Size exclusion chromatography (SEC) is a technique often used to resolve large molecules based off their hydrodynamic radius (or size). This technique is widely used for the analysis of biopharmaceuticals and proteins. In this application note, SEC was demonstrated on the capillary scale using the Axcend Focus LC®, a compact capillary-scale HPLC. Using a flow rate of 2.7 µl/min, the system was able to resolve a monoclonal antibody from its high molecular weight aggregate, as well as perform a multi-run repeatability study on a protein ladder. During the repeatability study, all peaks had <1% RSD for retention times, and ≤3.09% RSD for peak areas. These experiments show the viability of SEC as a separation mode on the Axcend Focus LC. The reduced flow rates required for capillary-scale liquid chromatography also make this a more cost effective and environmentally friendly alternative to existing workflows.
Size exclusion chromatography is a technique used for the separation of compounds based on their hydrodynamic radius (or size). SEC is commonly used for the determination of protein mass, aggregation, and fragmentation at nondenaturing conditions. In this application, capillary-scale SEC was used to separate high molecular weight aggregates from the monoclonal antibody (mAb) monomer. Additionally, a protein ladder was analyzed to determine repeatability across a wide mass range demonstrating the robustness of the technique on the Axcend Focus LC. Due to the reductions in solvent and sample consumption associated with capillary scale, this method offers cost effective and environmentally friendly alternatives to existing analytical scale workflows.
For analysis of mAb aggregation, a mAb standard (reference material 8671) was acquired from the National Institute of Standards and Technology (NIST). This sample is sold at a concentration of 10 mg/mL in 12.5 mM L-histidine, 12.5 mM L-histidine HCl pH 6.0 buffer1.
A Waters BEH200 SEC protein standard mixture was used for a protein ladder. This is a 5-component mixture of Thyroglobulin (3 mg/mL 660kDa), IgG (2 mg/mL 150 kDa), BSA (5 mg/mL 66.4 kDa), Myoglobin (2 mg/mL 17 kDa), and Uracil (0.1 mg/mL 112 Da). The sample was prepared in 100 mM Sodium phosphate buffer pH 6.8 as per the manual2. This sample was made fresh the day of analysis to avoid potential degradation.
| Column | YMC-SEC MAB 0.5 x 150 mm 3 µm 250 Å particles |
| Mobile phase | 100 mM Sodium Phosphate with 200 mM NaCl pH 6.8 |
| Flow rate | 2.7 µL/min |
| Injection volume | 40 nL |
| Temperature | Ambient |
| Detection wavelength | 275 nm |
| Method | Isocratic for 14 minutes |
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