A toolbox of amino acids for mAb separations
Size exclusion chromatography (SEC) is well established for mAb aggregate analysis. As the technique has been used since the early days of monoclonal antibody (mAb) development for pharmaceutical purposes, various method improvements have evolved. For instance, the benefits of arginine on analytical SEC of mAb aggregate samples are well-known. A new application note shows how SEC of mAb aggregate samples may take advantage of other amino acid additives in the mobile phase.
Recently, various approaches to improve analytical SEC have focused on reducing the analysis time. This can be achieved by staggered injection protocols or increased linear flow rates. The mobile phase composition itself leaves less room for method improvement, compared to other chromatographic modes. As soon as a certain ionic strength (inhibiting electrostatic interactions without causing hydrophobic interactions) and the pH of the mobile phase (ensuring structural integrity of proteins and the stationary phase) are set, one might think that the analysis solely depends on the particle size, packing quality and column length. However, the mobile phase composition is not at the end of its rope, in case the mentioned parameters have been set. For example, arginine and other amino acids can be added to the mobile phase to affect the aggregate recovery in SEC.
A mAb was aggregated by incubation at 75 °C for 5 min. Subsequently, the sample was analyzed via TSKgel UltraSW Aggregate 7.8 mm ID x 30 cm L with different mobile phases, all of them using virgin columns. 0.2 M lysine, arginine, proline, glutamine or sodium sulfate were added to 0.1 M sodium phosphate buffer, pH 6.7, respectively. A flow rate of 1 mL/min was applied, and 20 µl (100 µg) of the aggregated mAb samples were injected onto equilibrated columns. Figure 1 illustrates the results on aggregate recovery. Glutamine and proline show a similar behavior: the aggregates are hardly recovered for the first two injections, while the aggregate peak suddenly appears for injection #3 and #4. The rise is not as sudden for sodium sulfate, but the aggregate peak will only achieve its full size for injection #10. In opposition to these results, lysine shows an even improved aggregate recovery compared to arginine. The inter-injection variability is low, depicting the complete aggregate content for all of the injections.
