Pittcon 2019

Philadelphia, Pennsylvania
March 17 – March 21

Waters Posters, Orals, and Contributed Sessions at Pittcon

See details below for date and session information. Downloadable versions will be available for many of Waters technical presentations, after they are presented. Be sure to come back to visit!

Monday
Tuesday
Wednesday
Thursday

Monday, March 18, 2019

ORAL PRESENTATION

Session: 560
The Thirtieth James L. Waters Symposium on Ultra High Pressure Liquid Chromatography (UHPLC)

1:30pm – 4:45pm
Location: 118B
Organizer(s): Annette S. Wilson, Stephen Weber

If You Build It, They Will Come: The Birth of ACQUITY UPLC Technology
Presenter: Ed Bouvier, Waters Corporation 

Tuesday, March 19, 2019

POSTERS

10:00 am – 12:30 pm
Location: Expo floor

Session: 940-7
Small Scale Natural Product Isolation Using UPLC with Mass-Directed Purification
Author: Jo-Ann  Jablonski, Waters Corporation
Co-author: Andrew J Aubin, Waters Corporation

Throughout history, the benefits of adopting natural remedies for a variety of health-related maladies has been attractive, as the plants from which these therapeutics are obtained are often readily available.  Plant flavonoids, with their diverse biological properties, can be used as anti-allergenics, antivirals, and anti-inflammatories, or possess heart-protective vasodilating properties.  To fully understand the impact of specific flavonoids in living systems, material needed for experimental studies must often be isolated from very complex matrices.  Although traditional isolations have usually been accomplished using extraction followed by chromatography with UV detection, this long and arduous process is riddled with ambiguity due to the lack of specificity in target compound identification.  Tedious workup of fractions collected by UV-only detection requires time and resources.  Mass detection, with its high specificity and sensitivity, readily discriminates the product of interest from complicated sample mixtures, thereby reducing downstream sample processing time.  With improvements in column stationary phases and analytical instrumentation, good separations are more easily realized.  The fluidically-optimized flow path of the UPLC, combined with a specially-designed low dispersion fraction collector, enable the mass-directed isolation of sharp, narrow product peaks.  In this study, we illustrate the utility of the ACQUITY UPLC H-Class and Waters Fraction Manager-Analytical (WFM-A) Systems for the analysis and isolation of a natural product at the small scale.  Fast, targeted isolation increases purification efficiency by reducing unnecessary sample handling while generating just enough product for future experiments.

Application: Drug Discovery

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 940-22
Carryover Mitigation Using Needle Wash Solvent Chemistry and Autosampler Features of a UPLC UV/MS System
Author: Chris DesJardins, Waters Corporation
Co-authors: Zhimin Li, Patricia McConville, Waters Corporation

The greater sensitivity provided by mass detection, as compared to UV, can result in observed carryover or sample left over from a previous injection that may interfere or co-elute with analytes of interest. This phenomenon can interfere with accurate quantitation, particularly at low levels. While carryover has many potential sources, needle wash solvent chemistry and autosampler design both play critical roles in controlling instrument related carryover.  In this study, an ACQUITY UPLC H-Class PLUS system configured with both a photodiode array and a mass detector were used to examine the impact of needle wash solvent chemistry and wash mode on carryover of granisetron HCl, an antinauseant and antiemetic agent used in cancer therapy.

Application: Pharmaceutical

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 940-23
The Analysis and Quantitation of Itaconic Acid in Blood Serum Using HILIC (Hydrophilic Interaction Chromatography) Coupled to a Triple Quadrupole Mass Spectrometer
Author: Chris Henry, Waters Corporation
Co-authors: Mary Lame, Paula A Orens, Paul Rainville , Waters Corporation

Itaconic acid is a branched fatty acid which has been identified as a potential biomarker in alerting to a number of medical conditions including:  gestational diabetes mellitus (GDM) and rheumatoid arthritis. Early detection and quantitation of this analyte in blood could enable earlier medical intervention and improve patient health.

Itaconic acid being a small polar molecule is not easily retained on reversed- phase liquid chromatography and has in the past been analysed using gas chromatography coupled to a mass spectrometer with a derivatisation step required in the sample preparation as well as by a number of ion-pair liquid chromatography methods. While both of these methodologies have proven to be successful they both have draw backs due to their laborious nature and the need for dedicated instruments.

Hydrophilic liquid chromatography (HILIC) has been shown to enable the retention and quantitation of highly polar compounds by utilising an aqueous layer on the surface of the analytical column which interacts with polar molecules and elutes analytes in order of increasing hydrophilicity.

