OR WAIT 15 SECS
Volume 36, Issue 10
Topics that stood out at HPLC 2018 include advances in large-molecule separations, multidimensional separations, 3D printing, chiral separations, and HILIC. Also, many speakers addressed separation fundamentals, including ways to increase speed, generate higher throughput, and add selectivity.
The 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2018), chaired by Norman Dovichi, was held from July 29 to August 2 in Washington, D.C. This installment of "Column Watch" covers some of the highlights observed at the symposium. In addition, trends and perspectives on future developments in HPLC noted from the conference are presented.
The 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, or HPLC 2018, was held in Washington, D.C., at the Marriott Wardman Park Hotel and Conference Center from July 29 to August 2, 2018. This symposium, which continues to be the premier event bringing together leading scientists in the field of liquid chromatography and related techniques, attracted 870 delegates from 34 countries. The attendance for US-based venues continues to fall short of European sites; however, the US attendance has remained fairly consistent for a number of years (Table I). The well-organized conference was chaired by Professor Norman Dovichi of the University of Notre Dame. As noted by the chair, the program had a strong focus on separations in the pharmaceutical industry, including interesting sessions on continuous manufacturing and forward-looking pharmaceutical analysis. Other preconference highlights included presentations on three-dimensional (3D) printing, micropillar array technology development, and a special focus on the fast-rising cannabis industry.
The conference included 203 talks (of which 12 were tutorial presentations), 335 poster presentations, nine short courses and nine vendor technical workshops. In this installment of "Column Watch," observed highlights and trends from the conference are reported.
In a similar fashion to the previous HPLC review articles (1,2), several colleagues in attendance at the symposium were asked for their insights regarding the most interesting topics they observed at the event. Much of what follows is a synopsis of their responses along with some personal views.
In reviewing highlights from the past several symposia, multiple areas of interest stood out, namely 3D printing, advances in large-molecule separations, multidimensional separations, and chiral and hydrophilic interaction liquid chromatography (HILIC). Reported progress in 3D printing seemed to wane in 2017; however, at the 2018 symposium, developments in 3D printing were again at the forefront of discussions. Attention toward large-molecule separations seemed to be even greater than in previous years, perhaps because of an overall conference focus on pharmaceutical analysis. Advances in HILIC and chiral chromatography continued to be presented this year, and general topics in separation fundamentals that promise to increase speed, generate higher throughput, and add selectivity continue to drive discussions at the symposium.
As indicated by the name of the conference-the International Symposium on High Performance Liquid Phase Separations and Related Techniques-liquid chromatography was not the only separation technology present at the symposium. Like many symposia before, the meeting often takes on the personality of its chair. It was not unexpected then that electrophoresis (CE) was a significant topic at HPLC 2018. Several sessions were dedicated to CE, where much of the discussion centered on biomolecule separations.
The characterization of large molecules requires a variety of different chromatographic approaches, including reversed-phase, ion exchange, size exclusion, and hydrophobic interaction chromatography. HPLC 2018 included many presentations on techniques dealing with different aspects of large molecule separations. Matthew Lauber presented on a new polyphenyl column designed to improve efficiencies and resolution of targeted large molecules such as monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) (3). Lauber demonstrated that the novel column chemistry is capable of performing separations of mAbs and ADCs at lower temperatures and with lower concentrations of ion-pairing reagents than existing columns, thus minimizing on-column degradation of biotherapeutics. The more "protein friendly" reversed-phase chromatography was stated to be a result of the high surface coverage of the rigid phenyl moiety, which prevents access of the analyte to the underlying base silica particle.
Superficially porous particle (SPP) technology continues to be adopted for large molecule separations. Barry Boyes used a combination of C4, C18, and a new diphenyl stationary phase based on 1000-Å SPPs to develop methods for correct structure assignment of mAbs and ADCs (4).
Selectivity changes in large-molecule separations can be affected by mobile phases, as well as by alternative surface chemistry and particle technology. Kevin Schug showed how changes in mobile-phase components and pH may elicit different protein conformations, and thus producing alternative interactions with the stationary phase (5). The approach may have important implications in the realm of multidimensional separations of intact proteins.
