Improved reversed phase analysis of intact proteins

4th March 2013

Michael McGinley, Deborah Jarret, Jeff Layne and Dirk Hansen demostrate how new core-shell particles with their engineered particle design help improve the analysis of very complex and demanding bioseparations.

In the frame of the development of modern therapeutics more and more large biomolecules like oligonucleotides or proteins are in the focus of the researchers.In addition the first generation of biopharmaceuticals like EPO is off patent and an increasing number of companies are in the process of developing biosimilars. Both trends have increased the demand for reliable and robust bioanalytical separations.

Kinetex core-shell columns have introduced ultra-high performance by decoupling column efficiency from high backpressures. This results in small molecule applications with reduced run times and increased throughput without the need for new UHPLC instrumentation.

For protein separations speed is in most cases not the most critical factor. Here, very often the analyst has to separate impurities from the main component with very similar chromatographic behaviour. For this reason the new Aeris core-shell particle was designed to address modern protein analysis.

The analysis of intact recombinant proteins allows to quantitate the purity of the protein and allows to potentially identify any specific impurities in the sample. The typical impurity for most purified proteins is a post-translationally modified version of the protein or an improperly folded species of the protein. Such post-translational-modification (PTM) impurities are in most cases chemically similar to the intact therapeutic protein, and thus present a challenge in achieving chromatographic separation by HPLC or UHPLC between multiple species. Separation and quantitation of PTM proteins can be especially difficult for larger proteins since chemical differences induced by a single modification have a smaller net effect on the retention of large biomolecules compared to peptides or small molecules.

Aeris WIDEPORE utilises a particle morphology designed to reduce peak broadening resulting from slow protein diffusion in and out of the porous layer of the column. In addition, by using a large (3.2µm) silica core, the resultant particle is 3.6µm in diameter which allows for longer columns at lower backpressures. The following examples show how these material characteristics deliver significant benefits for the analysis of intact proteins. The first example is shown in Fig. 1, where degraded myoglobin is analysed with an Aeris WIDEPORE 3.6µm column. Note the large number of resolved impurities for the core-shell column. This good separation of the impurities allows a better and more robust quantitation.

A second example of improved resolution of an intact interferon sample with the Aeris WIDEPORE column is shown in Fig. 2. Also in this case the better resolution allows for a more accurate quantitation of impurities in the intact protein sample. While resolution is not complete between the interferon peak and modified components, one can see the additional component resolved by the column leading to more accurate quantitation of the impurities present. For an even better resolution of the components one could use a longer column and a shallower gradient. The 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. Thus, 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. This, in combination with a well bonded inert surface and an optimised diffusion path, can lead to 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. This 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. The different applications show the utility of Aeris WIDEPORE columns for obtaining data for intact protein applications.

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





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