Applications of Flow NMR and EPR in Pharma and Beyond

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1 Applications of Flow NMR and EPR in Pharma and Beyond Anna Codina, Director Pharmaceutical Business Unit, Bruker BioSpin PANIC 04Mar18 Innovation with Integrity

2 Applications of Flow NMR Expanding the InsigthMR portfolio

3 Online Reaction Monitoring by NMR InsightMR flow tube InsightMR software Online process monitoring in real-time under real conditions Process understanding: yield, mechanistic insights and reaction kinetics = > Savings Intuitive workflow making NMR an accessible PAT tool

4 Pharmaceutical Applications Dunn A.L., Codina A., Foley D.A., Zella M.T., Eur. Pharm. Rev., in Press Foley, D.A, The Advantages of Reaction Monitoring by NMR in the Pharmaceutical Industry, SelectScience, July 2015 Foley D.A., Bez, E., Codina A., Colson K.L., Fey M., Krull R, Piroli D., Zell, M., Marquez, B.L., Anal. Chem., 86, (2014) Dunn A.L. et. al, MRC, DOI: /mrc

5 Applications to Catalysis and More!

6 Xpress The Tesla of Reaction Monitoring

7 When do we Need to go Faster? On Demand Webinar Professor Guy Lloyd-Jones, FRS cation-training/webinars/nmrwebinars/improving-mechanisticunderstanding-of-fast-reactionsusing-rapid-reproducible-in-situanalysis-by-stop-flow-nmr-andir.html

8 When do we Need to go Faster? 1. Fast Reactions (seconds) 2. Repeating the reaction varying conditions Labour - intensive Time consuming Conc (M) mix insert lock start Time (s) t ½ 5 s

9 Automated in-situ Monitoring The Solution for Fast Reaction Monitoring and Optimisation + + Reactor 1. Flow Unit 2. High speed delivery pumps 3. NMR system Professor Guy Lloyd-Jones, FRS

10 Stop-Flow Xpress E Reference: Prof. Lloyd-Jones, FRS Rapid, streamlined process optimisation and understanding that can also monitor reaction in the time scale of seconds! times increase of productivity when screening reaction conditions SHARPER (Sensitive, Homogeneous And Resolved PEaks in Real time) POSTER 44 Jones, Ariana B.; Lloyd-Jones, Guy C.; Uhrin, Dusan Anal. Chem., 2017 DOI: /acs.analchem.7b02437

11 Cell Real-time Monitoring of Living Cells

12 Monitoring Cell Activity with InsightMR Cell Insights into cell activity with real-time NMR monitoring Unique structure information about compounds consumed and produced by cells Two modalities so far: 1) On-line: the cell culture flows from the bio-reactor to the NMR spectrometer 2) Cells are fed with nutrients and/or treated with drugs in the flow tube, where the cells are kept alive

13 Monitoring Yeast Fermentation Cell On-line analysis (modality 1) InsightMR to monitor living cell metabolism Saccharomyces Cerevisiae culture (20 ml) for 6h Flow tube (where the metabolite are detected by NMR) Peristaltic pump to flow the culture to the NMR probe and back Transfer line could be fully temperature controlled 600MHz Magnet InsightMR software for NMR acquisition and kinetic analysis Courtesy of Dr Andrei Bunescu

14 Monitoring Yeast Fermentation Cell On-line analysis (modality 1) 6h Glucose consumption Acetate formation Ethanol formation Simplified Saccaromyces Cerevisias biochemical pathway Glucose consumption measured for 6 hours Ethanol and Acetate metabolites formation detected Courtesy of Dr Andrei Bunescu

15 Monitoring Lymphophatic Leukemia Cells Cell On-line analysis (modality 2) Real time NMR with primary cells using the Bruker Insight NMR system NMR provides the unique opportunity to study the metabolism of primary human cells in real time. This has recently been demonstrated by Günther s group at the University of Birmingham by embedding primary chronic lymphophatic leukemia cells into agarose to measure the kinetics of metabolic turn over over up to 24h [1]. This experiment has now been considerably improved by adapting Bruker s Insight flow NMR system for use with mammalian cells. The Insight system keeps cells at 37 C, keeps O2 and CO2 pressure constant by flowing through an incubator and allows the addition of drugs or other contents to the media during the experiment. This offers unprecedented opportunities to study metabolism of human cells and is directly applicable to cells derived from human blood including cells from blood cancers. [1] Katarzyna M. Koczula, Christian Ludwig, Rachel Hayden, Laura Cronin, Guy Pratt, Helen Parry, Daniel Tennant, Mark Drayson, Christopher M. Bunce, Farhat L. Khanim, Ulrich L. Günther. Metabolic plasticity in CLL: Adaptation to the hypoxic niche. Leukemia 30(1):65 73 (2015). doi: /leu