Columns used for large-molecule separations are often characterized using traditional small-molecule probes and systems. Jennifer Field presented an interesting talk regarding the development of a column characterization protocol for large-molecule, reversed-phase columns (6). Field and colleagues developed a series of peptide probes and a set of conditions to characterize several commercially available, wide-pore reversed-phase columns. Field's protocol may lead to column characterization information that is much more useful to the large-molecule separation scientist than has previously been available.
Ion-exchange chromatography (IEX), size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) all provide important separation capabilities that are complementary to reversed-phase separations. The high salt content often employed in these techniques, however, precludes the use of mass spectrometry (MS) for detection. Jonathan Bones presented the analysis of mAbs for charge variants and aggregates by ion-exchange–MS and SEC–MS using a novel, low-salt mobile phase that enables these modes of chromatography to be coupled directly to MS (7). The research demonstrated the ability to identify unique C-terminal lysine variants and mAb fragments that would have been missed using conventional IEX and SEC methods.
HIC is a widely used non-denaturing technique that separates analytes based on their surface hydrophobicity. Similarly to IEX chromatography, the traditional use of non-volatile salt buffers in performing HIC have prevented direct coupling to MS. Bifan Chen reported on a novel combination of stationary-phase materials with volatile mobile phases based on ammonium acetate and some organic solvent (8). The resulting HIC–MS method was demonstrated with the analysis of intact proteins and more recently for mAbs. According to the authors, the mass spectra were characteristic of proteins with native structure, primarily featuring low charge states. For mAbs, the HIC–MS method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles, as well as sequence and structural characterization of the complementarity-determining regions in an online configuration. For ADCs, the same methods and conditions cannot be applied directly, because of the fragile nature of the molecules. Method development becomes a balance between generating enough retention for the drug-to-antibody ratio (DAR) zero variant, but not so much as to denature the high DAR species (DAR6 and DAR8) by the stationary phase or organic solvent. Using an ADC mimic, the total ion chromatogram and the mass spectra demonstrated that different DAR species were separated successfully and, more importantly, remained intact during the chromatography. The group noted plans to submit their manuscript on the ADC analysis for publication soon.
Advances in particle designs continue to highlight HPLC symposia. Ta-Chen Wei gave a talk on the synthesis of an SPP that has a "dual-pore" architecture (9). In 2015, the group presented a new SPP with ordered elongated pores through a process called pseudomorphic transformation (PMT) (10), and later demonstrated the new particle provides 18–30% more efficiency than conventional SPPs for small-molecule separations (11). The improved efficiency is due to the anisotropic diffusion from the ordered pore structure, resulting in a much reduced B term. At HPLC 2018, Wei presented on a second generation of the PMT material that has dual pore structure on a single particle (9). The inner region has ordered elongated straight pores for small-molecule separations, and the outer region has large conical pores for large-molecule separations. As a result, a single column packed with the new dual-pore SPPs can be used for both small- and large-molecule separations. In addition, the columns show improved efficiency and resolution over current conventional SPPs in their preliminary study. Figure 1 illustrates the progression of the PMT particle technology.
Figure 1: Progression of superficially porous particle technology. (a) Conventional SPPs. (b) First-generation pseudomorphic transformation SPPs. (c) Second-generation pseudomorphic transformation SPPs.
The quest for speed is ever present at the HPLC meetings. A much noted highlight was Dan Armstrong's presentation, "Practice and Ramifications of Ultrafast LC and SFC" (12). The talk centered on the idea that current practices in analytical chromatography will soon be obsolete as a result of combining fast chromatography (with appropriate columns) and peak processing. Armstrong believes that by performing HPLC in the optimal ultrafast mode and using columns above their peak capacity along with proper peak processing and resolution enhancement approaches, it may be possible to have 100 baseline or near baseline peaks in <60 seconds. Further, Armstrong predicted that identification (via retention time) and quantitation (via peak area measurement) using this approach will be comparable to, or better than, the typical best-practice separations today. The combination of advanced peak processing and fast LC is powerful. Armstrong went on to note that spectroscopy has been utilizing advanced processing techniques for decades and chromatography is behind the curve, but when the separations community catches up, the results will be impressive and profound.
