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Temperature

Following digestion, especially with trypsin, the resulting peptides as a rough approximation are 2 orders of magnitude smaller than the intact parent mAb – 1,500 Da compared with 1,500,000 Da. However, they are still relatively large, especially in terms of traditional pharmaceuticals, and for RPLC. Larger molecules suffer from hindered mass transfer kinetics, the ‘C-term’ in the van Deemter equation, and, as such, this can lead to broader peaks.

There are a number of factors that can improve the mass transfer kinetics of larger solutes; such as temperature, pore depth (typically by way of particle size), and linear velocity or flow rate.

Increasing the temperature not only reduces the viscosity of the mobile phase, meaning solutes can diffuse through them more easily and quickly, but also increases the amount of inherent energy of the solute. Solute molecules have to diffuse down through the particle pore, and back out, under their own volition as mobile phase movement through the pores is negligible.

The trastuzumab peptide mapping chromatograms clearly show a slight change in selectivity, but the main change as temperature is increased is an improvement in chromatographic efficiency (Figure 1). This will in turn produce a positive impact on resolution and sensitivity. Typically, the highest temperature where both the solutes and the stationary phase/particles are stable should be chosen.

This is approximately 60 °C, much above this can lead to hydrolysis of the stationary phase, dissolution of the silica support and/or thermal degradation of the solutes. With run times in excess of 30 minutes, later eluting solutes will be exposed to these temperatures for a prolonged time period.

Trastuzumab peptide maps at differing temperatures

Figure 1: Trastuzumab peptide maps at different temperatures.

 
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