Plenary Lecture
Dr. Pankaj Aggarwal, Kimber Barnett, James Morgado, Dave Fortin Pfizer, Groton, CT
Advancements in Liquid Chromatography Method Development for Pharmaceuticals
Systematic LC method development approaches provide information that can be used to establish robust method conditions with controls that ensure consistent method performance throughout method lifecycle. These approaches are well established in pharmaceutical industry and have evolved based on scientific advances to meet the needs of increased speed and accuracy at lower cost. The retention time data from these systematic approaches may be used for structure based predictions or data driven robustness predications by implementation of modern day data visualization tools and statistical analysis. The data driven retention time models developed from method optimization data can serve as a substituent for experimental DOE to establish method robustness. Structure based prediction using the screening data can reduce the need of method re-development to accommodate the changes in product development. Structure based predictions can serve as feedback loop for the method development approaches making it much more cost and time effective.
Speakers/Title/Abstract (No particular order)
Professor Attila Felinger Depart. of Analytical and Environmental Chemistry, University of Pécs, Pécs, Hungary
What van ’t Hoff Plots do not tell us About the Thermodynamics of Separations
van 't Hoff analysis is extremely popular in chromatographic studies to characterize the thermodynamics of chromatographic separations.
Those characteristics are usually believed to be important for the understanding of retention mechanisms in every areas of chromatography. The value of the enthalpy or entropy change is, however, limited by a number of factors. Stationary phases are often heterogeneous – for instance on the surface of a chiral stationary phase several enantioselective and achiral interactions can be identified. The enthalpy and entropy changes determined for those systems are definitely apparent values and cannot directly be related to the true thermodynamic parameters.
Furthermore, the real value of the stationary phase is never determined, thus the true phase ratio is never used in van ’t Hoff analyses.
We provide a detailed evaluation of the limits of van ’t Hoff analysis, demonstrating that it is very difficult to give a good thermodynamic characterization of the separation mechanism via the temperature dependence of the retention factor.
Attila Felinger is a Professor of Analytical Chemistry at the University of Pécs (Hungary), in addition to serving as President of the Hungarian Society for Separation Sciences. He graduated with a degree in chemical engineering from the University of Veszprém (Hungary) and obtained his Ph.D. in chemistry in 1988 from the same university; he also holds a D.Sc. degree from the Hungarian Academy of Sciences. His research interests focus on the fundamentals of chromatography including nonlinear, preparative, and analytical separations, as well as the statistical analysis of measurements by analytical chromatography. Prof. Felinger has published some 100 scientific papers and two books. He serves on the Editorial Board of the Journal of Chromatography A and LC-GC Europe.
Dr. Nelu Grinberg Grinberg Consulting New Milford, CT
HPLC ENANTIOMERIC SEPARATION OF AROMATIC AMINES USING CROWN ETHER TETRACARBOXYLIC ACID
Ling Wu1, Sherry Shen1, Shengli Ma1, Cristina Manolescu1, Nelu Grinberg2
1Boehringer Ingelheim Pharmaceuticals, Inc. Ridgefield, CT
2Grinberg Consulting, New Milford, CT
Separation of an aromatic amine is presented using Chirosil RCA(+), which consists of a chemically bonded crown ether tetracarboxylic acid stationary phase. To separate this amine, we used as mobile phase aqueous-organic mobile phases consisting of 0.1% HClO4 in water and acetonitrile or methanol. Plots of ln k vs. the volume fraction of the organic modifier showed a non-linear behavior which prompted us to investigate non-chromatographic techniques. In particular, we pursued vibrational circular dichroism in order to elucidate the interactions between the chiral amine and the crown ether tetracarboxylic acid. The enantiomeric separation of several other aromatic amino alcohols molecules was also performed. The relationship between the retention, enantioselectivity and the structure of the molecules is discussed.
Dr. Nelu Grinberg recently retired following a career in research chemistry in the pharmaceutical industry. His research in analytical chemistry has an emphasis on chromatography, spectroscopy, and chiral separations. He was a Distinguished Research Fellow in the Chemical Development Department at Boehringer Ingelheim Pharmaceuticals in Ridgefield, CT until January, 2017. Prior to this, he worked for sixteen years in the Analytical Department at Merck Research Laboratories in Rahway, NJ, where he was a Senior Research Fellow. Dr. Grinberg has authored and co-authored over 150 publications, including articles and book chapters, and has lectured and conducted courses worldwide. He is currently Editor-in-Chief of the Journal of Liquid Chromatography and Related Techniques, Editor of the Chromatographic Science book series, Editor of Supramolecular book series, and Co-editor of the Advances in Chromatography series. He is also a member of the Board of the Connecticut Separation Science Council and was a recipient of a Koltoff Fellowship of the Hebrew University of Jerusalem. Dr. Grinberg obtained his Ph.D. in Chemistry from the Technical University of lasi in Romania. He conducted postdoctoral research with Professor Barry Karger at Northeastern University in Boston, Massachusetts, and with Professor Emanuel Gil-Av at The Weizmann Institute of Science in Rehovot, Israel.
