Sophisticated antibody analysis by GPC/SEC

Monoclonal antibodies (mAB) are increasingly growing in importance for the diagnosis and therapy of various diseases, including cancer as well as autoimmune and inflammatory disorders. One essential parameter to define their quality is the content of aggregates (dimers, trimers and higher aggregates). These aggregates can be formed during processing and purification or are the result of long-term storage. Due to the aggregation antibodies lose their pharmaceutical efficacy and can facilitate an immune response.

Antibody fragments, which lack the Fc region, can be used for the treatment of diseases. Also, they can be the result of degradation of full length antibodies. Therefore, a GPC method that offers the opportunity to analyse antibodies and their aggregates as well as antibody fragments simultaneously with superior resolution is worthwhile, in addition to offering highly sensitive detection.GPC/SEC analysis was performed on a PSS SECcurity GPC System, equipped with a PSS SECcurity SLD1000 light scattering detector, using the following conditions:Figure 1 shows an overlay of elugrams obtained for a full length antibody and antibody fragments analysed on one set of columns.

Fig 1. Separation range of the column combination. Red curve shows the UV signal of a full length antibody and its dimers plotted against the elution volume. The blue curve is the elugram of antibody fragments and their high level aggregates.

All three detector signals for the analysis of a monoclonal antibody are shown in Figure 2. The light scattering signal shows improved sensitivity for high aggregates compared to the other signals.

Fig 2. Sensitive analysis of antibody aggregates. The light scattering signal for the dimer is relatively high compared to that of the mABs due to molar mass dependency and provides improved sensitivity for the detection of high aggregates (inset).

The GPC/SEC method including UV, RI and RALS can be used for the simultaneous determination of aggregate content of monoclonal antibodies and antibody fragments. The column combination used covers the separation range for all three types but nonetheless provides a high resolution for the determination of the dimer content. Due to its molecular weight dependency, the PSS SLD1000 RALS detector offers high sensitivity for small quantities of high aggregates and also allows the determination of the absolute molecular weight of the antibodies.

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