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BIOTECHNOLOGY 49


Fig. 2. Separation of Interferon alpha-2a with a 150 x 4.6mm Aeris WIDEPORE C8 column. Conditions: mobile phase A 0.1% TFA in water, mobile phase B = 0.1% TFA in acetonitrile, gradient from A/B (70:30) to A/B (35:65) over 30 minutes, flow rate 1.0 ml/min, detection UV at 214nm.


a shallower gradient. Te Aeris WIDEPORE core-shell media is significantly less hydrophobic than most fully porous media, so proteins will tend to elute at a lower per centage organic. Tus, to improve resolution on existing protein methods, one should look to lower the initial per centage organic and potentially use shallower


gradients when transferring a method to Aeris WIDEPORE core- shell columns.


An additional benefit of the low hydrophobicity of the column is better recovery of hydrophobic proteins. Tis, in combination with a well bonded inert surface and an optimised diffusion path, can lead to


Fig. 3. Analysis of Human Epidermal Growth Factor (EGF) with 150 x 2.1 mm Aeris WIDEPORE 3.6µm C4 column and a fully porous 3.5µm C3 column. Conditions for both columns: mobile phase A 0.1% TFA in water, mobile phase B = 0.1% TFA in acetonitrile, gradient for 3 minutes at A/B (97:3) then over 45 minutes to A/B (35:65), flow rate 0.3 ml/min, detection UV at 214nm.


dramatic differences when compared to fully porous 300 Å columns. An example of this is shown in Fig. 3.


A third example to demonstrate the utility of Aeris WIDEPORE columns for intact biogeneric


protein analysis is shown in Fig. 4. In this example, RNase is reduced with DTT and different mixtures of the reduced and native protein are overlaid in the figure. Analysing and quantitating the folding state of a recombinant protein is primarily done by reversed phase chromatography of the intact protein. Tis example shows how the core-shell column can easily resolve folded and unfolded forms of the RNase protein making it an ideal solution for analysing intact proteins.


For obtaining useful quantitation of post-translational modifications of biogeneric proteins it is critical to maximise resolution between proteins and their modified impurities. Te different applications show the utility of Aeris WIDEPORE columns for obtaining data for intact protein applications.


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Fig. 4. Separation of intact and reduced RNase with a 150 x 4.6mm Aeris WIDEPORE C4 column. Conditions: mobile phase A 0.1% TFA in water, mobile phase B = 0.1% TFA in acetonitrile, gradient for 3 minutes at A/B (97:3) then over 30 minutes to A/B (35:65), flow rate 1.2 ml/min, detection UV at 210nm.


Michael McGinley, Deborah Jarret, Jeff Layne and Dirk Hansen are with Phenomenex Inc, Aschaffenburg, Germany www.phenomenex.com


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