James Grinias presented a talk on the transfer of standard methods to ultrahigh-pressure liquid chromatography (UHPLC) systems (13). Grinias investigated the effects of cyclic thermal gradients on retention reproducibility. Because of the pressure cycles in gradient chromatography, variable viscous heating results in column temperature cycles. The impact can be described as start-up effects in which the first few gradients are different from the later ones. Further details can be obtained from the Grinias group's published work (14). Grinias went on to discuss the transfer of HPLC methods to UHPLC methods as applied to the modernization of pharmacopoeial methods. Grinias concluded that modern UHPLC instrumentation and columns utilizing SPP technology can be used to greatly increase throughput of pharmaceutical analyses based on monograph methods.
Miniaturization continues to be a topic of interest in liquid chromatography. Milton Lee's (15) and Luke Tolley's (16) presentations on miniaturized LC instruments with high performance were considered highlights by many. Both talks centered on a promising new miniaturized LC instrument being developed by a company called Axcend. An interesting note from Lee's talk was that the mobility of an instrument may be as useful within a laboratory as it is for remote usage.
Among the highlights using microfabricated devices, Qun Fang presented on performing single-cell analysis using droplet-based microfluidic techniques (17). Fang and colleagues developed a device that can sample and load different solutions on a microplate. As an example of an application for this system, Fang showed the purification and detection of µRNA species that are easily bound to proteins. To remove proteins from the sample, Fang performed "in-droplet" tryptic digestion to unbind and remove protein from the µRNA prior to nano-LC–MS detection. The group intends to further develop the system using capillary electrophoresis-mass spectrometry (CE–MS).
Another notable highlight was a presentation given by Attila Felinger entitled, "Reversed-Flow Liquid Chromatography" (18). Felinger described a technique whereby flow is stopped such that the analyte is still within the column. The flow is then reversed either by physically turning the column around or by the use of a valve system. The technique allowed the researchers to investigate band broadening contributions from frits, understand trans-column dispersion effects and probe the column sections for imperfections. Interestingly, it was found that the center of the column was more efficient than either of the ends.
The topic of 3D printing columns was once again a highly discussed topic at HPLC 2018. At the forefront of the discussions were two presentations by Simone Dimartino, one of which was a tutorial (19,20). Dimartino described employing monomers with the functionality desired to construct the columns, rather than employing post printing chemical techniques to alter selectivity. Printed columns are still not capable of generating efficiencies as high as those of modern packed-bed columns, but the technology is progressing rapidly. In addition to being used to print columns, 3D printing has also been shown to be useful for device optimization. Theodora Adamopolou presented a talk showing computationally derived 3D printed devices to aid in multidimensional separations (21).
HILIC was once again a much discussed topic at the HPLC meeting. At the head of the line again was Andrew Alpert, who presented a talk on the importance of salt selection when developing HILIC or electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) methods for phosphopeptides (22). Alpert provided examples in which the choice of both cation and anion elicited differential selectivity of phosphopeptides. The more hydrated the cation was, the more retained the negatively charged phosphopeptides were. Alpert also demonstrated evidence of molecular reorientation of peptides at the water–organic boundary based on salt type. This information could be used for applications involving glycopeptides and for intact proteins with different degrees of phosphorylation. A second HILIC talk that garnered attention was given by Jonathan Shackman. His talk, entitled "HILIC to the Rescue: Pharmaceutical Development Case Examples," showed that HILIC can be a useful and reliable techniques for small-molecule pharmaceuticals when traditional methodologies such as reversed-phase LC fail to provide adequate results (23).
Advances in chiral separation technologies were once again a topic of discussion at HPLC. Ravindra Hegade presented the use of stationary phase optimized selectivity, or the coupling of stationary phases, as an approach to develop chiral separations in both LC and supercritical fluid chromatography (SFC) (24). In another highlight, Csaba Horváth Young Scientist Award winner Martina Catani discussed mass transfer phenomena and thermodynamic properties of modern porous particles for efficient enantioseparations (25). One interesting note from the talk was that advantages normally observed for SPP particles over fully porous particles in reversed-phase separations were not clearly evident in the chiral systems investigated.
As in years past, the poster session at HPLC 2018 was an important part of the overall symposium. There were 336 posters presented across 21 session topics. Compared to HPLC 2016 in San Francisco (the last time the conference was held in the United States), there was a 25% increase in the number of posters presented at the conference.
