Highlights from the 52nd International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2024)

The 52nd International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2024), chaired by Susan Olesik, was held from July 20 to 25 in Denver, Colorado, USA. This “Column Watch” installment presents many of the highlighted topics and trends observed at this symposium.

The 52nd International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2024) was held in Denver, Colorado, and chaired by Susan Olesik (The Ohio State University). Continuing a long-standing trend, the HPLC symposium is the premier gathering of separation scientists in liquid chromatography and related techniques. The conference was composed of high-quality oral presentations from leading scientists, engaging poster sessions with scientists and students from all levels, and numerous opportunities to network and (re)connect with colleagues and friends.

In this installment of “Column Watch,” notable trends and highlights from the conference are described. Akin to previous symposia reviews (1–6), many colleagues in attendance were asked for their expert opinions on the conference, and what “stuck out to them” the most in terms of emerging, sustaining, or fading trends in the science. The article that follows is a distillation of these highlighted topics and some personal musings on the conference. For another perspective on the symposium, please also check out the “Analytically Speaking” podcast, where Dwight Stoll and James Grinias provide their analysis of the symposium (7).

Analysis of Oligonucleotide (and Other) Biopharmaceuticals

Oligonucleotides were by far the most discussed topic at HPLC 2024. The promise of this class of biomolecule being used to treat the most difficult diseases has driven the exponential increase in interest in the synthesis, development, and analysis of oligonucleotides. Many talks at the symposium centered around sample preparation, chromatographic analysis, and oligonucleotide purification. In addition, “legacy” research topics in the biopharmaceutical industry continued to be discussed at the show, including monoclonal antibody (mAb) analysis, antibody-drug conjugate (ADC) analysis, and peptide separations. Finally, several talks fell into the “omics” category, including proteomics, metabolomics, and glycomics. Indeed, the quest to use high-performance liquid chromatography to better understand the pharmaceuticals used to treat disease and the biochemistry occurring in our bodies was well-represented at HPLC 2024. A few of the talks on the topic of biopharmaceuticals and omics applications are presented here.

The first talk of the first day of the symposium made clear the importance of oligonucleotide analysis in the biopharmaceutical industry. Claus Rentel (Ionis Pharmaceuticals) delivered a comprehensive talk on oligonucleotide therapeutic analysis. After a review of the current critical quality attributes (CQAs) in oligonucleotide analysis, Rentel discussed the challenges in impurity analysis of oligonucleotide therapeutics. Several of the impurities that may be present in an oligonucleotide formulation are positional isomers or species that only differ by one dalton; these impurities cannot be resolved by mass spectrometry (MS) alone. To achieve resolution of these impurities, Rentel’s work highlighted two-dimensional (2D) weak anion-exchange ion-pair reversed-phase chromatography (WAX-IP-RPC) methods used to resolve the species. Rentel also provided several strategies for characterizing silencing ribonucleic acid (siRNA) duplexes via size-exclusion chromatography (SEC), hydrophilic-interaction liquid chromatography (HILIC), ion-pair reversed-phase chromatography (IP-RPC), and strong anion-ex- change (SAX). Finally, Rentel explained to the audience that in certain situations for Good Manufacturing Practices (GMP) in industry, spectral clarity and quality trumps chromatographic resolution, underscoring the fact that even though advanced methods and instrumentation may be available, ease of adoption into manufacturing and quality laboratories is more desired.

Characterizing biotherapeutic formulations for purity has been the central application driving interest in new methods and tools in protein chromatography. However, as noted in an interesting talk by Fred Regnier (Novilytic), appraising the function of the purified protein (mAb) is of equal importance. Regnier introduced the concept of mobile affinity selection chromatography (MASC) to examine the function of monoclonal antibodies (mAbs) in crude solution. In this mode of chromatography, an affinity selector is added to the mobile phase which binds to the Fc receptor of a mAb. The chromatographic trace then provides two pieces of data to the analyst: a sharp peak indicating efficient capture of the target antibody and a negative baseline peak indicating that the Fc function is working (as the Fc portion of the antibody is binding affinity selector). This technology, commercialized by Novilytic, was then applied to resolving mAb monomers from aggregates and fragments, and further extended to the analysis of different mAb subtypes and bispecific antibodies (bsAbs).

