Crystalline product assisted by intensified CO2 process – CYPRES

In order to meet the challenges proposed by the CYPRES project, three complementary experimental approaches was implemented:

(i) conventional crystallisation with in-situ diagnostics,

(ii) crystallisation assisted by supercritical CO2 (scCO2) in a microfluidic reactor,

(iii) crystallisation assisted by supercritical CO2 in a macroscopic reactor.

The approach consisted firstly of identifying and studying the behaviour of chiral molecules and co-crystals under conventional crystallisation conditions. Certain systems were fully characterised from the point of view of their thermal behaviour (establishment of phase diagrams), in particular to determine whether the system crystallises in the form of a conglomerate or a racemic compound.When a conglomerate was detected in conventional crystallisation, a preferential crystallisation process was optimised in solution and used as a basis for studying the transposition to crystallisation tests in scCO2.

At the same time, a screening of the manufacture of cocrystals by crystallisation under scCO2 was carried out. This crystallisation, mainly carried out here in antisolvent mode, involves adding CO2 to a mother liquor to reduce the solubility of the species and cause them to precipitate or co-precipitate. The major challenge of this semi-continuous process is to control crystalline purity and enantiomeric excess, characteristics that depend on the homogeneity in space and time of the reactor and on thermodynamics. The use of microfluidic microreactors has enabled better control of the mixing between the CO2 and the solvent, while providing direct optical and spectroscopic (Raman) access to information at the crystal scale. The larger volume reactor enabled the production of the quantity required for phase identification. The development of in situ analysis is currently underway for classical crystallisation.

- Screening of co-crystal production by crystallisation in antisolvent CO2: of the 13 systems tested, 5 co-crystals were produced, including 3 not described in the literature. 3 cocrystals obtained by an organic solvent-free process in which CO2 itself acts as a solvent for the compounds. The speed of the process (2h to obtain pure, dry cocrystals from a solution), combined with the low temperatures, helped to promote CO2 as an eco-friendly alternative.

- Identification of 2 chiral molecules (Proxyphylline PXL, Diprophylline DPL) crystallising with the coformers salicylic acid (SAL=2HBA) and 4 Hydroxybenzoic Acid (4HBA) in the form of racemic compounds and having the possibility of forming a conglomerate in the presence of water (hydrated cocrystal conglomerate).

Identification of solvent composition domains (water/organic solvent mixture) enabling the implementation of a preferential crystallisation process for 3 systems: PXL/SAL, DPL/SAL and PXL/4HBA. (Fig 1)

- Production of a crystalline phase that combines the molecules PXL, 4HBA and CO2, via crystallisation under compressed CO2, impossible to produce otherwise. CO2 plays a key role in the crystal lattice and this phase evolves spontaneously with time and/or temperature increase towards the anhydrous phase obtained by more conventional crystallisation: the stable PXL:4HBA cocrystal phase. (Fig 2)

- Transfer of the ‘conventional’ preferential crystallisation process (Nefiracetam-mandelic acid system) to a pressurised CO2 process: the 2 enantiopure cocrystals crystallise simultaneously in 2 separate zones of the reactor, unlike preferential crystallisation in solution in which only one enantiomer precipitates (Fig 3).

Establishment of a protocol for introducing germs into the pressurised reactor

- Development of a high-pressure microfluidic platform for in situ monitoring of diffusion phenomena and then nucleation-growth of crystals by supercritical anti-solvent effect. Set up under a microscope (+ camera), it can acquire images and video of crystallisation events in real time. Equipped with a Raman probe, it can be used to track species during the process (diffusion, nucleation, growth). Applied to chiral molecules, crystallisation on a high-pressure microchip gives results on the precipitation time of the RS and S forms of the active ingredient. The Raman images/spectra obtained can be processed to determine the growth kinetics of the active ingredient crystals. The combination of experimental observation and numerical simulation of species diffusion phenomena in the reactor has made it possible to determine the growth rate in situ and in real time (Figs 4 and 5).

- Implementation of an enantiopure crystallisation detection analysis using SHG (Second Harmonic Generation) spectroscopy.

The microfluidic platform with microwells for studying crystallisation processes under pressurised CO2 is operational and equipped with in situ characterisation techniques (microscopy, Raman spectroscopy). This is a very powerful tool that can be used to meet a wide range of analytical requirements. DRX coupling could also be envisaged, as the microsystems are compatible with this type of analysis, as demonstrated in other studies performed at synchrotron.

The characterisation of chiral crystalline phases by in situ SHG analysis could be extended under supercritical CO2 conditions, in particular with the CO2 microfluidic platform for rapid screening of phases obtained under pressure.

The experimental conditions for preferential crystallisation under CO2 will be optimised.

A fundamental study of the parameters influencing crystallisation conditions in solution (solvation, intermolecular interactions, nucleation/growth kinetics) in these multiconstituent systems (= solvated cocrystals in the solid state) will be initiated in the following of this project.