In the work presented here, we describe a HILIC method using an 100mm x 2.1mm, sub 2um Amide column with a UHPLC instrument coupled to a triple quadrupole mass spectrometer. Itaconic acid was extracted from blood serum using an anion exchange solid phase extraction cartridges, and analysed with internal standard with no derivatisation step required in sample preparation.

LLOQ’s of approximately 1ng/mL were achieved along with linearity of between 3-4 orders of magnitude.

Application: Bioanalytical

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 940-24
Evaluation of an Open Ambient Ionisation Source Coupled to a Portable Mass Detector as a Tool for the Rapid Detection of Undeclared Active Ingredient in Online Health Supplements
Author: Chris Henry, Waters Corporation
Co-authors: Paul Rainville , Waters Corporation

Dietary supplements are used by millions of consumers to improve health, maintain wellness, or to support a more challenging lifestyle. Some of these supplements address conditions that many regard as shameful, awkward or otherwise difficult to discuss with a physician, like sexual dysfunction or excessive weight gain.

Consumers can sometimes choose supplements because they want a ‘safe’ and ‘natural’ alternative to drugs that are contraindicated for health reasons – such as a heart condition. Almost exclusively, FDA approved erectile dysfunction medication are phosphodiesterase type 5 (PDE5) enzyme inhibitors2, e.g. Sildenafil (Viagra, Pfizer). This class of drug can be fatal when taken with nitrate vasodilators like nitroglycerin.

The DART QDa System integrates the Waters ACQUITY QDa Mass Detector with the DART Ion Source from IonSense to enable a rapid, sample preparation-free solution screening of uncontrolled dietary supplements. The DART ion source combined with the ACQUITY QDa Detector generates easy to interpret mass spectral information in seconds.

Within this body of work we tested a variety of dietary supplements obtained online, claiming 100% herbal treatments for a variety of conditions including impotence, obesity and rheumatism. All but one of the examples was cited on the FDA website as containing undeclared pharmaceutical ingredients.  We were able to detect undeclared compounds in all cases using in-source fragmentation for additional specificity.

Application: Consumer Products

Methodology: Mass Spectrometry

Session: 960-4
The Effects of SFC Preparative Scale-Up on Throughput, Purity and Recovery of an Impurity in an API Mixture
Author: Catharine Layton, Waters Corporation
Co-authors: Andrew J Aubin,  Jo-Ann Jablonski,  Jacquelyn Runco, Waters Corporation

Scale-up of SFC analytical methods to preparative scale allow laboratories to generate purified bulk quantities of target compounds. In some laboratories, users are provided with an analytical method from which an isolate of a specified purity and quality must be generated within strict timelines. The success of achieving this task depends directly upon the accuracy of the scale-up procedure. In this poster we will describe the preparative scale-up of an analytical method for isolation of milligram (mg) to gram (g) quantities (per run) for a mixture of an API and its associated impurities. A cost and time analysis is provided after scale-up as to demonstrate the relationship between column size and throughput.

Application: Pharmaceutical

Methodology: Separation Sciences

Session: 960-13
Small Scale Purification of Fractions from a Complex Pharmaceutical Formulation Using an Analytical Fraction Collector and a UHPLC-MS System
Author: Fadi Alkhateeb , Waters Corporation
Co-authors: Ronan Cleary,  Paul Rainville, Waters Corporation

Isolating active pharmaceutical ingredients from excipients can be challenging especially in complex pharmaceutical formulations.  Researchers with such challenging samples frequently use multidimensional systems to separate analytes of interest from other components. While the use of such systems is normally sufficient, the setup may not always be available or practical. In this study we demonstrate the use of a microscale analytical fraction collector in conjunction with an ultra-high pressure liquid chromatography/mass spectrometry system to purify, separate and identify several components of a model complex pharmaceutical formulation.  An over-the-counter cold and cough syrup (DayQuil) was used as a model complex pharmaceutical formulation in this work. Multiple modes of fraction collection will be demonstrated. The advantages of using mass directed collection of fractions will also be presented and discussed.

