Optimized purification of mono‐PEGylated lysozyme by heparin affinity chromatography using response surface methodology

Abstract

BACKGROUND: The 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. RESULTS: Protein adsorption of PEGylated and native lysozyme on Heparin Sepharose 6 Fast Flow resin was described byLangmuir 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).

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

“…The protein mixture tested at condition A (flow at 0.8 mL min ‐1 and 13 CVs, Fig. (A)) represents lysozyme PEGylation separation at the optimal conditions for the purification of mono‐PEGylated lysozyme using a linear salt gradient found by our group in a previous study . The relative error for the retention time of native lysozyme in mixture A (0.51%) was lower than that in the individual standard (1.62%), while for the mono‐PEGylated lysozyme the error increased slightly (0.87 vs 1.41%).…”

“…In our study, di‐PEGylated lysozyme was not included in the PEGylation protein mixture because in our previous work this protein was shown not to be retained in the heparin support at dynamic conditions and it was also shown that a mixture of mono‐PEGylated lysozyme and native lysozyme in a 4:1 ratio represents the separation observed in a lysozyme PEGylation reaction. Presently, model predictions based on pure proteins provide a good approximation to the real separation …”

“…Resin was packed into a 5/5 HR column (5 cm length, 0.5 cm diameter, Pharmacia Biotech) and chromatographic experiments were performed in an Äkta Purifier 10 System (GE Healthcare, Uppsala, Sweden) equipped with a 200 μL injection loop using a linear salt gradient of 1 mol L ‐1 of sodium chloride as was pointed out by Mejía‐Manzano et al As mobile phases A and B, 20 mmol L ‐1 Tris‐HCl pH 7.5 and 20 Mm Tris‐HCl pH 7.5 containing 1 mol L ‐1 NaCl were used, respectively. The detection of native and mono‐PEGylated lysozyme was done at 215 nm.…”

“…Individual standards of the proteins were injected separately to obtain the elution curves at a flow of 0.8 mL min ‐1 and 13 column volumes (CVs). To represent the lysozyme PEGylation reaction, mixtures containing mono‐PEGylated and native lysozyme in mass ratio (4:1) were prepared at a total protein load of 1 mg mL ‐1 . These mixtures were analyzed in the Äkta Purifier 10 System at different combinations of flow and gradient length as shown in Table .…”

“…Despite the fact that diverse chromatographic modes in a packed bed column have been tested in one‐single step purification, such as size exclusion (SEC), ion exchange (IEX), reverse phase (RPC) and hydrophobic interaction (HIC), the yield and purity are usually low . Recently, it has been demonstrated that heparin affinity chromatography (HAC) is able to separate the products of the lysozyme PEGylation reaction with high yield and purity . However, purification of mono‐PEGylated lysozyme and any other PEGylated proteins at pilot production level demands knowledge about the mechanism governing the separation so as to understand, optimize, control, predict and scale‐up the chromatographic operation.…”