Dr. Michael Dong MWD Consulting, Norwalk, CT
Method Development and Quality Control Strategies for new Small Molecule Drug Candidates with Multiple Chiral Centers
Development of new chemical entities (NCE) with high chemical and chiral purity is a regulatory expectation in new drug development. Today, it is not unusual to have complex molecules with 3 or 4 stereogenic centers which show high affinity for disease targets. For the process development of these “multi-chiral” molecules, numerous analytical methods capable of separating all stereoisomers (enantiomers and diastereomers) must be developed rapidly to assess and control the stereochemistry of raw materials, intermediates and the final active pharmaceutical ingredient (API). Achiral reversed-phase (RP) methods, used to assess the overall chemical purity assays, can typically be developed to monitor the diastereomeric content in a single run. In many cases, these achiral methods often become the primary quality control (QC) and stability-indicating purity assay methods. This paper describes the method development and QC strategies used for these complex multi-chiral drug molecules, which include:
· Adoption of a 3-pronged HPLC method development template approach
· Development of a single RP method using multi-segment gradients for determination of overall purity and diastereomeric content
· SFC for rapid initial screening for chiral methods and determination of enantiomers
· 2-D LC for peak purity determination and achiral/chiral combinational assays
These strategies will be described with actual examples used during clinical development of several new drug candidates.
Figure 1. A single RP-HPLC method for determination of purity and diastereomeric content.
Dr. Michael W. Dong is a principal consultant in MWD Consulting focusing on consulting and training services on HPLC/UHPLC, pharmaceutical analysis, and drug quality. He was formerly Senior Scientist in Analytical Chemistry and Quality Control at Genentech, Research Fellow at Purdue Pharma, and Senior Staff Scientist at Applied Biosystems / Perkin-Elmer. He holds a Ph.D. in Analytical Chemistry from the City University of New York, and a certificate in Biotechnology from U. C. Santa Cruz. He has 120+ publications including a bestselling book on chromatography (Modern HPLC for Practicing Scientists, Wiley). He is an editorial advisory board member of LCGC magazine, American Pharmaceutical Review, and Chinese American Chromatography Association.
Dr. Atis Chakrabarti Tosoh Bioscience, LLC King of Prussia, PA
Analysis of the Fragments Generated from the Oxidation of Monoclonal Antibody Using Hydrophobic Interaction Chromatography
Chromatographic analysis plays an important role in studying the chemical degradation pathways as well as the oxidative stress of monoclonal antibody (mAb) drugs used to prevent various diseases and aging.
Oxidative stress is defined as an imbalance between the production of free radicals and the ability of the body to detoxify or neutralize by antioxidants. Inability to prevent oxidative stress may oxidative damage to the proteins within body. Scientists use a variety of oxidation reagents to mimic the fragmentation of mAb from chemical degradation and oxidative stress on the monoclonal antibodies. A variety of oxidation reagents are used as the mechanisms of these reagents is different. Hydrogen Peroxide (H2O2), 2,2'- azobis(2-amidinopropane) dihydrochloride (AAPH), t-butyl hydro peroxide(t-BHP) etc., are accepted in scientific community for simulating oxidative stress on the monoclonal antibody drugs. H2O2 and t-BHP primarily oxidize the two Met residues. 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), and H2O2 with Fe(II) oxidize both Met and Trp residues. Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Oxidation of mAb changes the overall hydrophobicity of the molecules, by changing the polarity of the oxidized form or the overall conformation of the fragments. Hydrophobic interaction chromatography (HIC) can therefore be used to characterize these oxidized mAb fragments under native condition based on the difference in hydrophobicity. Here we report the use of a 4.6 mm ID × 3.5 cm, 2.5 μm analytical HIC column with nonporous polymethacrylate base material for this study. Preliminary results show that this HIC column could successfully be used to separate the antigen binding Fab domain from the non-antigen binding domain as well as the other fragments obtained from further oxidation. The results were reproducible, required lower sample loading and with faster analysis time compared to other HIC columns. Mass spectrometric characterization of the oxidized mAb fragments is also reported.