16 Monitoring Lymphophatic Leukemia Cells Cell On-line analysis (modality 2) Over 30 extracellular metabolites present

17 Monitoring Lymphophatic Leukemia Cells Cell On-line analysis (modality 2) POSTER 45 Lactate production (left) Analysis of time course data shows a significant decrease in glucose and increase in lactate over time, corresponding to the Warburg effect

18 Reaction Monitoring by EPR EMXnano EPR EMXnano Flow-through Cell Making research-grade data accessible to a larger audience Analyse all EPR species - transition metals, antioxidants and free radicals Valuable information and insights to biological and chemical reactions Novel permanent magnet and an efficient new microwave resonator for unmatched sensitivity and stability Analyse short-lived radicals using spin trapping Enables quantitative EPR with the inclusion of Bruker s patented spin counting module

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20 Impurity Control by EPR electron h, T Orthogonally with NMR! chemical reactions Stable molecule Free radical Free radicals & transition metals Innovation with Integrity 20

21 Impurity Control by EPR Degradation and Stability Studies Determining the root causes of degradation in drug products Measuring the extent of degradation of APIs, excipients, and formulations Predicting long-term stability characteristics of the APIs, excipients and formulations Determining the antioxidant efficiency to quench free radicals with well established assays Degradation correlates with the EPR signal! Placebo EPR data confirm the API s susceptibility to degradation Innovation with Integrity 21

22 Impurity Control by EPR Paramagnetic impurities, transition metals: Detecting and identifying traces of transition metals Monitoring drug degradation processes that produce and involve free radicals Observing the production of free radicals catalyzed by transition metals or other impurities Transition metal impurity identification and control via EMXnano! Innovation with Integrity 22

23 Impurity Control by EPR Case study: Tween 20 autoxidation Polysorbate 20 used in drug formulation as a stabilizer undergoes autoxidation Autoxidation is catalyzed by transition metals and results in side-chain cleavage and free radical formation The EMXnano can detect, identify and quantify the free radical impurities Lam X.E. et al.(genentech Inc.), Site-specific tryptophan oxidation induced by autocatalytic reaction of polysorbate 20 in protein formulation, Pharm. Res. (2011) Innovation with Integrity 23

24 Post Sterilization Quality Control Sterilization can generate radicals that need to be controlled EMXnano can help by: Characterizing the free radicals and identifying their source Gamma-irradiation of drugs in the solid-state (Captopril, Selegiline, Pentoxifylline) induces S-or C-centered free radicals. Providing easy go/no go decisions based on quantification of free radicals for quality control and assurance (QC/QA) Innovation with Integrity 24

25 Pharmaceutical Applications EPR On Demand Webinar Ralf T. Weber & Kalina Ranguelova cation-training/webinars/eprwebinars/pharmaceuticalapplications-of-electronparamagnetic-resonance-eprspectroscopy.html

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27 Contactless Check Weighing The NMR balance! At-line measurement of mass for expensive pharmaceuticals in their container (vials, syringes )

28 New Bench Top Solid Form Quantification

29 New Methods for Reaction Monitoring and Understanding You will learn how: Online monitoring enhances reaction understanding, hence confidence when transferring process from lab to plant. Stop-flow solutions enable monitoring of fast reaction and rapid screening of reaction conditions. Real-time monitoring of cellular metabolism by NMR offers unprecedented insights into cell activity and potentially their response to drugs. Educational Webinar 21Mar am ET i= &tp_key=127f8b4cc4

30 Acknowledgments InsightXpress InsightCell EPR InsightMR Ted King Prof. G. Lloyd-Jones Ruth Dooley Ariana Jones Dusan Uhrin Matteo Pennestri Mark Garvey Peter Neidig Martin Hofmann Ulrich Braumann David Foley Mark Zell Andrei Brunescu Jenny Roberts Ulrich Guenther Matteo Pennestri Martin Hofmann Aitor Moreno Yizhou Liu, Merck US Williams H.E. and Claybourn M. AZ, UK Kalina Ranguelova, Bruker Ulrich Hintermair Andrew Hall John Lowe

31 Innovation with Integrity Copyright 2018 Bruker Corporation. All rights reserved. INTERNAL USE ONLY