41


digest providing very narrow peak widths, excellent sensitivity and unique selectivity. Additionally, the optimised chromatographic conditions including use of formic acid ion-pair enabled 100% sequence coverage (Figure 6) and identification of 43 unique glycopeptides (Figure 7) [9]. This 34-minute LC/MS method resulted in 100% sequence coverage of the rhEPO protein.


Evaluating Column Ruggedness


The mobile phases routinely used for reversed phase analysis of proteins are acidic (low pH), containing either trifluroacetic acid or formic acid. A densely bonded phase will deliver exceptional peak shapes and reproducible performance under these conditions. Figure 8 shows chromatographic overlays of 200 injections, with consistent column performance using low pH conditions using such a column (Agilent AdvanceBio Peptide Mapping).


Conclusions


Superficially porous columns are a useful tool for delivering ultra-fast high-resolution protein and peptide separations. In combination with optimised chromatographic methods, difficult to manage proteins such as IgG1 and EPO can be efficiently resolved during fast run times, with separations achieved at HPLC system pressures.


It has been shown that the selection of the correct column will ensure phase stability during operation at low pH and elevated temperature. The conditions described here are highly compatible for LC/MS analysis and provide a robust solution for achieving repeatable, rapid and robust biopharmaceutical analyses.


Figure 8: Agilent AdvanceBio Peptide Mapping column performance over 200 repeated injections at pH 2.2 at 55°C – continuous HPLC operation for 2.5 days with pressures under 400 bar.


References:


1 F. Gritti, I. Leonardis, J. Abia, G. Guiochon, J Chrom., A 1217 (2010) 3819-3843 2 F. Gritti, Chromatography Today, June 2012 4-11


3 D. Cabooter, A. Fanigliulo, G. Bellazzi, B. Allieri, A. Rottigni, G. Desmet, J. Chrom. A, 1217, (2010) 7074-7081


4 Xiaoli Wang, William E. Barber, Peter W. Carr, J. Chrom. A, 1107 (2006) 139-151


5 M.J. O’Hare, M.W. Capp, E.C. Nice, N.H.C. Cooke, B.G. Archer, High-performance Liquid Chromatography of Proteins and Peptides, 1983, Pages 161-172


6 X. Wang, W. E. Barber, W. J. Long, J. Chrom. A, 1228 (2012) 72-88


7 L.W. Dick Jr., D. Mahon, D. Qiu, K-C. Cheng, J. Chrom. B, 877 ( 2009) 230-236


8. Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N- linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer, Lim, Amareth, et. al., ScienceDirect Analytical Biochemistry, 375 (2008), 163 – 172.


9. High Resolution Glycopeptide Mapping of erythropoietin (EPO) Protein by an Agilent AdvanceBio Peptide Mapping Column, Martosella, et. al , Agilent pub #5991-1813EN, Feb. 2013


10. Characterization of Glycosylation in the Fc Region of Therapeutic Recombinant Monoclonal Antibody, Martosella, et. al., Agilent pub # 5991-2323EN, April, 2013.


A New Approach for Sample Adsorption and Extraction of Volatile and Semi-Volatile Components in Food Analysis


MonoTrap™ is a state-of-the-art tool for the extraction and analysis of volatile and semi-volatile components by MMSE (Monolithic Material Sorption Extraction). MMSE using MonoTrap offers a number of key advantages over alternative methods for adsorption and extraction (eg SBSE, SPME). Conventional media


for sampling generally exhibit low surface area and thin polymer coatings, which may lead to poor recovery and the need for prolonged extraction times. Based on state-of-the-art silica monolith technology, MonoTrap has a large surface area due to the pores within the silica skeleton, whilst the through-pores reduce the resistance to flow. The result is a medium that requires no pre-conditioning, exhibits fast adsorption and extraction and offers complete desorption with ease.


These advantages have demonstrated excellent benefits in food analysis and many other areas. A range of applications has been identified including the analysis of red wine, coffee, tea, mango juice, curry powder, bread, mushroom fragrance, soy sauce, sesame oil, fresh and spoiled cabbage, seaweed, cigarettes, maple syrup and others. Copies of all application notes are available on request from Hichrom Limited at technical@hichrom.co.uk.


Sample adsorption with MonoTrap has been demonstrated to work using headspace sampling, direct contact sampling and passive sampling techniques. Sample extraction can be achieved using either solvent extraction or thermal desorption.


Please contact Hichrom Limited for further information on MonoTrap on Tel: +44 (0) 118 930 3660 or email: technical@hichrom.co.uk


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