Atis Chakrabarti earned a Ph.D. from North Bengal University, India on the design and synthesis of anti-proliferative probes. His post-doctoral research at the Indian Institute of Science focused on the molecular biology and biophysical characterization of bacterial thioredoxin. In 2000, he joined Thomas Jefferson Medical College researching diagnostic and therapeutic probes for PET imaging while utilizing different types of chromatography for the purification of the probes. Atis joined Tosoh Bioscience during July 2008 as a member of the Technical Service team supporting different modes of chromatographic research. Since October 2011, he became the Manager of the Technical Service. His group is responsible for customer support of the TSKgel HPLC columns, Toyopearl and TSKgel process media, for the purification of biopolymers and synthetic polymers using all modes of chromatography. He is also an ex-adjunct faculty member in the department of chemistry at Camden County College, NJ and Scranton University, teaching organic chemistry and toxicology. In leisure time he likes to travel and spend time with his family.
Derek Lohmann PSS USA Inc, Amherst, MA
Light Scattering without Refractive Index Increment - A New Approach to Calibrate SEC-Light Scattering Setups
D. Lohmann1, W. Radke2, J. Preis2, S. Lavric3
PSS USA Inc, Amherst, MA, PSS GmbH, Mainz, Germany, Melamin d.d., Kocevje, Slovenia
Size exclusion chromatography (SEC) with light scattering detection (SEC-LS) has become a popular method for polymer characterization. In contrast to conventional SEC, which yields molar masses only relative to a calibration curve, SEC-LS can provide absolute molar masses at each elution volume. This allows determining true molar mass distributions and molar mass averages. SEC-LS requires use of a light scattering instrument in conjunction with a concentration detector, typically a RI detector. The primary information obtained by the detectors are voltages, which have to be converted to the respective physical property measured. At present, calibration of SEC-light scattering detectors is either achieved by calibration using a reference liquid of known Rayleigh ratio, e.g. toluene, or by using well-characterized polymer standards for calibration. It needs to be understood that true molar masses are obtained by SEC-light scattering, even if the standards used to calibrate the SEC-light scattering setup are not of identical chemical structure as the analyte. This in in contrast to conventional SEC. If calibration is performed using a polymer standard, the molar mass and the specific refractive index increment, dn/dc, of the standard needs to be known. For molar mass determination of unknown analytes, knowledge of their refractive index increment, dn/dc is also required. Unfortunately the correct refractive increment is often unknown in the solvent applied, or its determination is difficult, e.g. in mixed solvents or in solvents containing salts or additives. The present contribution will describe an alternative approach to calibrate an SEC-LS setup and to determine the molar mass of a unknown analytes, requiring neither the refractive index increment of the sample, nor of the polymer used for calibration. Only the molar mass of the calibrant and the concentrations of the calibrant and the unknown samples are required. The new calibration approach does not require any refractive index increment. Consequently, experimental difficulties arising from preferential solvation as they are present in ternary systems (mixed solvent, salt containing eluents) are eliminated. Besides the theoretical approach, the contribution will provide experimental results, in organic as well as aqueous solvent, proving the suitability of the new approach.
Session: Current Approaches for Characterizing Recombinant Proteins, mAbs, ADCs, Protein Aggregates
Jared Auclair Biopharmaceutical Analysis Training Laboratory, Northeastern University Innovation Campus
Burlington, MA
Regulatory Convergence for Biologics through Capacity Building, Training and (other) Research
John de la Parra, William Hancock, and Jared Auclair
Over the last several decades the number of biologics, including biosimilar, approvals by international regulatory bodies has been on the rise. Currently, there is diversity in how biologics are approved around the world. Several international organizations, such as the ICH and others, are developing guidelines to be applied to the approval of biologics. To ensure successful implementation of these guidelines, training and capacity building is necessary. One goal of the Biopharmaceutical Analysis Training Laboratory (BATL) is to be the leader in regulatory convergence efforts for biologics through capacity building, training and research. Here, we will discuss the current state of regulatory convergence through training and capacity building as well as state-of-the-art research topics being applied to the approval of biologics. Finally, we will discuss current research projects under way at BATL.
Jared R. Auclair is currently an associate teaching professor in the department of chemistry and chemical biology at Northeastern University. In addition, Dr. Auclair is the Director of Biotechnology and Bioinformatics, as well as the Director of the Biopharmaceutical Analysis Training Laboratory, the Asia-Pacific Economic Cooperation Center of Regulatory Excellence in Biotherapeutics, and oversees the International Council for Harmonisation training. These latter appointments allow Dr. Auclair to collaborate with both academic researchers and industry in the area of biopharmaceutical development and analysis. He has expertise in mol. biology, protein biochemistry, analytical chemistry, protein crystallography, and biological mass spectrometry; and is interested in understanding the molecular mechanisms of neurodegenerative diseases as well as advancing diagnostics for Women’s health.
