<p>PEGylation, referring to the covalent attachment of poly(ethylene glycol) or PEG to protein, has become the most established technology for improving pharmacokinetic behavior of native proteins, especially the prolongation of circulation half-life <em>in vivo</em>. This thesis focuses on the synthesis and purification of PEGylated proteins.</p> <p>The conventional way to synthesize PEGylated proteins is in liquid phase batch reaction, which usually causes the formation of significant amount and high diversity of by-products (i.e. di-, tri-, and/or higher-PEGylated forms of a protein). Many chemical and physical ways have been explored to increase the specificity of mono-PEGylated protein. Chemical ways involve manipulation of operating conditions towards site-specific PEGylation. Understanding reaction kinetics is helpful in optimizing conversion and specificity of mono-PEGylation. In this thesis, the PEGylation reaction kinetics between a model protein and PEG NHS ester under various operating conditions was investigated.</p> <p>In the physical perspective, the key point is to gain degree of control on reactant addition instead of one-time addition as in liquid phase batch reaction. Herein, two novel reactor systems were developed. One is solid phase PEGylation bioreactor, bringing free protein to react with immobilized PEG on a membrane surface; the other is Hollow-fiber Membrane Reactor (HMR), distributing PEG into the fiber lumen (where protein is flowing) through the pores on the fiber wall. Greatly improved conversion and specificity of mono-PEGylated protein were observed in both systems, compared to liquid phase batch reactor.</p> <p>An effective and efficient purification technique is very essential because purification step accounts for a significant portion of total cost. In this thesis, the use of hydrophobic interaction chromatography with environment-responsive microporous membranes was examined for the fractionation of different PEGylated proteins. The capability of this technique was demonstrated by obtaining mono-PEGylated protein in a pure form and observing well-resolved chromatographic peaks for different PEGylated proteins.</p> / Doctor of Philosophy (PhD)