Simulation of mono-PEGylated lysozyme separation in heparin affinity chromatography using a general rate model

Abstract

BACKGROUNDThe bioavailability of therapeutic proteins is improved through PEGylation. This chemical modification involves the production of isomers with different numbers and sites of attached PEG chains, which are difficult to separate efficiently. Their purification with chromatography requires an understanding of the operation and the evaluation of different operational conditions. The General Rate Model (GRM) was applied for modelling the linear salt gradient elution of mono-PEGylated and native lysozyme in Heparin Affinity Chromatography (HAC) considering mass balance equations for proteins in the bulk-fluid phase, in the particle phase and the kinetic adsorption.

RESULTSThe model was able to simulate the individual proteins and the separation of these in a PEGylation reaction using as proof-of-concept a mono-PEGylated and native lysozyme mixture under changes of operational parameters such as the gradient length (5, 13, 25 column volumes) and flow (0.8 and 1.2 mL min(-1)) with a relative error in retention times of less than 6% and correlation coefficients greater than 0.78.

CONCLUSIONSimulation of the elution curves of PEGylated lysozyme in HAC was performed in this work and the diverse information generated by the model is explained through the physicochemical protein properties. This simulation represents a tool for optimization, prediction and future scale-up of PEGylated proteins purification, which would reduce the investment in time and resources to test several operating conditions. (c) 2017 Society of Chemical Industry