Daniel P. Donnelly Northeastern, University Boston, MA
Daniel P. Donnelly, Joseph P. Salisbury, Matthew G. Dowgiallo, Krishna C. Aluri, Jared R. Auclair, Steven A. Lopez, Roman Manetsch, Jeffrey N. Agar
Affiliations: Department of Chemistry and Chemical Biology, Northeastern University
Barnett Institute of Chemical and Biological Analysis, Northeastern University
Cyclic Thiosulfinates and Cyclic Disulfides Selectively Crosslink Thiols While Avoiding Modification of Lone Thiols
Current thiol crosslinking tools form “dead-end” modifications of lone thiols and are toxic, which prevents in-vivo use. We have developed new chemical tools, cyclic disulfides and cyclic thiosulfinates, that can selectively form crosslinks between thiol-pairs but avoid these terminal “dead-end” modifications. In a proof-of-concept study, we target the thiol pair on adjacent monomers of the Cu/Zn-superoxide dismutase (SOD1) native dimer (8 Å distance) with cyclic disulfides and cyclic thiosulfinates--including the drug and nutritional supplement lipoic acid—to form covalent crosslinks. Using an intact protein mass spectrometry assay, we show that in vitro crosslinking efficiency is high (100%) and fast (half-life ≈ 3 min). We determined, through cell-dosing experiments using HepG2 cells lines, that the EC50of our lead compound approaches the low µM range (1-5 µM) and that crosslinking proceeds in the presence of cellular reducing agents. LCMS of purified SOD1 from mice treated with these compounds confirms promising in vivo activity. The potential application of thiol-pair specific crosslinkers extends to many fields including protein/peptide-stabilizing therapeutics, quaternary structure elucidation, and polymer/hydrogel development.
Ewa Folta-Stogniew W. M. Keck Foundation Biotechnology Resource Laboratory
Yale School of Medicine Yale University, New Haven, CT
Application of Light Scattering in a Core Facility Setting
This talk will present several case studies of the application of Size Exclusion Chromatography (SEC) coupled with multi-angle Light scattering (MALS) in analysis of protein structure-function relationship, carried out at the Biophysics Resource of Keck Biotechnology Research Laboratory at Yale University School of Medicine. The examples include determination of stoichiometry of protein nucleic acid repressor complex, application of SEC/MALS for measurement of dimerization constant, as well as analysis of integral membrane proteins and application of three detector analysis for deciphering the oligomeric state of modified proteins or stoichiometry of protein complexes.
Roxana E. Iacoband John R. Engen Northeastern University, Boston, MA
Applicability of Hydrogen Deuterium Exchange Mass Spectrometry to address questions of higher-order structure for biotherapeutics
Hydrogen / Deuterium Exchange Mass Spectrometry (HDX-MS) is a critical tool for analyzing protein conformation and dynamics and has been widely utilized in the biopharmaceutical industry for higher-order structure characterization of protein therapeutics in both research and development. A strong attribute of HDX-MS is the ability to study proteins in solution in a more “native like” environment. Due to the speed, sensitivity and resolution of MS, all sorts of proteins can be interrogated; with the addition of ion mobility spectrometry, even mega-Dalton complexes are possible. One of the more sought-after utilities of HDX-MS is the investigation of protein-protein, or protein-ligand interactions. In this presentation I will show examples on how HDX-MS can be successfully employed to study small or large molecule binding to target proteins such as interleukins, kinases and deubiquitinating enzymes.
Amiss Sadiki Ph.D. candidate (Prof Sunny Zhou, Dept. of Chemistry and Chemical Biology)
Northeastern University, Boston, MA
Artifacts in the analysis of protein pharmaceuticals
Analysis is critical in the assurance of quality and compatibility of protein pharmaceuticals, such as biosimilars. However, given the complexity of biotherapeutic products and the analytical processes, analytical artifacts abound. Moreover, many of these artifacts are often overlooked, even when the process and results are reproducible. In this presentation, common and crucial artifacts will be reviewed, their mechanisms discussed and potential solutions proposed. I will discuss various artifacts, both false positives and false negatives, existing in the analysis of protein pharmaceuticals. An emphasis will be placed on attributes in relation to chemical transformation (e.g., deamidation and oxidation during sample preparation), labile nature of certain protein modification (such as glycation and trisulfide), unknown or not pre-defined proteins modification (such as crosslinking), and artifacts of analytical methods.
