Enhancing Aggregation Analysis by Size Exclusion Chromatography (SEC)– Inclusion of Light Scattering Detection

December 2, 2012
Andrew Coffey

The Application Notebook

The Application Notebook, The Application Notebook-12-02-2012, Volume 0, Issue 0

Agilent Technologies

Aggregation of proteins is a relatively common occurrence, but often poorly misunderstood. It can have dramatic consequences in biotherapeutic molecules, such as antibodies. Formation of dimers, trimers or larger aggregates can lead to reduced bioavailability and reduced shelf life, and to severe immunological response, including anaphylactic shock. Consequently, the need to monitor and quantify aggregation is of paramount importance and size exclusion chromatography (SEC) provides the ideal tool.

There are many factors to take into consideration when developing SEC methods for determining the extent of aggregation. In particular, stress is a key factor in protein aggregation, therefore the SEC experiment itself can potentially affect the results. Parameters such as buffer choice, sample concentration and temperature can all influence the outcome of the experiment.

Conditions

Column: Agilent Bio SEC-5, 500 Å, 7.8 × 300 mm SS

Mobile phase: 300 mM sodium phosphate, pH 7.0

Gradient: Isocratic

Temperature: 30 °C

Injection volume: 50 µL

Sample: Bovine gamma globulin

Sample conc.: 2.0 mg/mL

Detection: UV 280 nm + RI + LS 90°

Instrument: Agilent 1260 Infinity Bio-inert Quaternary LC system, with Agilent 1260 Infinity MultiDetector GPC/SEC system.

Results and Discussion

SEC separates molecules on the basis of size in solution (in the mobile phase), but it is often assumed that the separation mechanism is based on molecular weight. Most proteins are globular in nature and tend to elute in decreasing molecular weight order, but it is also necessary to perform a column calibration with clearly defined standards each time the column is changed, or each time the mobile phase is altered. By including a light scattering detector, this practice can be eliminated since the detector is mass responsive. It is therefore relatively straightforward to identify the absolute molecular weight of an unknown peak, and to ascertain if an aggregate is a dimer, trimer or higher combination.

As shown in Figure 1, the effect of excessive heat can cause an increase in aggregation. This is particularly obvious when comparing the 90° light scattering signal which gives a much larger response to the higher molecular weight components.

Figure 1: Effect of excessive heat on a sample of bovine IgG causing an increase in aggregation.

A factor often overlooked in the method development process is the temperature of the sample. In Table 1, the effect of autosampler temperature on the degree of aggregation was investigated by integrating the peak area of the monomer, dimer and higher aggregate peaks.

It may come as a surprise that although excessive temperature leads to an increase in aggregation, moderate temperatures can lead to a decrease in the aggregation level. In order to avoid the possibility of the analytical method influencing the degree of aggregation, a methodological approach using a light scattering detector can help identify those parameters of most importance.

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