The Chromatography Forum

of Delaware Valley

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Abstracts:

Non-Targeted Discovery of Volatile Stem Cell Growth Metabolites by GCxGC/TOF MS

John Dimandja (1), Chris Heist (2), Milad Navaei (2)

(1) GaTech, School of Mechanical Engineering, Atlanta, GA

(2) GaTech Research Institute, Atlanta, GA

In the past couple of years the US Food and Drug Administration (FDA) has approved a number of personalized therapeutic protocols that target blood and bone marrow cancer. While these approvals constitute a significant milestone in the field of immunotherapy research, there exist some significant challenges to the expansion of stem cell drugs to other disease areas. Issues related to standardized characterization, quality control and stability are in need of further development, and the current technologies employed to quantify the molecular and cellular characteristics of various stem cell cultures are either time-consuming, destructive, or qualitative in nature.

In this presentation we will report our recent efforts to develop a new method for the chemical profiling of stem cell cultures. Due to the complexity of the culture media and the low intensity of the volatile organic molecules in the sample matrix, the high-resolution chromatographic technique of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOF MS) was used. An overview of the figures-of-merit of this technology will be given, and results from the non-targeted analysis of a cohort of Mesenchymal stem cells (MSCs) from a variety of sources will be presented. The preliminary investigation of the variations in the MSCs via multivariate analysis data processing techniques (such as principal component analysis and hierarchical clustering) will also be presented that demonstrate the validity of this work to be pursued to help the overall goal of improving the efficient manufacturing of stem cells.

Advancing chromatographic investigations of pharmaceutical significance: Identification of co-eluting impurities and solutions to water dewetting from RPLC columns

Fabrice Gritti(1); Martin Gilar (1); Qi Yan (2)

(1) Waters Corporation, Instrument/Core Research/Fundamental, Milford, MA USA

(2) Pfizer, Inc., Groton, CT 06340 USA

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). Batch chromatography, steady state recycling (SSR), or simulated moving bed (SMB) chromatography are routine methods to prepare this material. However, they fail when the trace impurity nearly co-elutes with the concentrated API for the lack of resolution.

In the first part of this presentation, based on a classical twin-column recycling chromatography process (RCP) coupled to a fraction collector (FC), an easy-to-use and automated LC technology combining high-resolution and semi-preparative capabilities is described. The performance of the prototype RCP is first validated for the challenging separation of shape isomers (chrysene/benzo[a]anthracene, selectivity factor a=1.025), chiral compounds (g-phenylbutyrolactone, a=1.013), and deuterated benzene isomers (H6, -D3, and -D6, a=1.020) using standard HPLC columns (4.6 x 150 mm, 3 mm) below 400 bars. Next, it is demonstrated how the RCP-FC system can automatically prepare 1 mg of the unknown targeted impurity present in trace amounts (a few mg/L) in the actual stock solution of the API (10 g/L of estradiol). The capability of the FC-RCP to cope with severe sample diluent/eluent mismatches in elution strength and viscosity causing undesirable viscous fingering and band distortion, respectively, is discussed in terms of production rate of the process.

In the second part, the undesirable phenomenon of water dewetting from hydrophobic stationary phases used for the analysis of highly polar impurities and API is investigated from an experimental viewpoint. The study involves the role of pore size, temperature, and surface chemistry on dewetting kinetics. Water porograms (intrusion/extrusion of water) are directly measured by chromatography to determine the advancing and receding contact angles of water. The resulting receding contact angles onto Symmetry-C18 (92.6o) and Cortecs-C18 (91.6o) stationary phases are found smaller than those onto Symmetry-C8 (93.6o) and Cortecs-C8 (93.9o) phases, respectively, in correlation with the faster dewetting of water from C8 surfaces than that from C18 surfaces. Practical and chemical solutions to eliminate water dewetting will be discussed.

Overview of Cannabis Testing by HPLC

Craig Young

Shimadzu Scientific Instruments Columbia, MD USA

Cannabis products for medical and recreational use are enjoying an unprecedented surge in popularity. Accurate cannabis analysis is required in order to ensure customers get what they pay for. Since cannabis is often used as medicine, additional analysis is required to protect consumers from potentially harmful microbiological contamination, and chemical residues such as pesticides. This talk course will give an overview of the basics of cannabis and the industry, as well as some of the state-of-the-art testing methods currently available, particularly in regards to the testing of cannabinoids, called potency.

Part I. Brief overview of cannabis and cannabis industry background.

The cannabis plant and its use and cultivation will be presented. The plant's medical use, growing techniques, and final product formulation will be briefly introduced. The history and current legal status of cannabis will be summarized. The testing lab industry and the problems which have led to the need for testing will be emphasized.

Part II. Potency testing by HPLC.

Accurate cannabis potency testing is of the utmost importance for establishing safe and effective dosages, optimizing growing conditions, and preventing fraud in the cannabis industry. A detailed workflow of potency testing by HPLC will be presented.

