48 BIOTECHNOLOGY
Improved reversed phase analysis of intact proteins
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.
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n 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. Tis 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.
Te analysis of intact recombinant proteins allows to quantitate the purity of the protein and allows to potentially identify any specific impurities in the sample. Te 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. Te following examples show how these material characteristics deliver significant benefits for the analysis of intact proteins. Te 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. Tis 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
Fig. 1. Separation of degraded Myoglobin with a 150 x 4.6 mm Aeris WIDEPORE XB-C18 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 1.5ml/min, detection UV at 210nm.
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