Session: Chromatography Method Development for Pharmaceuticals
Laura Blue Amgen, Thousand Oaks, CA
Platform Method Development Approaches to Enable Speed to Clinic for Synthetic Molecules
Laura E. Blue1, Kelly Nadeau2, David Semin1, Helen Yan1, Gang Xue2
1Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320
2Amgen Inc. 360 Binney St., Cambridge, MA 02141
As the demands for break through therapies and the competitive landscape continues to grow, the need to move molecules to the clinic more quickly is paramount. The average time from the start of pre-clinical studies to IND filing takes approximately five years, but new industry trends are pushing to cut this time in half. Additionally, as resources decrease and the number of molecules in the pipeline increase the need for gains in efficiency are apparent. The number of molecules reaching first in human (FIH) studies has increased 13% each year for the past five years, but typically no additional resources have been allocated. While decreases in timeline and resources continue, the responsibility to control the quality attributes is unchanged which demand novel approaches to method development.
In order to meet the timeline demands and ensure quality, platform method development approaches have been developed for assessing the quality attributes of synthetic molecules. Platform method development approaches have been implemented for achiral purity, chiral purity, and residual solvent content. Based on the assessment of a set of reversed phase columns for orthogonality, four columns have been selected for the achiral method screen in combination with mobile phases with varying pH. This workflow can be completed in 18 hours of run time and 2 hours of processing time, which has dramatically decreased the method development time for achiral impurity methods. This approach has provided suitable method conditions for 90% of the molecules assessed (n=17). Similar to the achiral approach, a platform method screening approach has been developed for chiral method development. A series of eight columns are screened under both reversed phase and normal phase conditions. Furthermore, a platform GC/FID method has been implemented to cover the most common residual solvents used in synthetic processes. These platform approaches have enabled improved speed to clinic while maintaining product quality.
Laura Blue received her PhD. in analytical chemistry from the University of North Carolina at Chapel Hill under James Jorgenson in 2012. Her work focused on the development of superficially porous particles for high efficiency separations. Following graduation, Laura joined the GC R&D group at Agilent Technologies. Currently, Laura is a Sr. Scientist at Amgen Inc. (Thousand Oaks, CA) in the Pre-Pivotal Attributes Sciences group within Process Development. Her current work focuses on quickly moving projects to FIH while maintain analytical control and quality.
Yuanyuan Li Mersana Therapeutics, Boston, MA
Analytical Characterization of Dolaflexin-ADCs
Antibody-drug conjugates (ADCs) are a promising class of oncology drugs that combine the tumor cell targeting ability of the antibody with the potent cytotoxicity of a small molecule drug. High drug-to-antibody ratio (DAR) antibody-drug conjugates offer the potential for greater efficacy than ADCs with lower DAR, but they are difficult to synthesize using conventional linkers and conjugation strategies. Mersana uses its unique Dolaflexin® platform which allows conjugation of 10 to 15 molecules of Auristatin F-hydroxypropylamide to the antibody via a biocompatible, biodegradable polymer linker. Dolaflexin is a semi-synthetic polymer composed of a hydrophilic polyacetal backbone functionalized with sidechain substituents for payload and maleimide linker attachment. The material is heterogeneous with controlled polydispersity index (PDI). The quality of Dolaflexin directly impacts the process and quality of the resulting Dolaflexin ADCs. This talk will introduce the analytical methodologies used to assess selected critical quality attributes of Dolaflexin.
Dr. Yuanyuan Li is currently employed with Mersana Therapeutics as a Senior Scientist in the CMC analytical team. She obtained her PhD degree from Prof. Milton Lee’s lab at Brigham Young University (BYU), where her research work was focused on development of monolithic capillary columns for biomolecules analysis. After graduation from BYU, she had a post-doc training with Dr. Jack Henion working on innovative sampling technique and method development for biological samples from pre-clinical and clinical studies. Before joining Mersana in 2017, she was a process analytical scientist at AbbVie North Chicago site.
Jinjian Zheng Merck, Kenilworth, NJ
Fast development of robust HPLC methods utilizing modelling software
A robust HPLC method is necessary to ensure that accurate and precise results are generated throughout the lifecycle of a pharmaceutical product. However, development of a robust HPLC separation can be very challenging due to complex sample composition, different instrument specification, changing laboratory environment and varying analyst skill levels. In this presentation, development of robust HPLC methods utilizing modelling software will be discussed. The topics include: 1) Screening of columns and mobile phases; 2) Optimization of chromatographic separation; 3) DOE evaluation of method robustness; and 4) Knowledge management. The results demonstrate that efficient development of fast and robust HPLC methods can be achieved by leveraging the power of software simulation.
Dr. Jinjian Zheng is currently a principal scientist at Analytical Commercialization Technology department, Merck & Co. Inc.. Dr. Zheng has developed and validated numerous HPLC methods to support the development and commercialization of both drug substances and drug products. He has extensive hands-on experience in HPLC method development using modeling software including ACD/Labs, ChromSword, DryLab and Fusion QbD. He is currently leading the efforts at Merck to design an efficient workflow for HPLC method development. Dr. Zheng is also interested in other separation techniques such as gas chromatography (GC) with vacuum UV (VUV) detection and field flow fractionation (FFF).
