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European Biopharmaceutical Review

Analytical Methods: UHPLC

The increased complexity of protein therapeutic drugs, as well as ever-increasing regulatory requirements, has demanded improved analytical tools for protein identification. Advances in HPLC technology have brought improved speed, sensitivity and quantification of protein and peptide therapeutics.

The physical analysis of biopharmaceutical drugs over the last decade has seen dramatic changes due to advances in analytical technologies, as well as improved methodologies for analysing protein therapeutics. While the 1990s saw advances in sensitivity and throughput due to innovations in detection technologies such as mass spectrometry, the last decade has been more influenced by advances upstream of the mass spectrometer with significant improvement in both HPLC instrumentation as well as column technology. New HPLC instrumentation features very low system volumes and high performance specifications. Such improvements minimise the loss of chromatographic performance due to instrumentation limitations, with extra column system volume standing out as the most negative influence on separations. Higher performing HPLC systems (often called UHPLC) maximise the performance of next-generation UHPLC columns, which demonstrate improved resolution and higher peak capacity versus older 1980s-era protein columns (1,2).

For protein therapeutics this new paradigm of reversed phase chromatography has been especially helpful. Second generation protein therapeutics are typically larger than the first generation, generating more complex separations, especially for peptide map applications where large proteins can generate hundreds of peptide peaks demanding separation. Modern protein therapeutics are often Immunoglobulin G based (IgG), an extraordinarily complex protein with multiple types of post translational modifications (PTMs) (deamidation, glycosylation, glycation, and so on) that require maximum chromatographic separation to quantify any minor species present (3). Additional analytical challenges for modern protein therapeutics include PEGylation, where large poly-disperse polyethylene glycol is added to a protein or peptide to increase in vivo half-life and improve bioavailability. While such modifications improve the drug ability of a protein, from an analytical characterisation point of view such modification makes characterisation extremely difficult; this is right at a time where regulatory agencies require increasing amounts of information on protein therapeutics.

Adaptation of UHPLC column technologies to protein and peptides comes at an opportune time to address the increasing analytical requirements for protein therapeutics. With the development of small, fully-porous type-B (low metal) silica particle columns, as well as improved instrumentation, increased resolution and throughput has been realised for reversed phase separation of small molecules and peptides. A further development in the last three years has been the introduction of geometrically structured silica particle columns (also known as core-shell, superficially porous, or Poroshell) that have provided equal or better chromatographic performance for peptide and peptide mapping applications without pressure and instrumentation limitations of small particle, fully-porous media (see Figure 1) (4). Most recently we have seen the introduction of peptide applicationspecific and protein application-specific core-shell media specifically designed for the difficulties in analysing proteins and peptides. Surprisingly, separation principles and operating parameters for separating proteins and peptides are significantly different despite both being polymers of amino acids (proteins being considered greater than 10KDa molecular weight versus peptides which are less than 10KDa) (5). Key to successful method development for these separations is understanding the requirements for each application.

Peptide Mapping and Peptide Separations

The primary application that analytical biochemists perform to characterise proteins is called tryptic, enzymatic or peptide mapping, where a large protein is digested with a proteolytic enzyme into smaller peptide fragments. This mixture of peptides is chromatographically separated by a reversed phase HPLC column and individual peptides are positively identified by tandem mass spectrometry. Peptide mapping allows for identification and quantification of very low level impurities and post-translational modifications present in a biotherapeutics sample, provided that the peptide can be chromatographically separated from other peptides in the mixture. For firstgeneration protein therapeutics, which were typically smaller in size and less complex, separating the small number of peptide fragments generated in a peptide map was feasible with older column technologies. However, large protein therapeutics like IgG monoclonal antibodies can generate hundreds of peptide fragments, making resolution of PTM indicating peptides difficult using low-resolving HPLC methods.

Recent UHPLC column technologies, such as sub-2μm porous media and core-shell media columns, provide substantial benefit for analysing complex peptide maps. An example of the improvement that new UHPLC columns can provide for peptide mapping applications is shown in Figure 2, where significant increases in peak capacity and separation are realised by using a core-shell peptide mapping column versus older fully porous 3μm or 5μm columns. Key reasons for performance improvement relates to understanding the behaviour of peptides and how column parameters influence separations. Peptide retention of less than 10KDa is primarily influenced by the chemical composition of the amino acids that make up the peptide chain (charged, polar or non-polar). Many peptides are moderately polar with only minor differences in hydrophobicity, thus a fairly hydrophobic stationary phase with high methylene selectivity is necessary to separate chemically similar peptides. High efficiency media is required to maximise resolution of any chemical separation the phase achieves. UHPLC column technology (sub-2μm and coreshell) decreases the diffusion distance of peptides in-and-out of the porous media, resulting in sharper peaks, which increases resolution, peak capacity and sensitivity of any peptide mapping method.

