also concentrate the PEGylated protein. With diafiltered and concentrated PEGylated proteins, the higher loading capacity is achieved and UV interference removed during ion exchange chromatography. We will discuss the characterization of PEGylated proteins and the development of an effective process for purification of PEGylated proteins.

Impact of protein concentration on the size of PEGylated proteins and their adsorption in ion exchange chromatography

2019 Spring National Meeting

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