Popularized by the success of the COVID-19 vaccines, messenger RNA (mRNA)-based biotherapeutics were the subject of various presentations at HPLC 2024. Alexandre Goyon (Genentech) delivered such a presentation where he discussed strategies for analyzing mRNA and plasmid-based biotherapeutics. Plasmids can exist in several different forms, including supercoiled, open-circular, linear, or as multimers, thus leading to a complex characterization challenge. Certain modes of chromatography, like anion-exchange (AEX), can underestimate certain forms (i.e., open-circular); therefore, particular attention needs to be taken in developing the analytical method. Goyon documented how plasmids can degrade or be affected depending on the storage diluent. In addition, Goyon documented how certain impurities can affect therapeutic efficacy more than others, such as how open-circular forms affect knock-in of the plasmid into cells more than multimers. Nevertheless, larger multimers need to be detected, and Goyon highlighted how SEC can be used to characterize mRNA multimers ranging from 1000–5000 nucleotides (nts). Finally, in a novel use of multidimensional chromatography, Goyon demonstrated the use of columns with immobilized enzymes bonded to the stationary phase to perform online nucleotide mapping of mRNA species.

As the few highlighted talks above indicate, the biopharmaceutical industry is continuing to evolve and create more complex biotherapeutic modalities. These new formats require advanced characterization techniques that continue to drive innovation in LC instrumentation, detectors, and stationary phases. Future instances of this symposium are primed to witness continued advances in the field of separation sciences for biotherapeutics.

Sustainability Applied to Liquid Chromatography

Sustainability and the “greening” of liquid chromatography continued to be a trend at HPLC 2024. This observation was validated by the number of talks that either centrally focused on the concept of sustainability or applied aspects of green chemistry to their research.

Robert Kennedy (University of Michigan) delivered a talk on innovations in capillary liquid chromatography (LC). Kennedy used a 5 mm x 0.3 mm I.D. capillary column with electrospray ionization (ESI) MS for high-throughput analysis. According to Kennedy, one of the purposes of this paper was to reduce the amount of acetonitrile used in LC methods. Kennedy used a flow rate of 70 μL/min and employed a novel injection technique called “droplet injection,” which can continuously inject samples in series. This technique allowed Kennedy to process hundreds of samples enabling real time monitoring of organic reactions. Each chromatographic run lasted only 6 s. At the end of the talk, Kennedy showcased how his method ranked in terms of greenness by utilizing a calculator from the American Chemical Society (ACS) (8). His Analytical Method Greenness Score was ranked high, indicating a method that is sustainable.

Continuing the trend of using capillary LC to minimize sample consumption and solvent use, Samuel Foster (Rowan University) presented results obtained with a portable capillary LC instrument. In addition to the sample and solvent savings of using capillary flow LC, additional “green benefits” of this method include the fact that the instrument is brought to the sample (on-site analysis) instead of sending the sample to the instrument (which entails emissions from transporting the sample to the laboratory). Foster noted that additional advances in instrument and detector design (from Axcend Corporation) will improve the sensitivity of the measurements.

Elia Psillakis (Technical University of Crete) delivered a comprehensive presentation on Circular Analytical Chemistry. This approach to defining sustainability builds upon the concept of Green Chemistry through 12 goals, as illustrated in Figure 1. Psillakis walked the audience through each goal, and she emphasized the benefits that could be achieved if end-users and vendors adopted these principles. Many attendees commented that the presentation was a welcome invitation to changing long-held mindsets in analytical chromatography and chemistry that is worth addressing.

Figure 1: The twelve goals of Circular Analytical Chemistry.

Based on these few highlighted talks, and other discussions observed at HPLC 2024, the topic of sustainability is here to stay. Larger corporations are adopting internal policies to look for ways to become more sustainable, and the analytical chromatography community has several tactics that can be employed to facilitate the industry’s transition to more sustainable practices.