Application: Pharmaceutical

Methodology: Liquid Chromatography/Mass Spectrometry

Tuesday, March 19

2:00 pm – 4:30 pm
Location: Expo floor

Session: 1280-1
An Efficient Method for the Determination of Trace Excipient Impurities in Biotherapeutic Drug Products Containing Polysorbate
Author: Robert Birdsall, Waters Corporation
Co-authors: Brooke Koshel, Ying Qing Yu, Ximo Zhang, Waters Corporation

Polyoxyethylene sorbitan-based nonionic surfactants such as PS-80 and PS-20 are commonly used in formulated biopharamceuticals to reduce protein denaturing, aggregation, and adsorption to surfaces. Degradation of surfactants in formulated drugs can decrease overall product efficacy and safety, thus requiring methods to demonstrate drug products are safe and efficacious. HPLC-based separations using wide-bore columns often lack the sensitivity and efficiency needed by today’s standards when considering factors such as post-column dispersion and long run-times and their impact on assay performance. Efficient methods that are robust and can incorporate technology that offer improved sensitivity and throughput would be beneficial in ensuring drug product and safety over the products lifecycle.

The degradation of PS-80 and PS-20 through radical autoxidation or enzymatic processes results in the cleavage of the hydrophobic ester tail to produce a fatty acid that is UV-active and amendable to MS detection. A 5 minute assay was developed on a UPLC platform to minimize system dispersion under isocratic conditions for improved separation performance of the fatty acid. Using standards, assay L.O.D. was determined to be 0.24 ppm for UV-based detection and 60 PPB for MS-based detection.  Hydrolysates of infliximab (PS-80 containing) and trastuzumab (PS-20 containing) were used as representative samples to test the applicability of the proposed method. Using an internal standard as a single-point calibration, PS-80 was calculated to be present in the infliximab sample at a concentration of 0.065 mg/mL (% RSD = 4.07) based on fatty acid extract. Similar results were calculated when using a multi-point calibration curve to assess assay robustness (0.059, RSD = 4.40).This study demonstrates a robust method for the determination of fatty acid degradants and assessment of PS-80 and PS-20 concentration in drug products for straightforward deployment in a laboratory setting.

Application: Pharmaceutical

Methodology: New Method

Session: 1240-16
Chiral Purification of Iridium (III) Complexes by SFC
Author: Catharine Layton, Waters Corporation
Co-authors: Andrew J Aubin, Waters Corporation; Jacquelyn Runco, Thar Process

Iridium (III) complexes are well studied phosphorescent materials used in organic light-emitting diode applications.  Octahedral iridium complexes bearing at least two bidentate ligands exhibit intrinsic metal-centered stereochemistry.  Interestingly, different photophysical properties exist between enantiopure and racemic emitters, which affect device performance.  During synthesis of Iridium (III) complexes, stereoisomers are formed, and in order to obtain enantiopure complexes, chiral resolution is required to isolate the pure enantiomers.

The chiral separation of Iridium (III) complexes has been previously accomplished by normal phase chiral HPLC.  SFC, however, has many advantages over HPLC, especially for chiral separation and purification.  Here, chiral purification of Iridium (III) complexes using SFC will be demonstrated.  Method screening and development using the Waters ACQUITY UPC2 system will be discussed along with scale-up strategies to improve method transfer.  Purification using the Waters Prep SFC 150 Mgm System and fraction analysis on the Waters ACQUITY UPC2 System will be shown.

Application: Material Science

Methodology: Liquid Chromatography

Session: 1240-21
Chiral Purification of Iridium (III) Complexes by SFC
Author: Zhimin Li, Waters Corporation
Co-authors: Paula Hong, Patricia McConville, Waters Corporation

In various industries, liquid chromatography (LC) laboratories are constantly challenged to analyze more samples in less time.  Some common practices to shorten LC analysis time include increasing flow rate, using a shorter column and reducing column re-equilibration time. However, there are limits to the extent that these parameters can be modified without impacting chromatographic performance. Parallel column regeneration is a solution where the total analysis time is significantly reduced with no impact to the methods being run.

By adding an additional pump and utilizing the switching valves of the column compartment, a standard LC system can be configured to perform parallel column regeneration. In this study, we will use a 2D (two-dimensional) LC system which is equipped with two binary pumps and switching valves within the column manager. A food application will be used. Sample analysis is alternated between two identical columns with identical flow paths. For example, one sample is injected and separated on the first column, while the second column undergoes washing and re-equilibration. Once the separation is complete, the valves switch positions, and the subsequent injection is directed to the second column for analysis while the first column undergoes washing and re-equilibration.  With this configuration, the washing and re-equilibration steps can be excluded from the cycle time. Depending on the particular LC method conditions, including column dimensions, programmed gradient time and flow rate, the overall analysis time can be reduced up to 50%.  With reduced analysis time, increases in productivity or throughput are realized.