Intact Protein Analysis by UHPLC

While one might assume that protein separations are similar to peptides because of their similar amino acid composition, separation rules are actually substantially different. While peptides are partitioned based on their amino acid sequence, protein selectivity is significantly influenced by the three-dimensional structure of a protein. As proteins get larger, size and shape play a significant role in the retention of a protein. Large proteins diffuse much more slowly than peptides; to maximise protein separation performance, a much shorter diffusion path is required. For fully porous silica media this requires very small particles (sub 2μm) and for core shell media this requires a much thinner porous shell than peptidespecific columns. In addition, mean pore size for protein columns must be larger (approximately 250Ĺ plus) to allow accessibility to the larger protein and out of the porous layer. The lower surface area of such media results in a less hydrophobic stationary phase, which is beneficial for intact protein separations where recovery is always a major concern. An example of the improved performance that a next generation UHPLC core shell column can demonstrate is shown in Figure 3, where an IgG monoclonal antibody was loaded on either a fully porous 5μm 300Ĺ C18 column or a core shell 3.6μm C18 column. Note the greater efficiency, recovery, and resolution for the core shell column.

Intact protein analysis of large proteins can provide useful information about a protein therapeutic and is complementary to peptide mapping applications in understanding the purity and chemical stability of a protein therapeutic. While peptide mapping is most useful for determining site-specific modifications at a certain amino acid, peptide mapping struggles to provide information about the global threedimensional structure. Intact UHPLC analysis, on the other hand, can provide some of this information as protein folding and global structure both influence retention characteristics, thus intact protein analysis can elucidate global structural changes that peptide mapping misses. N-Terminal and C-terminal truncations and/or readthroughs can often be identified and quantitated better by intact protein reversed phase HPLC analysis. Indeed, the regulatory requirements for most protein therapeutics demand intact reversed phase and gel filtration analysis (for aggregate quantitation) as well as peptide mapping in most regulatory filings. Next generation UHPLC columns like core shell deliver more accurate, sensitive, and higher throughput solutions for protein characterisation that are application specific and characteristically different from peptide UHPLC columns.

Conclusion

As protein therapeutics have become larger and more complex, existing analytical analysis solutions have proven inadequate to fulfil the characterisation requirements of regulatory bodies. Fortunately, analytical instrumentation and separation devices have realised significant technological advances that meet current challenges. In addition, performance advances in chromatography have reached a level where using a 'one size fits all' approach can no longer provide the separation requirements for all separations. Protein-specific and peptide-specific UHPLC columns have been developed by multiple vendors to meet the contrasting requirements of each applicationj and address future analytical needs.


References

1. Mellors JS and Jorgenson JW, Anal Chem 76(18): pp5,441-5,450, 2004

2. van Deemter JJ, Zuiderweg FJ and Klinkenberg, A Chem Eng Sc 5: pp271-289, 1956

3. Gadgil H, Pipes G, Dillon T, Treuheit M and Bondarenko P, JASMS 17(6): pp867-872, 2006

4. Gritti F, Leonardis I, Shock D, Stevenson P, Shalliker A and Guiochon GJ, Chromatogr A 1217(10): pp1,589-1,603, 2010

5. McGinley M, Jarrett D, Layne J, Chitty M and Farkas T, Chromatography Today, pp33-36, November 2011

6. Meirovitch H, Radcousky S and Scheraga HA, Macromolecules 13: pp1,398-1,405, 1980

7. Davies PA, J Chromatogr 483: pp221-237, 1989


Acknowledgment

The author would like to thank Jeff Layne and Tivadar Farkas for their input into this article.

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Michael McGinley is Senior Product Manager at Phenomenex, Inc, where he is responsible for technical aspects of Phenomenex's HPLC and bioseparation product portfolios. Prior to joining Phenomenex in 2002, Michael was at Amgen where he held several positions over a 12-year period, including Associate Scientist, Project Manager and Laboratory Head. Michael received his BSc in Biochemistry from the University of California at Berkeley. His expertise includes HPLC method development, proteomics, biofuel analysis, oligonucleotide chemistry, protein characterisation and protein purification, and he is the author of more than 100 publications in these fields.
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