Characterization of PEGylated Lysozyme by Size Exclusion and Ion Exchange Chromatography

Werner Conze, Jessica Christel, Achim Sprauer, Volker Noedinger and Egbert Mueller, Tosoh Bioscience GmbH.

PEGylation, the process by which polyethylene glycol (PEG) chains are attached to protein and peptide drugs is a common practice in the development of biopharmaceuticals to prolong serum half-life and improve pharmacokinetics of a drug. There is increasing demand for chromatographic methods to separate the modified isoforms from the native protein. This application note describes the use of size exclusion and ion exchange chromatography for the characterization of PEGylated lysozyme.

Introduction

PEGylation of proteins is a well established method to decrease in vivo clearance rate and reduce toxicity and immunogenicity of therapeutic proteins.¹ While the addition of PEG groups to a protein or peptide improves its properties as a therapeutic, the addition also complicates both the separation and purification of such PEG/protein conjugates from the "non-PEGylated" protein species.^2,3

The polymeric modification changes the biochemical and physical properties of the protein and will, therefore, influence the behaviour during chromatographic analysis and purification. To facilitate method development for both, analytical and preparative applications, it is important to know the influence of a covalent PEG modification on the performance of a protein on HPLC columns and chromatographic bulk media.

Size exclusion (SEC) and ion exchange chromatography (IEC) are common methods for the separation of proteins. Both modes were used to characterize the PEGylation of hen white egg lysozyme, which was used here as a model protein. The amino acid structure of lysozyme shows six lysine residues, which can serve as PEGylation reaction sides (Figure 1).

PEGylation and Chromatographic Analysis

PEGylated lysozyme was produced out of methoxy-PEG-aldehyde (with a molecular weight of 5 kDa, 10 kDa and 30 kDa) and chicken white egg lysozyme in a phosphate buffer (0.1 M, pH 6.0) in the presence of sodium-cyano-borohydrid (NaCNBH3) as a reducing agent.⁴ The product mixture was analysed by SEC on a TSKgel G3000SWXL HPLC column and by IEC on TSKgel SP-5PW (20). Fractions were analysed subsequently by MALDI-TOF-MS.

Chromatographic Conditions:

SEC-HPLC:

Column: TSKgel G3000SWXL (7.8 mm i.d. × 30 cm L, 5 µm, 250 Å)

HPLC-system: Shimadzu LC-20A Prominence

Flow-rate: 1 mL/min

Mobile phase: 0.1 M phosphate buffer; 0.1 M Na2SO4, pH 6.7

Detection: PDA @ 280 nm

Injection volume: 20 µL

IEC-FPLC:

Column: TSKgel SP-5PW (20) (6.6 mm i.d. × 22 cm L, 20 µm, 1000 Å)

Flow-rate: 0.85 mL/min

Mobile phase: Buffer A: 25 mM phosphate buffer; 0.1 M Na2SO4, pH 6.0 Buffer B: A + 0.5 M NaCl

Detection: UV @ 280 nm

Injection volume: 100 µL

Results

The degree of PEGylation can be easily monitored by analysing the increase in molecular weight by size exclusion analysis, as every covalently linked PEG group adds 10 kDa to the molecular weight of the native protein. TSK-GEL SWXL HPLC columns are renowned for their high efficiency in protein separations by aqueous SEC.⁵ (1) DiPEG-, (2) MonoPEG- and (3) native Lysozyme species could be resolved by SEC-HPLC analysis on a TSKgel G3000SWXL column (Figure 2).

Figure 3 shows the result of the cation exchange analysis of the final product of the PEGylation process. The isomers of monoPEG lysozyme and diPEG lysozyme can be further separated by ion chromatography. Depending of the affected PEGylation site (Figure 1) the voluminous PEG group shields different areas of the protein. Resulting differences in surface charge distributions are then leading to partial separation of the isoforms in IEC. Fractions 1(a) to 5 were submitted to subsequent MALDI-TOF MS analysis, which confirmed the assignment of the IEC peaks to di-, mono-, and non-PEGylated lysozyme species.

Conclusion

Size exclusion HPLC with TSK-GEL SWXL columns is a fast and efficient tool to monitor time, concentration and temperature depending synthesis of PEGylated lysozyme species. The SEC chromatograms show different elution volumes for monoPEG, diPEG and native lysozyme respectively. PEGylation with other molecular weight polyethylene glycols (e.g., 5 kDa or 30 kDa) could be monitored as well by using the same method parameters (data not shown). The cation exchange resin TSKgel SP-5PW(20) was successfully applied to separate various isoforms of mono-PEGylated and of di-PEGylated lysozyme. The degree of PEGylation in the collected fractions was confirmed by MALDI-TOF-MS analysis.

Acknowledgements

The authors thank Dr H. Lange and K. Darsow, Institute of Bioprocess engineering, University of Erlangen-Nuremberg, for MALDI-MS analysis.

References

1. J.M. Harris and R.B. Chess, Nature Reviews, 2, 214–221 (2003).

2. S.P. Monkarsh et al., Anal.Biochem., 247(2), 434–440 (1997).

3. S. Yamamoto et al., J. Biotechnol., 132(2),196–201 (2007).

4. A.S. Morar et al., BioPharm International, 19(4), 34–49 (2006).

5. B. S. Kendrick et al., Analytical Biochemistry, 299(2), 136–146 (2001).

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