Application: High-Throughput Chemical Analysis

Methodology: Liquid Chromatography

Wednesday, March 20, 2019

ORAL PRESENTATION

8:30 am – 8:50 am
Location: 121A

Session: 1460-1
Streamlined Method Development for Screening Active Pharmaceutical Ingredients in Cough and Cold Medication Using a Systematic Protocol
Presenter: Margaret Maziarz, Waters Corporation
Co-author: Paul Rainville, Waters Corporation

Method development is process of screening a range of chromatographic parameters to generate conditions for reliable separation. While many approaches to method development are adapted across laboratories, the ultimate goal is to develop a robust and reproducible method that generates accurate results.

While evaluating one-factor-at-a-time is an easy to perform approach, but can be time-consuming and may not provide a robust method. A quality-by-design (QbD) is a statistic based approach that generates design of experiments (DOE) to comprehensively study interactions of multiple variables to identify operating space region of the method. It provides the most information about method robustness, but requires a specialized software platform. A systematic approach is based a protocol with predefined experimental setup that consistently evaluates major selectivity factors during method development, which maximizes the understanding of method capabilities and robustness.

In this work, we employ a systematic protocol to develop a UPLC method for screening active pharmaceutical ingredients found in common multicomponent over-the-counter (OTC) cough and cold medication. The protocol includes scouting, screening, and optimization steps, each designed to investigate chromatographic parameters systematically. We will utilize both UV (PDA) and mass data to accurately identify and track all of the components during method development and to confirm peak homogeneity in the final method. We will demonstrate that a systematic protocol enables quick development of reproducible and robust methods, which increases chances of successful method validation.

Application: Pharmaceutical

Methodology: Liquid Chromatography

SYMPOSIA

9:45 am – 10:20 am
Location: 115C

Session: 1360-4
LC Column Innovations to Facilitate the Analysis of Pharmaceuticals
Author: Matthew A Lauber, Waters Corporation
Co-authors: Bonnie A Alden,  Cheryl Boissel,  Martin Gilar,  Jennifer M Nguyen,  Paul Rainville, Susan C Rzewuski, Kerri M Smith, Thomas H Walter,  Qi Wang,  Kevin D Wyndham, Waters Corporation

The analytical approaches for assaying pharmaceuticals have been forever changed by the availability and utility of liquid chromatography (LC).  Because of their effectiveness in profiling active ingredients, degradants and product related variants, LC separations now represent some of the most well established techniques for analyzing both small molecule drugs and biotherapeutics.  Facilitating this adoption rate has been the continuous improvement in LC column technology, wherein insights about particle construction, surface chemistry and mobile phase systems have led to numerous compelling innovations.

In the pursuit of higher and higher efficiency, stationary phase particles have been miniaturized and solid core morphologies have been adopted.  Shorter analysis times can now be achieved without sacrificing resolution.   Carefully optimized surface chemistries have also made it possible to minimize undesirable secondary interactions as well as to reduce the dependence of a separation on mobile phase additives or even afford an advantageous mixed mode retention mechanism.  Moreover, novel mobile phase systems have been developed so as to improve the quality, sensitivity and robustness of particular assays.

We will describe a number of these innovations that have been of impact to the analysis of pharmaceuticals.  A number of topics will be presented, ranging from the use of charged surface stationary phases for mixed mode retention of anionic small molecules to the development of a universally applicable pH gradient ion exchange separation for charge variant profiling of monoclonal antibodies.

Application: Pharmaceutical

Methodology: Liquid Chromatography

POSTERS

Wednesday, March 20
10:00 am – 12:30 pm
Location: Expo floor

Session: 1550-24
LC-MS/MS Analysis of Legacy and Emerging Perfluoroalkyl Substances (PFAS) in Environmental Water Samples

Author: Keil Brinster, Waters Corporation
Co-authors: Paula A Orens,  Kari Organtini, Ken Rosnack, Douglas Stevens, Waters Corporation