Quality by Design of chromatography methods that require transfer to global drug substance manufacturing sites – Risk mitigation and transfer troubleshooting

P. Tattersall, X. Xu, L. Li, M. Kanthasamy, Brent Kleintop and Johnathan Shackman
Bristol-Myers Squibb, New Brunswick, NJ, USA

Analytical methods evolve during the stages of clinical development to meet the control strategy needs for small molecule API syntheses chemistry. This culminates in the development of robust chromatography methods required for transfer at the late stage of a project to afford QC support for manufacturing and registrational stability studies. Here we will review strategies and approaches towards method development and the transfer of analytical method knowledge with specific consideration on timelines and challenges encountered during a highly accelerated (transfer to commercial site > 3yrs after entering the clinic) project. The methods we discuss were successfully transferred to sites at many different geographical locations. The discussion will focus on concerns with: i) instrumentation differences, challenging chromatography, and compendial methods; ii) validation, co-validation, and verification as transfer approaches; iii) method interpretation / documentation, communication and timelines; iv) expectations vs. outcomes as lessons learned. Specific examples of challenges and troubleshooting encountered will be described and include: 
• Instrumentation / geography effects on GC testing compared to the expert laboratory experiences and data. 
• Investigation into problems with impurity profiles and sample stabilization during preparation followed by the subsequent root cause determination. 
• Troubleshooting an in-process control’s accuracy concerns and sample stability where an incidental instrument setting had an impact on method performance. 
• Trace impurity testing of specific impurities where validation failure at the vendor required a change in method and analytical control strategy. 
• Attempts to bring about more consistent method development using a platform approach in development Experience with vendor to vendor method transfers will also be discussed. 

A Stroll through the Cannabis Field: Physiological Function and Therapeutic Targets of the Endogenous Cannabinoid System

Aron H. Lichtman, PhD

Department of Pharmacology and Toxicology

Department of Medicinal Chemistry

Virginia Commonwealth University

Richmond, VA

The scientific pursuit to understand how cannabis produces its pharmacological effects has led to enormous strides in basic and medical research. These advances include the discovery of the endogenous cannabinoid (eCB) system that consists of two cannabinoid receptors (CB1 and CB2), endocannabinoid ligands (e.g., anandamide; AEA and 2-arachidonylglycerol; 2-AG), and enzymes regulating the biosynthesis and catabolism of the endogenous ligands. The eCB system regulates myriad physiological systems within the central nervous system and throughout the periphery, which include synaptic plasticity, stress responses, immune function, renal function, and energy regulation. To date, the FDA has approved three cannabinoid-based medications, with others in the pipeline undergoing various preclinical testing and clinical trials with the intent to treat a wide variety of conditions, such as pain and inflammatory disorders, neurodegenerative diseases, psychiatric conditions, cancer, obesity, drug abuse and dependence, and a wide variety of other medical conditions. This presentation will communicate the history of medicinal cannabis use from antiquity to the present, as well as introduce the components of the eCB system, its general physiological functions, and potential medications. Finally, we will cover an unintended consequence of cannabinoid research; the subversion of synthetic cannabinoids as research tools to emerging abused substances that are associated deleterious public health consequences. At the conclusion of this presentation, the audience will gain understanding into how cannabis and its primary constituents produce their pharmacological effects, be familiar with major components of the eCB system as well as its physiological functions, and appreciate the need for evidence-based cannabinoid research

Application of Two-Dimensional Liquid Chromatography for Analysis of Pharmaceutical Compounds - More Knowledge in Less Time

Qinggang Wang, Jessica Conforti, Kaitlyn Frankenfield, Hua Chia Tai, Brian He, Jonathan Shackman and Brent Kleintop

Chemical and Synthetic Development, Bristol-Myers Squibb

Pharmaceutical companies have been under consistent pressure to bring safe therapeutics to patients faster. In the meantime, complex synthetic molecules, such as compounds with multiple chiral centers, peptides, and oligonucleotides, are continuing to increase in the pharmaceutical development pipeline. Due to factors such as large molecular size and complicated synthetic routes, these compounds usually have much more diverse impurity profiles compared with those of traditional small molecules, which poses challenges to developing stability indicating HPLC methods for monitoring impurities. These pressures have prompted the evolution of innovative analytical techniques and the development of fit-for-purpose HPLC methods to accelerate knowledge generation. Two-dimensional liquid chromatography (2DLC) has grown rapidly in the last two decades, and turnkey commercial instrumentation is now available from multiple vendors. Compared with conventional one- dimensional liquid chromatography, 2DLC can significantly improve separation power through unique selectivity pairings between the first and the second dimension LC conditions. In this presentation, a few applications of heart-cutting 2DLC to the analysis of pharmaceutical compounds will be demonstrated. The examples include peak purity evaluation using orthogonal RPLC-RPLC and RPLC-chiral LC methodologies; quantitative analysis of co-eluting impurities; impurity identification by 2DLC-MS; and on-column degradation investigations. The results of these studies demonstrate that 2DLC is a very powerful tool in knowledge generation for pharmaceutical development.

Call for Posters

All undergraduates, graduate students, and new professionals are invited to present work with relevance to the Chromatography Community, in either research or applied science, at the Chromatography Forum of Delaware Valley Symposium. Presentations need not be chromatography-centric, but chromatographic science should at minimum be an important component in achieving the objective of the project presented.

Free Admission to the Symposium** Registration fees are waived for students and new professionals presenting posters.

CASH PRIZES for outstanding posters will be awarded by the CFDV Executive Board.

Content, Completeness, and Aesthetics of the poster. Quality of writing and data analysis/interpretation. Outcome of the Interview with CFDV judges. Discussion of any method development aspects of the chromatography. Significance of the presenter’s contribution to the project. Applicability of the science to the practicing Chromatography Community.

Abstracts should be submitted by email to chromforumdv@gmail.com by April 5, 2019. Include the presenter’s name, contact information (email and phone) and association (school or company).

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