Using MALS-UV-RI Detection for Accurate Characterization of PEGylated Proteins

PEGylation involves covalent attachment of synthetic polyethylene glycol (PEG) chains to another molecule, usually a protein, peptide, or nucleotide. For a small therapeutic protein, PEGylation can offer significant advantages, including reduced renal clearance, improved immunogenicity, and enhanced solubility. It is not surprising that for over 30 years, PEGylation has been established as one of the most fundamental techniques in drug formulation.

Figure 1 - Molecular modeling of a protein covalently bonded to a branched PEG, courtesy of Dr. A. Ghose, Amgen (Thousand Oaks, CA). Both protein and PEG molecules have a similar MW (18.2 and 18.0 kDa, respectively), but their sizes are significantly different due to the conformational difference of each component.

However, PEG–protein conjugates exhibit considerable heterogeneity with regard to their molecular weight, composition, and structure, and thus are difficult to characterize using conventional analytical techniques. Complexity is further increased due to potential aggregation and degradation during PEGylation as well as incompete conjunction. As a result, there is a need for advanced analytical methodologies that can overcome these challenges.

Size exclusion chromatography

Size exclusion chromatography (SEC) has traditionally been used for the characterization of PEGylated proteins. In an ideal SEC run where column interaction is absent, the separation of molecules is achieved based on their hydrodynamic volume. In order to gain molecular weight information on samples from the SEC run, known molecular weight standards must be used to calibrate the column(s). In addition, an assumption is made that the sample of interest and the reference standards have the same conformation and elution. This assumption is not valid for PEG–protein conjugates, which have their own unique conformation that differs, sometimes substantially, from that of the protein and the PEG (Figure 1). As a consequence, a conventional SEC method with column calibration generates erroneous molecular weight results for PEG–protein conjugates.

Technological innovations

Recent technological advancements have allowed the coupling of SEC systems with multiangle light scattering (MALS) detectors to form a powerful platform capable of determining the absolute molecular weights of SEC fractionated species—all without regard to column calibration or reference standards. First, the method eliminates assumptions and the use of reference standards,1 and second, it measures the concentration of the polymer solution using either an on-line UV absorption detector or a differential refractive index (dRI) detector. UV detectors can detect only the moiety of proteins, whereas dRI detectors can detect the moieties of both proteins and PEGs. Using dRI and UV detectors in conjunction with a MALS instrument, it is possible to identify the stoichiometry of PEG–protein conjugates.

The absolute molecular weights of the conjugate, protein, and PEG can be determined subsequently using the stoichiometry and concentration data. To perform the analysis, it is necessary to know the specific refractive index increment (dn/dc) at the wavelength of the MALS detector, and the extinction coefficient at the UV detection wavelength for both the PEG and the protein.

Experimental

Figure 2 - Chromatograms of MALS (red), UV (green), and dRI (blue) detectors obtained from SEC of a PEGylated protein.

DAWN HELEOS MALS detector with an embedded dynamic light scattering detector, an Optilab rEX dRI detector (both from Wyatt Technology Corp., Santa Barbara, CA), and an Agilent 1200 UV detector (Agilent Technologies, Santa Clara, CA) were used to generate SEC chromatograms for a PEGylated therapeutic protein (Figure 2). A WTC-050S5 7.8 mm × 300 mm SEC column (Wyatt Technology Corp.) packed with 5-μm coated silica beads with a nominal pore size of 500 Å was used. The mobile phase was 10 mM succinate containing 3.5% (w/v) sorbitol and 0.3 M L-arginine at pH 5.5.

Figure 3 - Molecular weights from both PEG and protein measured by the MALS-UV-RI method are plotted against elution volume with UV trace overlaid.

The system was controlled by the ASTRA light scattering software platform (Wyatt Technology Corp.) , which has a protein conjugate template to determine the conjugate molecular weights of the peak that eluted from 9.5 to 10.4 min. The conjugate molecular weight was found to be homogeneous and approximately 60 kDa (Figure 3), while the conjugate consisted of PEG polymer chains at a total molecular weight of 40 kDa and a polypeptide chain at a total molecular weight of 20-kDa polypeptide chain. The peak that eluted from 8.5 to 9.1 min had an overall molecular weight of 120 kDa with the respective contribution of 40 kDa and 80 kDa from protein and PEG. Based on the measured molecular weights and the degree of PEGylation, it was concluded that the early-eluting peak is the dimer of the later-eluting monomeric PEGylated protein.

Hydrodynamic radius

Attaching PEG polymer chains to a therapeutic protein is of great benefit because it increases the size of the protein, subsequently decreasing its renal clearance rate. Therefore, it is important to perform accurate measurements of the hydrodynamic radius of the protein both before and after PEGylation. By coupling on-line dynamic light scattering (DLS) to MALS, simultaneous collection of static and dynamic light scattering data is achieved, enabling the determination of the absolute molecular weight as well as hydrodynamic radius.

Figure 4 - Hydrodynamic radii of three samples measured by on-line DLS overlaid with their dRI traces: protein without PEGylation (red), PEG (green), and PEGylated protein (blue).

In the second part of this experiment, MALS was used in conjunction with the embedded DLS detector to measure the hydrodynamic radii of a therapeutic protein, PEG, and the PEGylated protein that was investigated previously. The overlay of the dRI traces of these three samples obtained using SEC is shown in Figure 4. The hydrodynamic radii of the samples were plotted across the peaks. The hydrodynamic radii for the protein before PEGylation, and PEG and protein after PEGylation were 2.0, 3.7, and 4.7 nm, respectively, confirming the significant increase of the size of the therapeutic protein after PEGylation.

Field flow fractionation

Field flow fractionation (FFF) can be used as an alternative separation technique for PEG–protein conjugates, instead of SEC. This orthogonal fractionation technique offers many key advantages, including the capability to separate a broader range of conjugates while also reducing the risk of removing high-molecular-weight aggregates of the protein samples due to shearing or adsorption onto a column. Just like SEC, FFF can be effectively used in conjunction with MALS, DLS, UV, and dRI detectors.

Conclusion

PEG–protein conjugates play a vital role in drug formulation and delivery, but their successful characterization has proven to be a challenging task because of their inherent heterogeneity. The combination of MALS, UV, and dRI detection—following separation of the conjugates by either SEC or FFF—offers a viable solution for uncomplicated, rapid, and precise characterization of PEG-protein conjugates. The method can be complemented by on-line DLS, generating reliable measurements of the hydrodynamic radius of conjugates.

Reference

  1. Wyatt, P.J. Light Scattering and the Solution Properties of Macromolecules. In Handbook of Size Exclusion Chromatography and Related Techniques; Marcel Dekker: New York, NY, 2004; Chapter 21; pp 623–55.

Dr. Chen is Director of Analytical Services, Wyatt Technology Corp., 6300 Hollister Ave., Santa Barbara, CA 93117-3253, U.S.A.; tel.: 805-681-9009; fax: 805-681-0123; e-mail: mchen@wyatt.com.

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