PFAS are common, persistent environmental contaminants used in the production of many consumer products. Due to their amphiphilic properties, they are used as surfactants and for nonstick, stain, and water resistance coatings. PFAS are also a major component of fire fighting foams used for suppression of fuel fires. Global widespread use of these compounds over decades has led to their release into the environment and PFAS are classified as persistent organic pollutants (POPs).  Currently, there are no legal regulations pertaining to PFAS monitoring, although the most common PFAS (PFOS and PFOA) are included in many advisory guidelines. The United States EPA has established a drinking water health advisory level of 70 ppt (ng/L) for total levels of PFOS and PFOA. In Europe, the Water Framework Directive and Drinking Water Directive have set minimum quality standards of PFOS and PFOA which range from the ppb to sub-ppt levels. Such examples of monitoring guidelines demonstrate the need for highly sensitive analytical measurements to detect PFAS.  Typically to reach the sub-ppt levels, sample enrichment prior to LC-MS/MS analysis is required. Sample prep similar to that described in the ISO 25101 method is typically applied for enrichment of PFAS in water samples. It will be demonstrated that ISO 25101 can be expanded to a wide range of PFAS compounds. Data provided will demonstrate the use of a weak anion exchange SPE cartridge for the analysis of approximately 40 legacy and emerging PFAS compounds, including GenX. The method was assessed using four types of environmental water samples (surface, ground, influent and effluent). The extended method was found to be robust in all types of matrices tested with detection limits in the low to sub ppt range, making this method in compliance with the guidelines set in both the US and EU. Percent recoveries were within the prescribed range of 70 – 130 % and method repeatability was assessed with %RSDs below 15% over six replicates.

Application: Environmental

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 1560-25
Quantitative Analysis of Erucamide Slip Agent in Polyethylene by Convergence Chromatography

Author: Isabelle Vu Trieu, Waters Corporation
Co-authors: Dayong Sun, Henkel

Erucamide is a slip agent widely used in polyolefin films. A method was developed using the Waters UPC2 convergence chromatography system coupled with mass spectrometry to quantify erucamide slip agent in thin films of polyethylene. This method is significantly faster than traditional methods, enabling the quantitative analysis of erucamide in less than 2 minutes.

Application: Polymers and Plastics

Methodology: Liquid Chromatography/Mass Spectrometry

Thursday, March 21, 2019

SYMPOSIA

9:45 am  – 10:20 am
Location: 120B

Session: 1660-4
A Cyclic Ion Mobility – Mass Spectrometry Instrument with High Resolution and Multi-Function Capability
Presenter: Kevin Giles, Waters Corporation
Co-authors: Jakub Ujma, Waters Corporation

There is continued interest in the combination of ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) either from the added peak capacity provided by IMS or in the ability to determine collision cross section (CCS) values of analyte ions.  Along with a growth in application areas, there have been significant advances in hyphenated IMS-MS technology from both academic researchers and instrument manufacturers alike. Some of the key IMS technology advances have been in enhancing ion transmission, increasing resolution and more recently providing additional analytical functionality.  One such technology is a multi-pass cyclic ion mobility (cIM) separator which has been embedded in a Q-ToF instrument.  The cIM device not only provides higher mobility resolution through the multi-pass separation but also multi-function operation leading to IMS{n} capability whereby mobility selected species can be extracted, activated and further separated and so on, prior to mass analysis.  The cIM device is a stacked plate ion guide with in excess of 600 electrodes which provide a 100 cm path length around which travelling voltage waves pass to provide mobility separation.  A key component of the cIM device is a novel electrode array which is used to facilitate ion entry / separation / exit and is central to the IMS{n} capability. Here the design and performance of the cIM device will be presented with particular emphasis on the multi-function capability.

Application: Bioanalytical

Methodology: Mass Spectrometry

ORAL PRESENTATION

2:30 pm – 2:50 pm
Location: 126A

Session: 2010-4
Improving Glycan Profiling in Biopharmaceutical Process Development Using Spectral Library
Presenter: Ximo Zhang, Waters Corporation

Glycosylation is one of the most common and complex post-translational modifications on monoclonal antibodies. Due to its direct correlation to the efficacy of antibody-based drugs, glycosylation has been recognized as a critical quality attribute (CQA) of drug product. To ensure the accuracy and consistency of glycosylation profiles, the suitable characterization and monitoring method must be in place for product quality throughout the product lifecycle. To this end, methods that can increase the confidence in glycan profiling are highly desired for process development and control.

MS detection has been increasingly applied in regulatory environment as an orthogonal detection technique for its high sensitivity and specificity of measuring product quality attributes. In this study, a streamlined LC-FLR/MS method is developed to improve glycan profiling in process development of biotherapeutics through a spectral library matching algorithm. A multi-parameter data processing and reporting method was created for automated new peak detection and monitoring of glycans. By implementing orthogonal matching techniques using both retention time and MS information, highly robust spectral matching was achieved for identification of unknown glycans and co-elutions in glycan profiling. Together, the spectral library enabled by orthogonal techniques increased confidence and productivity of glycan profiling in process development of biotherapeutics.

