Lysozyme PEGylation is the covalent attachment of Polyethylene glycol (PEG) to Lysozyme, which is one of the most widely investigated PEGylated proteins. The PEGylation of proteins has become a common practice of modern therapeutic drugs, as the process is capable of enhancing solubility, thermal stability, enzymatic degradation resistance, and serum half-life of the proteins of interest. Lysozyme, as a natural bactericidal enzyme, lyses the cell wall of various gram-positive bacteria and offers protection against microbial infections. Lysozyme has six lysine residues which are accessible for PEGylation reactions. Thus, the PEGylation of lysozyme, or lysozyme PEGylation, can be a good model system for the PEGylation of other proteins with enzymatic activities by showing the enhancement of its physical and thermal stability while retaining its activity. Previous works on lysozyme PEGylation showed various chromatographic schemes in order to purify PEGylated lysozyme, which included ion exchange chromatography, hydrophobic interaction chromatography, and size-exclusion chromatography (fast protein liquid chromatography), and proved its stable conformation via circular dichroism and improved thermal stability by enzymatic activity assays, SDS-PAGE, and size-exclusion chromatography (high-performance liquid chromatography). Methodology PEGylation The chemical modification of lysozyme by PEGylation involves the addition of methoxy-PEG-aldehyde (mPEG-aldehyde) with varying molecular sizes, ranging from 2 kDa to 40 kDa, to the protein. The protein and mPEG-aldehyde are dissolved using a sodium phosphate buffer with sodium cyanoborohydride, which acts as a reducing agent and conditions the aldehyde group of mPEG-aldehyde to have a strong affinity towards the lysine residue on the N-terminal of lysozyme. The commonly used molar ratio of lysozyme and mPEG-aldehyde is 1:6 or 1:6.67. When sufficient PEGylation is reached, the reaction can be terminated by addition of lysine to the solution or boiling of the solution. Various profiles can result in the PEGylation of the protein, which includes intact mono-PEGylated, di-PEGylated, tri-PEGylated, and also possibly their isoforms. Purification Ion exchange chromatography Ion exchange chromatography is often employed in the first step, or capturing step, for the separation of PEGylated proteins as PEGylation may affect the charges of target proteins by neutralizing electrostatic interaction, changing the isoelectric point (pI), and increasing the pKa value. Due to the high pI of lysozyme (pI = 10.7), cation exchange chromatography is used. As the increased degree of PEGylation decreases the ion strength of the protein, the poly-PEGylated proteins tend to bind to the cation resin weaker than the mono-PEGylated protein or the intact form does. Thus, the poly-PEGylated