BACKGROUNDThe efficient, controlled and robust purification of conjugates from PEGylation has a growing demand in the biopharmaceutical's market. In general, the yield and purity reached through the conventional chromatographic modes are not particularly high or efficient. Affinity chromatography has so far scarcely been explored. The present work introduces the purification of mono-PEGylated lysozyme from a PEGylation reaction by heparin affinity chromatography (HAC) for the first time in a single step. Response surface methodology (RSM), particularly a Box-Behnken design (BBD) was employed to optimize the separation. RESULTSProtein adsorption of PEGylated and native lysozyme on Heparin Sepharose 6 Fast Flow resin was described by Langmuir isotherms, showing a relatively low affinity for the PEGylated proteins. From the experimental design, optimal elution conditions in a linear gradient of sodium chloride (NaCl) for the three response variables (yield, purity and productivity) were: gradient length of 13 column volumes (CVs), flow at 0.8 mL min(-1) and protein load of 1 mg mL(-1). Based on this optimization, a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme with approximately 100% yield and purity in comparison with 92.7% and 99.7% with the linear gradient. Productivity was c. 0.048 0.001 mg mL(-1) min(-1) using 0.05 mol L-1 NaCl for its elution. CONCLUSIONS Mono-PEGylated lysozyme was completely separated from a PEGylation mixture with high yield and purity using HAC for first time. Applying response surface methodology (RSM), adequate conditions for more than one requirement were found as well as optimal conditions for a linear gradient of NaCl. Based on this optimization a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme in one step with advantages respect to time, resolution, yield and purity compared with other chromatographic modes such as hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEX). (c) 2017 Society of Chemical Industry