Advances in HPLC: A Virtual Symposium presented by LCGC



In this one-day virtual symposium from LCGC, leading chromatographers present advances in high performance liquid chromatography (HPLC), addressing both theory as well as methods developed to tackle real-world problems.

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Event Overview:

In this one-day virtual symposium from LCGC, leading chromatographers present advances in high performance liquid chromatography (HPLC), addressing both theory as well as methods developed to tackle real-world problems. In the afternoon session, scientists from leading instrument manufacturers will present best practices, tips, and troubleshooting.

Key Learning Objectives:

  • Learn easy ways to reduce noise in your HPLC chromatograms
  • Discover a workflow for automated and unsupervised optimization of LC–MS and 2D-LC–MS methods
  • Gain the benefits of practical industry examples of HPLC use in pharmaceutical and biopharmaceutical analysis, including in PAT for a synthetic peptide and multiplexed impurity monitoring of antibody–drug conjugates

Morning session: 9:00 am – 12:15 pm EDT
Advances in HPLC: Theory and Applications

9:00 am EDT Reducing Noise in Chromatographic Data with User-Friendly Mathematical Techniques
M. Farooq Wahab, Research Engineering Scientist, Department of Chemistry and Biochemistry, University of Texas at Arlington
Almost all analytical signals are affected by unwanted noise. Although noise is inevitable in real chromatographic data, judicious use of mathematical techniques can always enhance signal-to-noise (S/N) ratios. In turn, this helps to integrate peaks more accurately, lowers the limit of detection, and improves data visualization. This presentation outlines several advanced mathematical methods that are easy to implement on real data. It is common in liquid chromatography to encounter signal-related problems such as peak shapes that overlap, a small peak riding on the tail of a larger one in the presence of noise, or poor signal-to-noise ratios. Various digital signal-processing topics will be covered to address baseline correction and spike removal using a median or Hampel filter. Following a description of existing denoising methods embedded in current HPLC systems, more advanced methods such as the "perfect smoother" method, Fourier windowing, and cross-correlation will be discussed with their advantages and pitfalls.

9:30 am EDT Closing the Loop: Automating 2D-LC-MS Method Development
Tijmen S. Bos (a,d), Jim Boelrijk (b,d,e), Stef R.A. Molenaar (c,d), Brian van ‘t Veer (c,d), Leon E. Niezen (c,d), Denice van Herwerden (c,d), Saer Samanipour (c,d), Dwight R. Stoll (g), Patrick Forré (b,e), Bernd Ensing (e,f), Govert W. Somsen (a,d), Bob W.J. Pirok (c,d,e,g) (presenting speaker)
(a) Division of Bioanalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, The Netherlands; (b) AMLab, Informatics Institute, University of Amsterdam; (c) Analytical Chemistry Group, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam; (d) Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, The Netherlands; (e) AI4Science Lab, University of Amsterdam; (f) Computational Chemistry Group, Van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam; (g) Department of Chemistry, Gustavus Adolphus College, Saint Peter, MN 56082, United States

Two-dimensional liquid chromatography (2D-LC) is a powerful technique for separating highly complex samples, such as for biopharmaceuticals, food, polymers, and small molecules. The great potential gains in separation power and analysis time that can result from rigorously optimizing LC–MS and 2D-LC-MS methods for routine measurements has prompted many scientists to develop computer-aided method-development tools, but most methods are still developed in a conventional manner, by analysts who rely on their knowledge and experience. In this presentation, a novel, closed-loop, and open-source algorithm is presented that can be used for the automated and unsupervised optimization of LC–MS and 2D-LC–MS separations by interfacing directly with the LC system. The developed workflow can support different optimization strategies using retention modelling and machine learning. This will be demonstrated by applications to two complex samples. The developments and challenges for automation of optimization of 2D-LC-MS separations will be discussed with experimental data.

10:00 am EDT Continuous Manufacturing of Tirzepatide Using Online UHPLC-Based PAT: An Enabling Technology for Commercialization of a Synthetic Peptide
Stephen R. Groskreutz, Senior Chemistry Advisor (presenting speaker), and Gordon R. Lambertus, Senior Director, Chemistry, both in Synthetic Molecule Design and Development, Lilly Research Laboratory, Eli Lilly and Company
Tirzepatide is a glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 (glucagon-like peptide-1) receptor agonist recently approved in the United States for the treatment of type 2 diabetes. Commercial manufacture of a complex synthetic peptide presents significant challenges to obtaining the desired product of high quality and yield. Recently, a hybrid solid-phase peptide synthesis/liquid-phase peptide synthesis (SPPS/LPPS) approach was demonstrated at scale to leverage the benefits of continuous processing and online UHPLC-based process analytical technology (PAT). The hybrid SPPS/LPPS synthesis of tirzepatide utilizes four high purity fragments synthesized via traditional SPPS methodologies and released as GMP intermediates. Fragments are coupled in three continuous LPPS reactions performed in dedicated plug flow reactors (PFR) with two nanofiltration-based impurity purge points between chemical reactions. Each PFR is equipped with an online UHPLC, and a Lilly developed process sampler to monitor reaction completion and residual starting materials. A brief discussion on how to effectively implement online UHPLC for a peptide synthesis with a PAT-directed control strategy for control of key impurities will be presented.