Bioinert LC and LC Consumables

As was also highlighted in the HPLC 2023 review, the importance of bioinert instrumentation and consumables was featured in many conference contributions throughout HPLC 2024. Many of the talks discussing oligonucleotide analysis made use of bioinert instruments and hardware owing to oligonucleotides’ proclivity to adsorb to stainless steel portions of the flow path. Several vendors have employed metal passivation strategies that minimize the presence of exposed stainless steel in the injection flow path. The industry has moved well past "simple" PEEK-clad columns and tubing, as noticed at HPLC 2024. As one attendee said, “If a vendor does not have bioinert consumables or instruments, we go elsewhere.” As new, complex biotherapeutic modalities and increasingly intricate, matrix-rich samples for environmental, clinical, or food analysis become more common in testing, the capabilities of current bioinert coatings to conceal secondary interactions and adsorption will continue to be challenged. It will be intriguing to observe the innovative soluti ns that arise to address sample loss and chromatographic performance deterioration.

Other Conference Highlights

Poster Presentations

The poster presentations continue to be an important aspect of the HPLC symposiums. The breadth of topics and techniques on display at the poster presentations covered the entire spectrum of the chemical industry employing liquid chromatography (LC). In alignment with the oral sessions, many posters included topics focusing on biotherapeutic analysis, including oligonucleotides, mAbs, ADCs, and others. Many posters also included supercritical fluid chromatography (SFC) as the analytical method of choice, which was an interesting approach to making methods more sustainable due to the limited use of solvent in the methods. It will be interesting to see if SFC continues to become more prominent at the HPLC symposiums in the future, especially with the renewed push for greener methods and with SFC instrumentation becoming more user-friendly.

HPLC Tube Competition

The HPLC Tube Competition is a scientific contest for the best video in which each author presents the impact of their research for society. The contest provided a fun and entertaining alternative approach to presenting scientific information whilst simultaneously highlighting the authors’ creative talents. As noted by several attendees, the HPLC Tube was a conference highlight and continues to be an invigorating and amusing part of the overall symposium program.

Conclusions

The HPLC 2024 symposium was well-organized, informative, and engaging. The analysis of oligonucleotides and their related impurities was a driving force behind many of the presentations. Continued advances in this area will drive overall innovation in chromatography and enable the use of the separation sciences to solve ever increasing challenges. Sustainability was also a central topic at the symposium. Whether through reduction of organic solvent in the mobile phase, use of capillary LC columns and instruments to minimize sample consumption, or adopting completely new mindsets to analytical chemistry (Circular Analytical Chemistry), the separation sciences are examining how they can play a role in green chemistry adoption. Several other topics were also repeated throughout the conference, including the use of bioinert instruments and consumables for biomolecule analysis, using SFC to improve sustainability, and multidimensional LC, among others. Innovation continues to be shown at the HPLC symposium, and what was presented at this edition of the symposium continues to validate the claim that many at the show echo: "This is the premier conference for separation scientists."

The next HPLC meeting will be in Bruges, Belgium from June 15th–19th, 2025.

Acknowledgments

Due to the sheer size of the conference, it is impossible to cover such a conference without assistance. The author would like to acknowledge the following individuals for their input and discussion: Dr. James Grinias, Dr. Dwight Stoll, Dr. David Bell, Dr. Michael Dong, Dr. Koen Sandra, Dr. Martina Catani, Dr. Elia Psillakis, Dr. Michael Ye, Dr. Bob Pirok, Dr. Deirdre Cabooter, Dr. Jesse Bischof, Dr. Harald Ritchie, and Dr. Stephanie Schuster.

References

(1) El Zahar, N. M.; Magdy, N.; El-Kosasy, A. M.; Bartlett, M. G. Chromatographic Approaches for the Characterization and Quality Control of Therapeutic Oligonucleotide Impurities. Biomed Chromatogr. 2018, 32 (1), e4088. DOI: 10.1002/bmc.4088

(2) Goyon, A.; Yehl, P.; Zhang, K. Characterization of Therapeutic Oligonucleotides by Liquid Chromatography. J. Pharm. Biomed. Anal. 2020, 182, 113105. DOI: 10.1016/j.jpba.2020.113105

(3) Roussis, S. G.; Koch, C.; Capaldi, D.; Rentel, C. Rapid Oligonucleotide Drug Impurity Determination by Direct Spectral Comparison of Ion-Pair Reversed-Phase High-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry Data. Rapid Commun. Mass Spectrom. 2018, 32 (14), 1099–1106. DOI: 10.1002/rcm.8125