Application: Pharmaceutical

Methodology: Liquid Chromatography/Mass Spectrometry

WORKSHOP

3:10 pm– 3:30 pm
Location: 120B

Session: 1970-7
Ultra-High Resolution Semi-Preparative Liquid Chromatography Application to Impurity Identification in Drug Samples
Presenter: Fabrice Gilles Gritti, Waters Corporation

As requested by regulations, a recurrent problem in the pharmaceutical industry is the unambiguous identification of unknown impurities present in a concentrated solution of active pharmaceutical ingredients (API). Standard separation methods are needed to isolate the impurity from the API and to collect about 1 mg of impurity (at least 90% purity) for complete structure elucidation by nuclear magnetic resonance (NMR). In some specific cases, the impurity peak nearly co-elutes with the main API: standard preparative methods fail and an alternative separation technique that combine both ultra-high resolution and semi-preparative capabilities is needed.

In this presentation, based on a twin-column recycling chromatography process (RCP) coupled to a fraction collector (FC), a new easy-to-use and automated LC technology, which combines high-resolution and semi-preparative capabilities, is presented. The principle of RCP is to insert a switching valve in between two identical columns connected in series enabling the user to transfer the separation zone from one to the second column and to further improve the resolution of the compounds at low pressures. First, the capability of the prototype RCP is demonstrated for the challenging separation of chiral compounds ([gamma]-phenylbutyrolactone, [alpha]=1.013) and deuterated benzene isotopes (H6, -D3, and -D6, [alpha]=1.020) using standard HPLC columns (3 [micro]m particles) below 400 bars. Next, the RCP is coupled to the FC and run automatically for the 1 mg preparation of an unknown impurity present in trace amounts in the actual stock solution of API (10 g/L). 100 [micro]L of stock solution are injected into 4.6 mm x 150 mm twin columns. Finally, it is shown that the FC-RCP happens to be advantageous in the presence of a severe sample diluent/eluent mismatch (elution strength and viscosity), which causes undesirable viscous fingering and band distortion.

Application: Pharmaceutical

Methodology: Liquid Chromatography

POSTER

2:00 pm– 4:30 pm
Location: Expo floor

Session: 2100-5
Meeting the Challenges of Implementing Accurate-Mass Mass Spectrometry for Biotherapeutic Development in Regulated/non-Regulated Environments
Author: Scott Jason Berger, Waters Corporation
Co-authors: Weibin Chen, Nilini Ranbaduge, Henry Shion, Ying Qing Yu, Ximo Zhang, Waters Corporation

Experienced MS users have long been required for instrument operation, data processing and interpretation when high resolution MS is deployed in biopharma. The desire to broaden capabilities to analysts more familiar with optical detection methods calls for a solution addressing inherent complexities of HRMS technology and lack of compliance for platforms capable of generating biopharma product attribute results. Here, we report on a novel high performance benchtop orthogonal acceleration time-of-flight (oa-TOF) LC-MS system with simplified and optimized operation modes to deliver automated, accurate, and reproducible mass measurements for proteins, peptides, and released glycans.

Assessment of the system for the three workflows (intact/subunit mass, peptide mapping/monitoring and released glycan profiling) was conducted with multiple biotherapeutic samples. Data acquisition, processing and result reporting were accomplished using a single method-driven, workflow-centric compliance-ready informatics platform.

Our results demonstrate that detection limits, mass accuracy and deconvoluted mass profiles of mAbs and ADC molecules were comparable to modern MS systems.  Average drug-to-antibody ratios for ADCs agreed with previous MS and HIC-UV results. Subunit data with low ppm mass accuracy were reproducibly generated from reduced mAbs or IdeS digests.  Tryptic maps yielded high (>90%) sequence coverage using both precursor accurate mass and confirmatory fragment ions. Released glycan analysis enabled profiling of glycans from mAbs and complex fusion proteins using Glucose Unit (GU) based retention and MS confirmation. Availability of these routine capabilities on a compact, accessible, and compliant-ready LCMS platform should fulfill biopharmaceutical industry desire to expand accessibility of higher information content techniques to more scientists and organizations in the effort to drive efficiencies during product development manufacturing and release.

Application: Pharmaceutical

Methodology: Mass Spectrometry

Session: 2100-12
Method Transfer and Routine Analysis of Protein and Peptide-Based Drug Products Using a Biocompatible UHPLC System
Author: Brooke Koshel, Waters Corporation
Co-authors: Robert Birdsall, Paula Hong, Stephan Koza, Zhimin Li, Corey Reed, Waters Corporation.

