SEC-HPLC Protocol
Principle
Size Exclusion Chromatography (SEC or SEC-HPLC), also known as molecular sieve chromatography, is a technique of purity analysis and macromolecular separation based on molecule size. SEC is a major mode of HPLC that employs porous particles in the column. It is usually applied to determine molecular weight distributions of proteins or separate proteins and polymers used in a wide range of industries.
SEC works by trapping smaller molecules in adsorbent pores ("stationary phase"). This procedure is often carried out within a column, which is made up of a hollow tube firmly packed with micron-scale polymer beads with varying pore diameters. Large molecules aggregates are blocked by most pores and therefore flow rapidly through the SEC column. Smaller molecules take longer to diffuse within the particle pores of the column stationary phase and take longer to elute.
Fig. 1 illustrates the theory behind size exclusion chromatography. Distributed under CC BY 2.5, from Wiki, without modification.
Materials
Reagents
- Mobile phase: 150 mM phosphate buffered saline, pH 7.0
- Sodium chloride, hydrochloric acid (HCl), and sodium hydroxide (NaOH)
- Nylon membrane
Instruments and Consumables
- A completely biocompatible Agilent 1260 Infinity Bio-inert Quaternary LC System
Methods
Making the Mobile Phase
1. Dissolve Na2HPO4.12H2O and NaH2PO4.2H2O in ultrapure water to make 1000 mL. The resulting solution should have a pH between 6.8 to 7.0.
2. Filter the mobile phase through a 0.22 µm Nylon membrane filter under vacuum to degas the solution and to remove solids that could plug the chromatographic column.
3. Degas the mobile phase via ultrasonication to avoid bubbles, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance.
Balance AdvanceBio SEC Column
4. Put a line in mobile phase solution and unscrew "Purge" valve, set flow rate to 5 mL/min for 3-5 min.
5. Adjusted flow rate to 0.5 mL/min and install chromatographic column, balance for 30-60 min until the baseline is stable.
Preparing Sample
6. Adjusted concentration of sample to 1 mg/mL.
7. Filter the sample through a 0.22 µm Nylon membrane filter.
8. Place a volume (~20 µl) of protein solution into sample vials and put it on the sample platform of Agilent 1200 Series.
9. Set up system parameters: Wash Location, Inject Location, Inject Volume, Sample Name, Column temperature and Wavelength.
10. Run the sequence.
11. Perform data analysis.
Application
- Protein Aggregation Analysis
SEC-HPLC is an essential tool in the characterization and quantification of protein aggregation, including soluble aggregates, partially folded intermediates, and insoluble aggregates. By applying molecules of different sizes to the SEC-HPLC column, SEC-HPLC separates the aggregates from the monomer.
- Purity and Characterization of Monoclonal Antibodies
SEC-HPLC is commonly used to assess the purity of monoclonal antibodies (mAbs) produced in large-scale manufacturing processes. It can also characterize mAb fragments such as Fab and Fc fragments. By comparing the protein profile of a sample with that of a reference standard, researchers can ensure the quality and consistency of mAbs.
- Determination of Molecular Weight and Size Distribution
SEC-HPLC allows the determination of the molecular weight and size distribution of biomolecules accurately which is critical to assessing the quality and activity of biomolecules. By calibrating the HPLC system with molecular weight standards, researchers can determine the molecular weight and size of target molecules exquisitely.
- Analysis of Polysaccharides and Other Macromolecules
SEC-HPLC is a powerful tool for separating, characterizing, and quantifying polymers, such as polysaccharides. SEC-HPLC can be used for the determination of molecular weight, size distribution, degree of polymerization, and other important properties, providing insights into the physical and chemical properties of these macromolecules.
- Protein-Protein Interaction Analysis
SEC-HPLC coupled with multi-angle light scattering (MALS) enables the analysis of protein-protein interactions. By measuring the change in the hydrodynamic radius of the complex, researchers can determine the stoichiometry of the complex and its binding affinity.
- Quality Control in Biopharmaceutical Manufacturing
SEC-HPLC is a powerful tool for monitoring the quality of biopharmaceutical products. It allows the characterization and quantification of impurities, including aggregates, by-products, and degradation products, ensuring the safety and efficacy of biopharmaceuticals.