The number of posters presented in each category was fairly evenly distributed. A breakdown of the number of posters in each session topic is shown in Figure 2. The four most popular poster session topics, in terms of number of posters presented, were stationary phases, method development, biopharmaceutical applications, and characterization of monoclonal antibodies, drug conjugates, and other protein-based drugs; this ranking mirrors both the oral presentation topics at this year's conference, and also the overall direction of growth in the LC markets. The popularity of the stationary phase and method development topics shows the ever present need to continue to improve and develop new separation media and analysis techniques. Within those two topics, and in several other session topics, there was an increased presence of posters on HILIC separations related to understanding and developing HILIC methods for a wide array of polar molecules that are difficult to retain on reversed-phase columns. One of the posters receiving an honorable mention in the poster awards competition was from Dennis Asberg on "HILIC Method Development in Pharmaceutical Analysis." Asberg outlined a comprehensive HILIC method development strategy to optimize the analysis of hydrophilic pharmaceutical compounds.
The twenty-one poster session topics were spread out across six different presentation sessions, two each day on Monday, Tuesday, and Wednesday. On Thursday morning, the finalists for consideration for poster awards presented their work again. The posters under consideration for awards were evaluated by a panel of international scientists and judged based on scientific contribution, originality of work, completeness of work, quality of experimental or theoretical execution, and lastly the readability and the presentation of the poster. The Best Poster Awards, sponsored by Agilent Technologies, were presented at the closing ceremonies of the conference. The winning posters, and those that received honorable mention, are listed in Table II.
Table II: Best poster awards
HPLC 2018 was a well-organized and lively symposium that engaged researchers interested in analytical science from around the globe. Many recent trends continued in 2018. A revival of progress in 3D printing of columns and devices was evident. There also seemed to be an additional surge of interest in innovative large-molecule separations. It is clear from the activity at the conference that research in the separations sciences is alive and well.
Coverage of such a large symposium is impossible without a great amount of assistance. The authors would like to acknowledge invaluable assistance from Andrew Alpert, Robert Kennedy, Xiaoning Lu, Daniel Shollenberger, Ken Broeckhoven, Ta-Chen Wei, Matthew Lauber, Mariosimone Zoccali, and Daniel Armstrong for providing notes, text, insights, figures, and fruitful discussions regarding the content of various sessions.
(1) D.S. Bell, LC-GC Europe30(4), 196-207 (2017).
(2) D.S. Bell, LC-GC North Am.34(9): 700-709 (2016).
(3) M. Lauber, J. Nguyen, S. Rzewuski, D. Walsh, J. Cook, M. DeLoffi, G. Izzo, and Y. Xu, "A Novel Phenyl-based RPLC Stationary Phase for High Throughput, High Resolution Characterization of Protein Therapeutics," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(4) B. Boyes, B. Libert, S. Schuster , B. Wagner, C. McHale, W. Miles, M. Schure, and J. Lawhorn, "Manipulating Protein Variant Separations using High Performance Large Pore Superficially Porous Particles," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(5) K. Schug and Y. Baghdady, "Altered Selectivity in Mass Spectrometry-Compatible Reversed Phase Separations of Intact Proteins," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(6) J. Field, P. Petersson, and M. Eureby, "Characterization of the Peptide Separation System: Development of a Column Characterization Protocol based on Peptide Probes," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(7) F. Fuessl, A. Trappe, K. Cook, K. Scheffler, and J. Bones, "Quality Profiling of Biopharmaceuticals as Intact Entities using High Resolution Native LC–MS," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(8) A. Alpert, Y. Ge, B. Chen, and Z. Lin, "HIC-MS of Intact Monoclonal Antibodies and Antibody-Drug Conjugates," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(9) T.C. Wei, "One-Fits-All HPLC Column: Synthesis of Superficially Porous Particles with Dual Pore Structure," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(10) Bell, D.S., X. Wang, and R.E. Majors, LC-GC Europe28(9), 500–505 (2015).
(11) Wei, T.C., et al., Journal of Chromatography A,1440, 55–65 (2016).
