Abstract
PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.
Highlights
During the last decades, several proteins have been used to treat di erent diseases where their high biological activity and high speci city have been demonstrated
Monolithic disks were PEGylated through reductive amination to separate the di erent PEGylated Ribonuclease A (RNase A) species obtained a er its conjugation reaction
Since EDA monoliths have primary amines and methoxy poly(ethylene glycol) propionaldehyde (mPEG) is activated with aldehyde groups, it is possible to gra the polymeric chains to the support using sodium cyanoborohydride as a reducing agent and methanol as protic solvent [19]
Summary
Several proteins have been used to treat di erent diseases where their high biological activity and high speci city have been demonstrated Some of these kinds of proteins have unfavorable properties such as low solubility, instability, and rapid clearance by the human body [1]. E di-PEGylated protein or those conjugates with higher PEGylation degrees are less biologically active mainly because the active site becomes hindered by the PEG chains [6, 7]. For this reason, it is highly desirable to separate the mono-PEGylated protein from the other species. Its separation is a hard issue and an interesting
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