A New Silica-Based Aqueous SEC Column for Analysis of Antibody Proteins

February 1, 2017
Showa Denko America, Inc.,

The Application Notebook

Volume 35, Issue 2



Antibodies are Y-shaped proteins predominantly produced by plasma cells and used by the immune system to neutralize pathogens. Antibodies glycoproteins part of the immunoglobulin superfamily, constituting most of the gamma globulin fraction of the blood proteins. Although the general structure of all antibodies are very similar, a small region at the tip of the protein drastically varies, allowing millions of antibodies with slightly different tip structures to exist.

The study of antibodies began in the late 1800s and has been continuing through today. Recently, there has been an increase of antibody research, causing a growing need for better antibody analysis. HPLC and Size Exclusion Chromatography (SEC) are ideal for determining molecular weight and size of the antibodies.

Shodex introduces the LW-803 column with a durable silica based packing material for antibody SEC analysis. This column has an adopted pore size suitable for analyzing proteins with a molecular weight of several hundreds of thousands.It also corresponds to the USP L20, L33, and L59. Shodex LW-803 is appropriate for proteins and specifically antibody analysis, and has demonstrated the determination of molecular weight for seven proteins.

Experimental Conditions

The analysis of seven proteins (Thyroglobulin, ?-Globulin, BSA, Ovalbumin, Myoglobin, Ribonuclease A, and Aprotinin) was accomplished with Shodex PROTEIN LW-803 (8.0 mm ID ? 300 mm, 3 µm), a silica-based column for aqueous SEC antibody analysis. Column temperature was kept at room temperature and flow rate was 1.0 mL/min. Eluent conditions were 50 mM sodium phosphate buffer (pH 7.0) with 0.3 M NaCl. The HPLC system was coupled with a UV detector set to 280 nm.

Results

Seven proteins with molecular weights ranging from 6,500 to 670,000 Da were analyzed on the PROTEIN LW-803 (Figure 1). From the seven proteins, a calibration curve could be formed. From this curve, unknown antibody proteins can be analyzed to determine the molecular weight.

Conclusions

Shodex PROTEIN LW-803, a Size Exclusion Chromatography (SEC) chromatography column is suitable for antibody protein molecular weight analysis.

Column: Shodex PROTEIN LW-803 (8.0 mm I.D. ? 300 mm)
Eluent: 50 mM Sodium phosphate buffer (pH 7.0) + 0.3 M NaCl
Flow rate: 1.0 mL/min
Detector: UV (280 nm)
Column temp.: Room temperature

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