(12) D.W. Armstrong, "Practice and Ramifications of Ultrafast LC and SFC," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(13) J. Grinias, "Increasing Througput for Pharmacopeial Monographs," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(14) J.P. Grinias, J.M.T. Wong, and R.T. Kennedy, Journal of Chromatography A,1461, 42–50 (2016).
(15) X. Xie, L. Patil, L. Tolley, P. Farnsworth, D. Tolley, and M. Lee, "Nanoflow LC using Serial Columns and Detectors," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(16) L. Tolley, X. Xie, T. Truong, L. Patil, P. Farnsworth, H.D. Tolley, and M. Lee, "Rapid Analysis in Portable Nanoflow Liquid Chromatography," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(17) Q. Fang, Z. Li, X. Guo, M. Huang, X. Wang, Y. Zhu, J. Li, and C.C.L. Wong, "Single Cell Analysis with Droplet-based Microfluidic Technique," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(18) A. Felinger, "Reversed-Flow Liquid Chromatography," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(19) S. Dimartino, "3D Printing in the Separations Sciences," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(20) S. Dimartino, "3D Print Your Own Chromatography Column," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(21) T. Adamopoulou, S. Deridder, S. Nawada, G. Desmet, and P.J. Schoenmakers, "Creating Devices for Multidimensional Separations based on Conputational Insights," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(22) A. Alpert, "A Major Increase in Selectivity for Phosphopeptides and Glycopeptides in ERLIC and HILIC Conferred by the Salt Selection," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(23) J. Shackman, "HILIC to the Rescue: Pharmaceutical Development Case Examples," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(24) R. Hegade, M. De Beer, and F. Lynen, "Enhanced Resolution of Stereoisomers through Stationary Phase Optimized Selectivity Liquid and Supercritical Fluid Chromatography (SOS-LC and SOS-SFC)," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
(25) M. Catani, O.H. Ismail, S. Felletti, C. De Luca, M. Morbidelli, F. Gasparrini, and A. Cavazzini, "Investigation of Mass Transfer Phenomena and Thermodynamic Properties of New Generation Porous Particles for Ultrafast High-Efficient Enantipseparations," presented at 47th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Washington, DC (2018).
ABOUT THE AUTHOR
Cory E. Muraco is a Senior R&D Scientist in the Liquid Separations R&D group at MilliporeSigma, in Bellefonte, Pennsylvania. Cory completed his graduate studies at Youngstown State University in 2013, focusing on the analysis and characterization of oxidized proteins. Upon graduation, Cory joined MilliporeSigma, first joining the chemical standards R&D group, then transferring to the liquid separations R&D group. Cory's current role at MilliporeSigma is to research, develop, and present on new particle technology for improved chromatographic separations of both small and large molecules and to develop new methodologies for characterizing biomacromolecules by several modes of chromatography. Cory has written about his research in several trade magazines and presented oral and poster presentations on his research at numerous conferences.
Connor Flanneryis a Product Development Chemist in the HPLC R&D group at Restek, in Bellefonte, Pennsylvania. Connor has a Bachelor's of Science in Chemistry, and joined Restek in 2014. Connor's work at Restek focuses on the development and characterization of new stationary phases and column-packing technologies.
ABOUT THE COLUMN EDITOR
David S. Bell is a director of Research and Development at Restek. He also serves on the Editorial Advisory Board for LCGC and is the Editor for "Column Watch." Over the past 20 years, he has worked directly in the chromatography industry, focusing his efforts on the design, development, and application of chromatographic stationary phases to advance gas chromatography, liquid chromatography, and related hyphenated techniques. His undergraduate studies in chemistry were completed at the State University of New York at Plattsburgh (SUNY Plattsburgh). He received his PhD in analytical chemistry from The Pennsylvania State University and spent the first decade of his career in the pharmaceutical industry performing analytical method development and validation using various forms of chromatography and electrophoresis. His main objectives have been to create and promote novel separation technologies and to conduct research on molecular interactions that contribute to retention and selectivity in an array of chromatographic processes. His research results have been presented in symposia worldwide, and have resulted in numerous peer-reviewed journal and trade magazine articles. Direct correspondence to: LCGCedit@ubm.com