Characterization of Lysozyme PEGylation products using polarized Excitation Emission Matrix (pEEM) spectroscopy

Abstract

The growing use of therapeutic proteins requires accurate measurement techniques for measuring biophysical and structural changes during manufacturing. This is particularly true for PEGylation of proteins, because characterization of PEGylation reactions and products can often be difficult because of the relatively small impact on protein structure, the lack of an accessible PEG chromophore, and the heterogeneous final product mixtures. Intrinsic fluorescence spectroscopy is one potential solution because of its relatively high sensitivity to small changes in protein structure and its suitability for online or at-line measurements. Here we use PEGylation of lysosome as a model system to determine the efficacy of polarized Excitation Emission Matrix (pEEM) spectroscopy as a rapid tool for characterizing the structural variability of the lysozyme starting materials and PEGylated products with PEG to protein ratio (PPR). Dynamic Light Scattering (DLS) showed that as PPR increased from 0 to 2.8, the hydrodynamic radius increased from ~2.2 to 4.8 nm. pEEM measurements provided several sources of information: Rayleigh scatter identified size changes and aggregate/particle formation, and fluorescence emission to assessed chemical and structural change. PEGylation induced sufficient physicochemical changes in lysozyme which produced changes in the pEEM spectra largely due to changes in hydrophobic environment for tryptophan residues close to a PEG attachment site. These significant spectral changes when modelled using conventional multivariate analysis methods were able to easily discriminate the raw product solutions according to degree of PEGylation and were also able to predict PPR with reasonable accuracy (RMSEC ~10%, REP