Many of the top-selling pharmaceuticals currently on the market and in the pipeline are biologics. To date, many of the methods used in development and quality control laboratories are HPLC-based. While this may be sufficient in some cases, there are noted advantages of updating legacy systems and methods with more modern instrumentation. By updating from an HPLC platform, better resolution, shorter run time, and greater peak capacity can be achieved.

The product lifecycle includes development and manufacturing activities as well as technology transfers. When adopting new technology, it is of critical importance that instrumentation be robust and easily deployed. In this work, a biocompatible UHPLC platform will be used to replicate legacy HPLC methods and demonstrate system equivalency across HPLC, UHPLC, and UPLC platforms. Furthermore, the benefits of laboratory modernization are shown through new column chemistries with smaller particle size and lower dispersion LC systems. Bio-inert flow paths aid in system robustness and performance, which is especially important when using the high salt or extreme pH conditions that are required for many of the mobile phases used in bioseparations. To this point, intra system repeatability is demonstrated for SEC, IEX, and HIC separations. Additional features of the UHPLC system including dual flow paths and a tool for modifying the gradient start time (without making changes to the gradient table) are employed to ensure consistent results are maintained across LC platforms. System features for aiding in the method development process are also highlighted in an IEX workflow for charge variant analysis in method transfer across different vendor platforms. Through purposeful LC system design, method transfer and development as well as routine bioseparations are streamlined to add confidence to a dynamic biotherapeutic environment.

Application: Pharmaceutical

Methodology: Liquid Chromatography

Session: 2100-17
Designing a New Particle Technology and pH Gradient Mobile Phase Concentrates for Robust, High Resolution Charge Variant Analysis of mAbs
Author: Paula A Orens, Waters Corporation
Co-authors: Stephan Koza,  Matthew A Lauber,  Justin McLaughlin,  Mike F Morris,  Bei A Niu, Susan C Rzewuski,  Stephen Shiner,  Qi Wang,  Mingcheng Xu,  Hua Yang, Waters Corporation

Ion exchange chromatography (IEX) is routinely relied upon to characterize and monitor the charge variants of protein-based therapeutics such as monoclonal antibodies (mAb), which can very often be flagged as critical quality attributes. There has long been a need to address resolution limitations and challenges related to method implementation and robustness.

A new IEX column technology based on a 3 µm non-porous sorbent, specialized polymerization reactions and a finely tuned sulfonic acid grafting has been developed to improve charge variant analyses. Optimization of particle size, particle morphology, and surface chemistry has produced a column with superior properties compared to leading technologies. Its performance has been confirmed to be universally applicable to multiple mAbs, and it has been proven to be reproducible and robust by way of observations on minimal column-to-column and batch-to-batch variation as well as column lifetime. This column also provides a high degree of loadability that is useful for purifying charge variants for characterization and structure-function studies.

In addition, a new pH gradient mobile phase system has been developed to provide universally applicable cation exchange separations of mAbs having a wide range of pI values. The composition and concentrations of buffer salts, as well as the pH range, were optimized using theory and empirical observations to achieve robust and high resolution separation of mAbs. The performance of this method has been confirmed to be reproducible from batch-to-batch and to afford column lifetimes up to and beyond 500 injections. Method implementation and optimization has been simplified while both throughput and resolution have been improved.

With its reproducibility and improved performance, it is believed that these new separation technologies will better serve biopharmaceutical scientists dealing with perplexing investigations and even the most rigorous examples of QC testing.

Application: Pharmaceutical

Methodology: Liquid Chromatography

Session: 2100-18
High Sensitivity LC-MS Profiling of Antibody Drug Conjugates with Difluoroacetic Acid Ion Pairing and a High-Coverage Phenyl-Bonded Stationary Phase
Author: Paula A Orens, Waters Corporation
Co-authors: Matthew A Lauber, Waters Corporation, Cristina Legido-Quigley, Systems Medicine, Steno Diabetes Center, Jennifer  M Nguyen, University of Copenhagen, Susan C Rzewuski, Waters Corporation, Jacquelynn Smith, Pfizer

Protein reversed phase chromatography, while preferred for LC-MS, is heavily dependent on the conditions under which it is performed.  Methods employing polymeric columns and trifluoroacetic acid (TFA) have been preferred but are inherently restricted to low pressure, low throughput analyses and compromised MS detection.