Ning Yang, Tawnya Flick and G. Charles Cheng Amgen, Cambridge, MA 02421
Advancing structural elucidation of pharmaceutical molecules by Ultra-high resolution and UVPD mass spectrometry
Recent advancement in instrumentation and software has enabled 1 million ultra-high resolution previously only accessible on FT-ICR platform to be available on an orbitrap platform. This breakthrough in resolution has opened the door for utilization of isotopic fine structure (i.e.15N, 13O, 32S, along with 13C) on pharmaceutical synthetic molecules and small peptides. Furthermore, UV Photodissociation (UVPD) in the same platform is achieved in the ion trap region, enabling an alternative fragmentation technique beyond CID/HCD that is less restrictive than ETD for fragmentation of singly protonated ions.
A Thermo Orbitrap Fusion Lumos Mass Spectrometer with 1 million resolution and UVPD was utilized for the study. Both direct infusion as well as LC-MS with generic gradient were utilized. Resolution of over 500,000 can be achieved at m/z 500 and lower using both direct infusion as well as in LC time scale. To maximize isotopic fidelity, MS data were typically acquired in SIM mode with 20 Da windows around the compound of interest. A 213nm solid state laser irradiate ions in the ion trap region prior to sending the fragment into the orbitrap. UVPD activation time ranges from 20 ms to 500 ms.
Pharmaceutical relevant molecules were evaluated on the Thermo Orbitrap Fusion Lumos MS with 1M resolution and UVPD options. Data from Eriocitrin, Adenine, and other pharmaceutical relevant molecules will be presented. Eriocitrin MS data were acquired in negative mode with 1M resolution, a resolution of close to 600,000 was achieved in SIM mode (Figure 1). At this resolution, 18O and 13C are well resolved for the M+2 peak (Figure 2). This allows isotope fine structures, along with mass accuracy and 13C relative isotope abundance to be utilized for structural elucidation.
In addition to manual interpretation, this level of isotopic fine structure can be readily assessed in high throughput manner with the FreeStyle software, which combines mass accuracy and isotopic fine structure to give a combined scoring. It is worth noting that in the case of Eriocitrin (Figure 3), the theoretical mass from the correct elemental composition is not the closest to the experimental data. Rather, the combination of mass accuracy and isotopic fine structure ranks the correct elemental composition as the top candidate.
Demonstration of UVPD as an alternative fragmentation for pharmaceutical relevant compounds will be presented. Preliminary data suggest UVPD can be a highly valuable tool for synthetic and peptide modality, in addition to peptide digest and intact applications. Examples shown in Figure 4and Figure 5are just two example of synthetic molecules where UVPD provides richer fragmentation information than HCD on the same instrument. The utilization of UVPD on LC time scale will also be demonstrated.
Figure 1. SIM spectrum of Eriocitrin with Elemental Composition of C27H32O15 for Neutral Molecule
Figure 2. M+2 peak for the MS spectrum
Figure 3. Scoring of top candidate for experimental MS data
Figure 4. Comparison of UVPD (50ms activation time) vs. HCD spectrum of Eriocitrin
Figure 5. Comparison of UVPD (500ms activation time) and HCD spectrum of Adenine
Session: Supercritical Fluid Chromatography
Mr. James Bradow Pfizer, Groton, CT
Achiral SFC: Yesterday, Today and Tomorrow
With the expansion of SFC into purification labs, the initial drive was towards fast and efficient chiral separations that would remove the need for high cost solvents, increase throughput, and increase purity. As this proved to be very successful, an interest in looking at achiral separations for SFC started to take hold. The initial issue that was encountered was what columns, solvents, and methods should be used to build an efficient screen that can cover a broad chemical space, but is efficient. With this in mind, our lab has gone through a progression of columns that has shown to enable a wide range of chemical space. How we move forward to discover new phases that will keep up with the ever changing landscape of medicinal chemistry compounds is the challenge. This presentation will focus on the diverse screening panel and methods we have developed to adapt to a changing chemical landscape.
Mr. Carl Griffin Agilent, Wilmington, DE
Method Development for Chiral HPLC
Supercritical Fluid Liquid Chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is often used for the separation of chiral compounds. SFC uses columns that are similar to standard HPLC systems. Methods are typically developed on one of four stationary phases. Each phase is specifically designed to react with the target analyte. Development of efficient and effective methods that result in effective separation of chiral compounds is often more challenging than conventional HPLC. Principles are similar to those of high performance liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. Chiral Poroshell columns have a wide variety of applications that typically can be run in less time than most chiral columns, and are suited for LC as well as SFC.
