Development of chromatographic platforms for IMERS characterization as prototypes for flow-chemistry applications

Enrica Calleri, Drug Sciences, University of Pavia, Pavia, Italy (enrica.calleri@unipv.it)
Marco Corti, Drug Sciences, University Of Pavia, Pavia, Italy
Gabriella Massolini, Drug Sciences, University Of Pavia, Pavia, Italy
Paola Conti, Germanyddepartment Of Pharmaceutical Sciences, University Of Milan, Milan, Italy
Marco Terreni, Drug Sciences, University Of Pavia, Pavia, Italy

Enzymes are increasingly used as biocatalysts for the production of fine chemicals and pharmaceutical products. In this context biocatalyzed reactions performed in a flow system can benefit from improved mass transfer, temperature control and, importantly, continuous substrate feed and product removal, thus limiting the possible substrate/product inhibition of the enzyme activity.

Flow reactors can be used for different scopes: analytical scale synthesis (i.e. for screening purposes and rapid optimization of the reaction conditions), lab scale synthesis for pilot studies or large scale synthesis (e.g., industrial production of APIs). In the recent years low-volume immobilized enzyme reactors (IMERs) and analytical IMER-based platforms have found several applications in the drug discovery field with a special emphasis on enzyme inhibitors screening and on-line proteolysis of proteins prior to analytical characterization. However, the potentiality of IMERs for reaction optimization studies on analytical scale as prototypes for biosynthetic purposes (very low sample consumption, short analysis time and the possibility of connecting them to different separation and detection systems) has not been fully exploited.

In this presentation two families of enzymes are selected: nucleoside phosphorylases and ω-transaminases. Nucleoside phosphorylases have shown their powerfulness in the synthesis of modified nucleosides with clinical relevance as antiviral and antitumor agents. The synthesis of these products can be of great interest for small or medium pharmaceutical industries because the low dosage of most of the antiviral and antitumor nucleosides (and nucleotides) necessitates small production sizes of high value products. On the other hand, ω-transaminases are powerful enzymes for the preparation of enantiomerically pure chiral amines, which represent useful synthetic intermediates for the preparation of several different classes of APIs. Enzymes belonging to the two different families were immobilized on different analytical supports (monolithic silica and poly-HIPEs supports) and used in chromatographic platform for IMERs characterization.

The flow-synthesis of nucleosides catalyzed by immobilized Purine Nucleoside Phosphorylases was carried out on analytical scale in an integrated systems for reaction control and product purification.

A chromatographic platform based on liquid chromatography has been also developed to facilitate studies on the biocatalytic properties of an ω-transaminase useful for chiral amines synthesis (ATA117). ATA117 was covalently immobilized on different supports. The developed enzymatic reactor was coupled through a switching valve to an analytical column for separation and quantitation of the hydrolysis products. The enantiomeric excess was determined to follow the kinetically controlled enantiomer-differentiating reaction.

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Abstract Reference & Short Personal Biography of Presenting Author

Curriculum Vitae

Prof. Enrica Calleri got her master degree in Chemistry and Pharmaceutical Technology in 1996 at the University of Pavia and in 2000 she defended her PhD thesis titled “development of new protein-based chiral stationary phases”. She spent part of her PhD course at Georgetown University, Washington DC (USA), under the supervision of Prof. I.W. Wainer. In 2006 she worked at the Gerontology Research Centre NIA/NIH, Baltimore, carrying out a research project aimed at the development and characterization of new stationary phases based on purinergic receptors for binding studies.

Since 2014 she is Associated Professor of Pharmaceutical Analysis at Pavia University. From 2000 to 2016 she was member of the scientific committee of the Summer School on Pharmaceutical Analysis supported by the Division of Medicinal Chemistry of the Italian Chemical Society and by the European Federation for Medicinal Chemistry. She has been a member of the organizing committees of the 10th and 14th Editions of the International Meeting RDPA (International Meeting on Recent Developments in Pharmaceutical Analysis). From 2011 to 2013 she was Member of the teaching board of the PhD course in “Chimica e Tecnologia Farmaceutiche” and from 2013 to 2016 Member of the teaching board of the PhD course in “Scienze Chimiche e Farmaceutiche” at the University of Pavia.

Prof. Calleri is author of more than 65 publications in peer-reviewed journals and she has a strong analytical background, mainly on the use of separation techniques for molecular recognition and drug discovery. Her scientific interests have been focused on:

1. Development and validation of novel HPLC and CE methods in pharmaceutical analysis;
2. High Performance Affinity Chromatography with immobilized receptors (membrane and nuclear receptors) for binding studies and affinity selection of new potential drug candidates;
3. Development of biochromatographic systems based on immobilized enzymes for proteomic applications, quality control of biopharmaceuticals and chiral resolution of drugs;
4. Development of bioreactor for the flow-synthesis of Active Pharmaceutical Ingredients.