Our investigations show that it is possible to achieve higher resolution separations when difluoroacetic acid (DFA) is used in place of TFA.  For separations of reduced, IdeS digested NIST mAb, up to 40% gains in peak capacity are observed.  Along with a column technology based on a novel phenyl surface chemistry, it has been possible to boost resolution and to accelerate analyses using high flow rates.

Additionally, DFA also confers notable gains in MS sensitivity versus TFA.  A 4-fold increase in MS signal has been observed when 0.1% DFA is used in place of 0.1% TFA.  Nevertheless, the use of DFA has presented a surprising challenge since a reagent of purity suitable for MS work is not commercially available.  We have addressed this issue by purifying DFA to a quality appropriate for MS analyses.

Coupling the phenyl-based column with DFA can also grant exceptional levels of protein recovery, resolution, and MS sensitivity for ADCs.  Recovery of subunits bearing multiple drug payloads, like Fd'(+3 payloads), can be greatly improved alongside the enhanced resolution of protein variants.  Furthermore, species resulting from on-column degradation can be minimized by using lower temperatures.

Using this pairing, we proposed a new platform LC-MS method for profiling ADCs.  The method facilitated quick assessments of drug-to-antibody ratio (DAR) and displayed robustness in method qualification.  Ultimately, we hope that this demonstration of obtaining highly attractive protein MS data from stringently-purified DFA will encourage broader use of this novel acid, including implementation in additional types of LC-MS work.

Application: Pharmaceutical

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 2100-19
Automated Sample Preparation for Hybrid LC-MS/MS Protein Quantification

Author: Paula A Orens, Waters Corporation
Co-authors: Mary Lame, Waters Corporation

Developing a highly sensitive and accurate LC-MS method for large molecule therapeutic quantification is quite challenging. This is especially true for proteins when using the surrogate peptide approach, which requires complex and time consuming sample preparation.  This workflow requires optimization of many steps and often includes highly selective protein level clean-up to achieve high sensitivity from limited sample volumes of complex biological matrices, often requiring a highly skilled scientist. Thus, there is a strong need for more simplified and standardized approaches, which can be automated to streamline the sample preparation workflow, reduce analytical variability, and increases lab productivity.

METHOD: Standard curve and QC samples for various mAb therapeutics were prepared in serum/plasma. Protein level sample clean-up of the prepared plasma was performed using generic and/or specific antibody purification.  This purification and enzymatic digestion was performed using a commercially available liquid handler and supplied script.  LC-MS/MS quantification of the resulting signature tryptic peptides was performed using low dispersion LC, coupled to a triple quadrupole MS system (ESI+).

RESULTS: A complete sample preparation workflow using a standardized and automated approach for the sensitive and accurate quantification of multiple mAb therapeutics from serum/plasma has been developed, yielding excellent quantification performance with LOQs <100 ng/mL, linearity of calibrators > 0.99, QC accuracies between 85-115% and mean CVs < 15%, indicating an accurate and reproducible analytical method. Based on this developmental work, proof of concept of a fully automated sample preparation workflow for mAb quantification from serum/plasma will be presented.

Application: Bioanalytical

Methodology: Liquid Chromatography/Mass Spectrometry

Session: 2100-20
Strategies to Evaluate and Monitor Forced Degradation Studies Using a Dual Detection (UV-MS) System

Author: Paula Hong, Waters Corporation
Co-authors: Patricia R McConville, Waters Corporation

Forced degradation studies are typically performed to understand the degradation pathway of pharmaceuticals.  Given the range of impurities and their chemical and physical properties, a single detection technique may not be adequate to accurately measure all of the degradants. Specifically, when ultra-violet (UV) detection is used alone, non-chromophoric species and/or co-elutions may be missed.

To address the challenges of measuring and quantifying degradants, a dual detection system consisting of a photodiode array (PDA) and a mass detector (MS) will be used to analyze a stressed drug substance.  While UV is typically used to assess and measure degradants, mass spectrometry allows for detection by an orthogonal technique and provides information to aid in characterization.  For example, mass detection will be used to measure any non-chromophoric degradants that may be produced. The impact of missing degradants will be assessed. In addition, orthogonal detection will also be used to illustrate the impact of co-elutions on mass balance determinations. By assessing peak purity using both MS and UV, the final separation for the API and its degradants can be optimized to ensure no co-eluting peaks. The addition of MS information, whether for non-chromophoric species or co-elutions, will allow for a more complete evaluation and more comprehensive understanding of the degradation pathway.

Application: Pharmaceutical

Methodology: Liquid Chromatography/Mass Spectrometry