Session: Metabolomics & Proteomics
Marcy Balunas, Ph.D. Associate Professor University of Connecticut Department of Pharmaceutical Sciences
Storrs, CT
Interaction-driven Molecule Discovery from Host-Microbe Symbioses
Microbial symbioses are increasingly recognized to play an integral role in host structure and function. Co-evolution of these benign and/or beneficial relationships has been the focus of numerous microbiology studies, although the role of secondary metabolite interactions has received considerably less attention. Small molecule interactions in these host-microbe symbioses are likely to contribute to the complex molecular conversations occurring between bacterial symbionts, eukaryotic hosts, and their pathogens/prey. Given that these host-microbe associations have naturally evolved to select for biologically active bacteria, they provide a source of secondary metabolites more likely to have potent medicinal activity and thus be poised for future preclinical drug development. We utilize several unique host-microbe symbioses to explore interactions between eukaryotic hosts and their associated bacteria, integrating natural products and analytical chemistry, medicinal and synthetic chemistry, microbial chemical ecology, and biological screening with advanced molecular biology to provide a comprehensive understanding of secondary metabolite production in these symbioses. Our long-term goal is to use insights gained from these host-microbe symbioses to discover and develop new druggable lead compounds for a wide range of human diseases. Recent developments from these studies will be presented including experiments to allow for competitive interactions to enhance metabolite production and further our understanding of microbial interactions.
Dr. Balunas is an Associate Professor of Medicinal Chemistry at the University of Connecticut. Dr. Balunas’s research group focuses on host-associated microbial communities as unique sources for natural products drug discovery, using these to search for new, targeted drug leads and to address critical questions within the broader field of chemical ecology. Her research ties together an in-depth understanding of how secondary metabolites function in their environment with the application of these insights to discover new medicines for a wide range of human diseases. Dr. Balunas received a dual B.A./B.A. in Chemistry and Biology from the University of Rochester, a M.S. in Plant Ecology from the State University of New York College of Environmental Science and Forestry, and a Ph.D. in Pharmacognosy from the University of Illinois at Chicago. She conducted her postdoctoral research on marine natural products drug discovery at the Scripps Institution of Oceanography at the University of California San Diego in conjunction with the Smithsonian Tropical Research Institute in Panama City, Panama.
Xudong Yao, Ph.D. Associate Professor University of Connecticut Department of Chemistry
Storrs, CT
Scaling of Proteome-wide Reactivity between 0 and 1: a versatile technology platform for quantitative chemical proteomics
Quantitative analysis of the proteome-wide reactivity to small molecule probes has broad utilities in drug development and precision medicine. The talk presents a modularly-designed technology that uses non-isotopic chemical probes for the quantitation of probe-reacted proteins proteome-wide, and a scaling analysis of the measurement results for reporting the protein reactivities to the probe regardless of reaction mechanisms.
Dr. Xudong Yao is an associate professor at the Department of Chemistry and the Institute for Systems Genomics of the University of Connecticut. His group develops novel separation and measurement technologies for mass spectrometric and omic analysis. Prior to working at the University of Connecticut, he held several biotechnological, pharmaceutical, and academic positions in China, Switzerland and the United States.
Daniel Schwartz, Ph.D. Associate Professor University of Connecticut Department of Physiology and Neurobiology
Storrs, CT
Analysis of Thousands of Eukaryotic Phosphorylation Sites in a Prokaryotic Organism
As mediators of intracellular signaling, protein kinases are among the most important cellular enzymes, and not surprisingly their dysfunction has been associated with a myriad of human disease conditions. Yet, our knowledge of kinase specificity, namely the determinants in primary structure associated with the recognition of substrates, remains limited. This talk will describe a fast and inexpensive proteomic methodology, called ProPeL, to query eukaryotic kinase specificity through the use of bacteria, which serve as living peptide libraries for thousands of simultaneous kinase reactions. Examples of using ProPeL to understand disease mutations, as well as the rational design of alternate kinase specificities, will additionally be discussed.
Dr. Schwartz is an Associate Professor in the Department of Physiology and Neurobiology at the University of Connecticut where his research focuses on the development of experimental and computational tools to discover and understand patterns in protein sequences. Software developed in the Schwartz Lab has been cited over 1000 times and has had over 40,000 users from approximately 130 countries. Since 2015 Dr. Schwartz has additionally served as the Director of the Center for Open Research Resources and Equipment (COR²E), a center with a mission to enhance research at UConn and break down academic silos by providing access to state-of-the-art research equipment and developing interactive research and collaboration software.