10:30 am EDT Multiplexed Small-Molecule Impurity Monitoring in Antibody–Drug Conjugates by Mixed-Mode Chromatography Paired with Charged Aerosol Detection
Tao Chen, PhD, Principal Scientist and Group Lead, Small Molecule Analytical Chemistry, Small Molecule Pharmaceutical Sciences, Genentech, Inc.
With advanced genetic engineering technologies and better understanding of disease biology, antibody–drug conjugates are emerging as a promising new generation biopharmaceuticals. These novel therapeutic modalities are carefully designed to possess desired features, such as enhanced selectivity and reduced toxicity. However, their high level of structural complexity with multiple components often leads to long development and complex multi-step manufacturing processes, through which a variety of potential small-molecule impurities can be introduced. To this end, an in-process assay was developed in which mixed-mode chromatography coupled with charged aerosol detection was utilized for multiplexed detection of nine reagents commonly used in development and manufacturing of antibody–drug conjugates. This method utilized a mixed-mode column with ion-exchange properties operated in the hydrophilic interaction chromatography (HILIC) mode. Various parameters were systematically optimized and under optimal conditions, the method demonstrated excellent specificity, sensitivity, linearity, precision, accuracy, and was successfully applied to determine residual impurities in multiple antibody–drug conjugate samples.

11:00 am EDT Revealing Migrating Chemicals from Reusable Plastic Bottles by LC–HRMS Non-Targeted Screening
Selina Tisler (presenting speaker) and Jan H. Christensen, Environmental Analytical Chemistry Group, University of Copenhagen
Reusable plastic bottles are popular, especially during sports activities, but little is known about the migration of chemicals from the bottles into drinking water. In this study, we investigated the migration of chemicals into drinking water stored for 24 hours in new bottles, used bottles, and bottles washed in the dishwasher. Non-targeted screening (NTS) by liquid-chromatography–high-resolution mass spectrometry (LC–HRMS) was performed to identify these compounds. We detected >3500 dishwasher-related compounds, with 430 showing migration even after subsequent flushing of the bottles. In addition, more than 400 plastic-related compounds were detected, with high peaks for oligomers suspected to originate from the biodegradable polyester polycaprolactone, and aromatic amines with unknown origin. Most of the identified compounds migrating out of the used bottles were plasticizers, antioxidants, or photoinitiators. The presence of photoinitiators is of particular concern, due to possible endocrine disrupting effects. Furthermore, diethyltoluamide (DEET) was detected, which may have been formed from the plasticizer laurolactam. Typically, the dishwashing process enhanced the leaching of plastic-related compounds, and even after additional water flushing, the average peak intensity of these compounds was only reduced by half.

Afternoon session, 2:00–4:00 pm EDT
Advances in HPLC: Best Practices, Tips, Tricks, and Applications from Our Sponsors

2:00 pm EDT How Advances in Material Design Have Allowed for UHPLC Development
Jacob Schmidt, Ph.D., Sciencix
Chromatographic separation methods can be more efficient and completed faster by increasing the operational pressure of the HPLC system. Traditional HPLC instrumentation was limited by the sealing components, such as piston seals and rotor seals. Continuous advances in the material design of these components have allowed for ultra-fast chromatography, virtually zero carryover, and increased instrument up-time, among other improvements. In this seminar we will explore the evolution of HPLC into UHPLC by focusing on the material improvements of several critical components of the chromatography systems.

2:20 pm EDT Intact Monoclonal Antibody Characterization: How to Optimize Your Mobile Phase to Improve Selectivity, Resolution, and Recovery in Reversed-Phase Separations
April Rachamim , Agilent Technologies
Monoclonal antibodies (mAbs) are the most common type of biological therapeutics. Development and manufacturing processes play a key role to ensure that protein biotherapeutics meet regulatory requirements for clinical trials and commercialization. These features are known as critical quality attributes (CQAs). This work analyzes the performance of some common chromatographic stationary phases (short alkyl chains to polyaromatic) designed to deliver high resolution and reproducible results for mAbs analysis. The effect of organic alcohols, different ion pair reagents and temperature have been investigated to obtain best analytical performance.

2:40 pm EDT Simultaneous LC–MS Analysis of Ultra-Short- Through Long-Chain PFAS Compounds (C1-C10) Using Multimodal Chromatography
Ron Benson , Showa Denko America
Perfluoroalkyl and polyfluoroalkyl compounds (PFAS) are a structurally diverse class of manmade chemicals utilized in a variety of manufacturing processes since the 1960s. Due to their links with adverse health effects, recalcitrance, and widespread occurrence in the environment, attention is focused on the ability to detect PFAS at low concentrations. Reversed phase methods generally target long-chain PFAS (for example, PFOS) and do not simultaneously retain polar 1-3 chain carbons species. Utilizing the Shodex VT-50 2D, a sensitive and repeatable assay of PFAS including ultra-short chain (C1-C4) though long chain (C8-C10) compounds was developed to explore the retention mechanism and MS sensitivity. The chromatographic separation utilized a polyvinyl alcohol solid support with quaternary ammonium surface functional groups, capable of multi-modal retention capabilities in buffered acetonitrile eluents.

3:00 pm EDT Addressing the Concerns with of Biomolecule LC Analysis
Nivesh K. Mittal, MBA, PhD, Shimadzu Scientific Instruments
The development of biologics was recently propelled into the limelight with the biopharma industry working relentlessly on the development of a vaccine for the SARS-CoV-2 virus. Most vaccines that were being developed were based on biomolecules such as oligonucleotides (mRNA sequences) or viral proteins (vectors and adjuvanted protein subunits). One of the challenges in the accurate and high efficiency analysis of such biomolecules is the limitation that UHPLC instruments need to have metal components to withstand the high pressures. However, biomolecules typically “‘stick”’/(adsorb) to the metal surfaces, resulting in inaccurate results. In this talk Dr. Mittal discusses a new UHPLC system that solves this issue by using PLS (PEEK lLined sStainless-steel) that is inert to biomolecules and able to withstand UHPLC ranges of operating pressures.

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