F. Ercicek, O. Nguyen, C. Harscoat-Schiavo, M. Marchivie, A. Erriguible, P. Subra-Paternault, S. Marre, Microchip co-crystallization with supercritical CO2, European Meeting on Supercritical Fluids, poster session, May, 2021, online.

Léa Nimod, Isabelle Ziri, Olivier Monnier. “CYPRES project – Enantiomeric resolution by using supercritical CO2 co-crystallization”, Sanofi internal «R&D week« Sanofi Montpellier, 2 décembre 2021.

F. Ercicek, O. Nguyen, A. Erriguible, C. Harscoat-Schiavo, P. Subra-Paternault, S. Marre. On-chip in-situ observations of crystallization events under supercritical CO2. Oral presentation. ISSF 2022, 13 th International Symposium on Supercritical Fluids, Montréal 15-18 may 2022.

F. Ercicek, M. Marchivie, C. Harscoat-Schiavo, S. Marre, P. Subra-Paternault. Cocristallisation d’actifs pharmaceutiques en milieu supercritique. Présentation orale. CRISTAL 10, 9-10 juin 2022, Lyon

F. Ercicek, C. Harscoat-Schiavo, M.Marchivie, S. Marre and P. Subra-Paternault. Investigations of pharmaceutical compounds co-crystallization for application to chiral resolution assisted by supercritical CO2. Présentation orale. BIWIC 2022, 27th International Workshop on Industrial Crystallization, 31st august – 2nd september 2022, Espoo Finland

«Cristallisation préférentielle d’un cocristal chiral ; une voie originale pour la séparation d’énantiomères.«, Chrystal LOPES, Nino PATRY, Clément BRANDEL, Yohann CARTIGNY, Congrès CRISTAL 10 Lyon, juin 2022. Poster presentation

COMPARATIVE STUDY OF TWO RELATED CO-CRYSTALS: CRYSTALLIZATION BEHAVIORS, CRYSTAL STRUCTURES AND PREFERENTIAL CRYSTALLIZATION, Clément BRANDEL, Nino PATRY, Chrystal LOPES, Nicolas COUVRAT, Yohann CARTIGNY, CGOM, Sept 2023, Brussel. Oral presentation.

Teaching at ENSIC Engineering School, Nancy: «Aspects fondamentaux et appliqués du polymorphisme des composés moléculaires et pharmaceutiques« Y. CARTIGNY juin 2022

Fatma Ercicek, Christelle Harscoat-Schiavo, Patrick Layrisse, Mathieu Marchivie, Yohann Cartigny, et al.. Naproxen-bipyridine cocrystallization assisted by pressurized carbon dioxide. Journal of Supercritical Fluids, 2023, 200, pp.105976. ?10.1016/j.supflu.2023.105976?. ?hal-04098643?

Fatma Ercicek, Christelle Harscoat-Schiavo, Patrick Layrisse, Mathieu Marchivie, Yohann Cartigny, et al. Cocrystallization of Naproxen and Bipyridine assisted by supercritical CO2.
CO2 solvent or CO2 antisolvent, what is the best process?
présentation orale. European Meeting on Supercritical Fluids, May 2023, Budapest
Yohann Cartigny. STUDIES ON SOLID-VAPOR EQUILIBRIA TO DEVELOP NEW SOLID MATERIALS: THE CASE OF HYDRATES. Congrès Société Chimique de France, Nantes 25 au 27 juin 2023

In the context of sustainable crystallization and process intensification, the overall objective of the CYPRES project is to evaluate the performances of supercritical CO2 at controlling the crystal purity through a better understanding of CO2-mediated crystallization mechanisms with special insight into time-related aspects. The targets considered here cover (i) the fabrication of co-crystals pure powders and (ii) chiral resolution, both implying to manage a selective crystallization. Though supercritical CO2 technology is increasingly proposed as medium in advanced manufacturing processes for simple and composite products, the control of the crystal phase and its purity of organic materials is far less investigated than particle generation whereas it is of vital importance to assure the desired effect specially for pharmaceutical active ingredient. Since selective crystallization is controlled by the interplay of phase equilibria and kinetics, the use of CO2 is foreseen here as a different way to manage those factors through the unique flexibility of varying conditions via pressure, the modality of contacting species with CO2 and the adaptable composition of the CO2:solvent mixture that are likely to impact the structural and purity of products.
To achieve the objectives, CYPRES relies onto five complementary partners’ expertises and three technologies that will contribute to the rationalisation of key parameters of the CO2-based process. Diagrams of phase co-existence and conventional crystallization monitored by in-situ X-Rays diffraction (XRD) at atmospheric conditions will provide the fundamental grounds for selected systems. Microfluidics in scCO2, undeveloped for organic molecules, will provide temporal and spatial informations of crystallization in CO2 media thanks to a better control of local conditions whilst numerical simulation will help at understanding the contribution of the involved phenomena. CO2 crystallization will optimize the production of the relevant crystals based on both conventional and microfluidic outcomes. The project aims at developing in-situ techniques for the CO2-based set-ups ( Raman, UV, RX) to get access to real time monitoring of crystallization events.