(4) Dalmijn, J.; Glüge, J.; Scheringer, M.; Cousins, I. T. Emission Inventory of PFASs and other Fluorinated organic substances for the fluoropolymer Production Industry in Europe. Environ. Sci. Process Impacts 2024, 26 (2), 269–287. DOI: 10.1039/d3em00426k

(5) Lardeux, H.; Guillarme, D.; D’Atri, V. J. Chromatogr. A. 2023, 1690, 463785. Comprehensive Evaluation of Zwitterionic Hydrophilic Liquid Chromatography Stationary Phases for Oligonucleotide Characterization. DOI: 10.1016/j.chroma.2023.463785

(6) Kawachi, Y.; Ikegami, T.; Takubo, H.; Ikegami, Y.; Miyamoto, M.; Tananka, N. Chromatographic Characterization of Hydrophilic Interaction Liquid Chromatography Stationary Phases: Hydrophilicity, Charge Effects, Structural Selectivity, and Separation Efficiency. J. Chromatogr. A. 2011, 1218, 5903–5919. DOI: 10.1016/j.chroma.2011.06.048

(7) Ikegami, T.; Taniguchi, A.; Okada, T.; Horie, K.; Arase, S.; Ikegami. Y. Functionalization using Polymer or Silane? A Practical Test Method to Characterize Hydrophilic Interaction Chromatography Phases In Terms of Their Functionalization Method. J. Chromatogr A. 2021, 1638, 461850. DOI: 10.1016/j.chroma.2020.461850

(8) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. New Procedure for the Use of High-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry for the Analysis of Nucleotides and Oligonucleotides. J. Chromatogr. A. 1997, 777, 3–21. DOI: 10.1016/S0021-9673(97)00256-2

(9) Guimaraes, G. J.; Bartlett, M. G. The Critical Role of Mobile Phase pH in the Performance of Oligonucleotide Ion-Pair Liquid Chromatography–Mass Spectrometry Methods. Future Sci OA 2021, 7 (10), FSO753. DOI: 10.2144/fsoa-2021-0084

(10) Hsiao, J. J.; Kennedy, A. P.; Van de Bittner, G. C.; Wei, T. LCGC Supplements Special Issues 2018, 36 (6), 30–35.

(11) Temsamani, J.; Kubert, M.; Agrawal, S. Sequence Identity of the n - 1 Product of a Synthetic Oligonucleotide. Nucleic Acids Res. 1995, 23 (11), 1841–1844. DOI: 10.1093/nar/23.11.1841

(12) Fearon, K. L.; Stults, J. T.; Bergot, B. J.; Christensen, L. M.; Raible, A. M. Investigation of the ‘n–1’ impurity in Phosphorothioate Oligodeoxynucleotides Synthesized by the Solid-Phase β-Cyanoethyl Phosphoramidite Method Using Stepwise Sulfurization. Nucleic Acids Res. 1995, 23 (14), 2754–2761. DOI: 10.1093/nar/23.14.2754

About the Column Editor

David S. Bell is the Lead Consultant and Owner at ASKkPrime, LLC. He specializes in separation science consultancy, is Editor of the Column Watch series of articles and serves on the Editorial Advisory Board for LCGC International. With over 30 years of experience, he has contributed significantly to chromatography advancements, focusing on stationary phase design, device development, and molecular interaction research. Dave’s work spans gas chromatography, liquid chromatography, sample preparation, and pharmaceutical analytical method development. He holds a PhD in Chemistry from The Pennsylvania State University, has presented research globally, and has authored more than 95 peer-reviewed and trade magazine articles.

About the Author

Cory E. Muraco is the Biomolecule Reference Material Product Manager at MilliporeSigma, the life science business of Merck KGaA, Darmstadt, Germany. After finishing his graduate studies at Youngstown State University (Youngstown, Ohio, USA), Cory started his career at MilliporeSigma in 2013. After holding several roles in both R&D and Marketing functions across the past decade, in 2024, Cory assumed his current role as the Biomolecule Reference Material Product Manager where he is tasked with designing, developing, and executing the marketing and R&D strategies around MilliporeSigma’s biomolecule reference material. Cory is the author of several manuscripts appearing in trade magazines and has delivered over 100 presentations at international conferences, round table symposia, and at various pharmaceutical and biopharmaceutical companies.