Latest Additions
Avinash Dalmia, PerkinElmer, Norwalk, CT
A Novel LC-MS/MS Method with Dual Electrospray and APCI Source for Analysis of All Pesticide & Mycotoxin Residues in Cannabis
Over 30 states in the U.S. have legalized the use of recreational or medical cannabis because of therapeutic benefits for ailments such as cancer, multiple sclerosis, and ALS. Recently, government in Canada passed a law to allow its residents to use cannabis for medical and recreation purposes. Apart from Oregon state regulatory limits for about 59 pesticides in cannabis, the state of California and Canada has issued more stringent regulatory limits for about 65 and 95 pesticides residues in cannabis flower, edibles and oil. Normally pesticide analysis in cannabis and other food matrices is done by both GCMS and LCMS since some nonpolar and chlorinated pesticides are difficult to ionize with electrospray and APCI ion sources used in LCMS systems. LCMSMS method with electrospray and APCI source was used for low level analysis of all pesticides (including the very hydrophobic and chlorinated pesticides analyzed by GCMS) and mycotoxins in cannabis. The overall sensitivity for pesticides and mycotoxins was between 1-500 ppb in cannabis, well below regulatory limits set by the Canada and the states of Oregon and California for pesticides and mycotoxins analysis in cannabis. The ability to screen and quantitate all pesticides, including the very hydrophobic and chlorinated GCMS amenable, in cannabis with LCMSMS only with dual ESI/APCI source makes this a novel way of screening and quantitation of pesticides in cannabis and different matrices with a single instrument. Long term stability data for pesticide analysis in cannabis was collected using a triple quadrupole mass spectrometer fitted with dual electrospray ionization source and atmospheric chemical ionization source ( APCI) and combined with a heated and self cleaning laminar flow interface. Long term stability data for pesticide analysis in cannabis showed that response RSD over 1 week for majority of pesticides was between 2 to 10 %. These results demonstrated that the heated self-cleaning interface with laminar flow interface in an LCMSMS system would reduce maintenance needs of the LCMSMS based method for pesticide analysis in dirty matrices such as cannabis. A simple solvent extraction method was used for extraction of pesticides and mycotoxins from dried cannabis flower. The recovery of all of pesticides and mycotoxins was between 70-120 % with RSD less than 20 %.
Avinash Dalmia received his PhD in chemical engineering in 1996 from Case Western Reserve University in Ohio. Dr. Dalmia is a Principal Scientist for the cannabis application group at PerkinElmer. During the last 10 years at PerkinElmer, he has been involved in carrying out studies for design and optimization of different mass spectrometry ionization sources such as electrospray, APCI and direct sampling analyzer and has developed more than 60 applications using HPLC and GC coupled with different MS instruments. His most recent research interest is in the analysis of low levels of pesticides in cannabis related matrices. During his career, he has presented his research accomplishments in research areas varying from electrochemical sensors, fuel cells, catalysts, thermal and fluid flow modeling, MS sources, HPLC/MS and GC/MS applications and has published 15 papers and 20 patents.
Eduard Rogatsky, Ph.D. Research Associate Professor, Albert Einstein College Department of Medicine (Endocrinology) Bronx, NY
A Journey from MRM Transition to Reproducible Clinical Assay of Peptide Biomarkers
The typical method of choice for quantitative LC/MS analysis of small molecules and tryptic digests is MRM (SRM). However, in our practice of routine quantitation of large peptide hormones/biomarkers, we have found that for trace level analytes measurement (e.g. low pg/ml), MRM does not provide satisfactory results compared to SIM. Post-translation modifications, an influence of tertiary structure on electrospray ionization and especially different fragmentation pathways of peptides by collision-induced dissociation (which generates many low-abundant fragments) are common factors reducing MRM-based assay sensitivity.
We have taken an alternative approach to MRM based assays, by implementing 2D LC-LC/MS with stable isotope labeled internal standards and have achieved highly reproducible quantitation of low fmole quantities on the column for large peptides/small proteins such as human insulin, glucagon, insulin C-peptide and proinsulin.
Dr Rogatsky is a Research Associate Professor of Medicine at Albert Einstein College of Medicine (NY, Bronx). He completed his M.Sc degree in physical chemistry at the Belarus State University in 1990. In 1998 he completed his Ph.D. in bioanalytical chemistry at the Bar-Ilan University in Israel. At the end of 1999, he started a post-doctorate project at Albert Einstein College of Medicine and joined the faculty in 2001. Since 2001, Dr. Rogatsky has been working in the field of clinical mass spectrometry. He has over 25 years of experience in the various fields of chromatography. Currently, he serves as the Editor-in-Chief for the Journal of Chromatography and Separation Techniques (OMICS publishing group). He has given over one hundred scientific presentations, invited lectures and publications mostly as the first author. Among various awards and honours, Dr Eduard Rogatsky received the Recognition Award from the New York State Commissioner for the Department of Health in 2016.