Analysis of PEGylated Protein by Tetra Detection Size Exclusion Chromatography

Reliably detecting low amounts of high molecular weight impurities during process development and characterization of biopharmaceutical products.
Aug 02, 2011

Size exclusion chromatography (SEC) provides critical information about the purity and oligomeric state of protein therapeutic products. SEC analysis is important for process development, formulation optimization, release testing, stability, comparability, and forced degradation studies SEC coupled with light scattering (LS), ultra-violent (UV), and refractive index (RI) detection offers detailed molecular weight information about proteins, protein complexes, and glycosylated proteins (1–3). The current study uses a tetra detection system (laser light scattering, viscometer, RI, UV) coupled with software that integrates data from all detectors to analyze a model PEGylated protein. The detection system provides accurate molecular weight, intrinsic viscosity, and hydrodynamic volume with minimal input of known parameters. High molecular weight (HMW) impurities observed in different stress and process samples were characterized by this method. This study examines the major and minor species present in samples of a model PEGylated protein encountered during biopharmaceutical development.


A model PEGylated protein was prepared for use in these studies. The protein of approximately 19 kDa was expressed in E. coli, purified, and then modified with methoxypolyethylene glycol aldehyde (mPEG) with a molecular weight of approximately 20 kDa. Samples representing process intermediates, PEGylation with different lots of mPEG, and force-degraded material were analyzed.

The Viscotek TDAmax system (Malvern Instruments) was used for the SEC analysis of the protein conjugates. This tetra detection system (TDA305) includes photodiode array UV, differential RI, right angle light scattering (RALS), low angle static light scattering (LALS) and four-capillary differential viscometer detectors. The system also includes a GPCmax VE 2001 liquid chromatography autosampler with an isocratic pump and degasser equipped with a Superdex 200 column (10 × 300 mm, GE Healthcare). The detectors were in the following order: UV, LS, RI, and viscometer. OmniSEC 4.7 software (Malvern Instruments) was used for the acquisition and analysis of SEC data.

Determination of the detector offsets, asymmetric band broadening correction, and detector constants was completed using bovine serum albumin (BSA) monomer peak in a mobile phase of PBS (150 mM phosphate and 100 mM NaCl) at 0.5 mL/min flow rate. 0.185 (1) and 0.66 (2) were used as (dn/dC) value and extinction coefficient (dA/dC or ε1% at 280 nm) of BSA, respectively. The same process was followed with the unmodified protein for comparative analysis. The dn/dC was set at 0.185 (experimentally confirmed) and the extinction coefficient (dA/dC or ε1% at 280 nm) was experimentally determined. An isocratic elution profile was used at a flow rate of 0.5 mL/min. Buffer solutions were vacuum filtered through a 0.2 µm nylon filter and degassed before use. Samples were run under the same chromatographic conditions. Injection volumes were adjusted to accommodate samples with lower protein concentrations (injection volume range = 20–100 µL).