Y. Rigaldie (1,2,3), A. Largeteau (2), G. Lemagnen (1), D. Larrouture (1), M. Abba (1,3), R. Haller (3), G. Demazeau (2), L. Grislain (1)
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(1) LTPIB (Laboratoire de Technologie Pharmaceutique Industrielle de Bordeaux), Faculté de Pharmacie, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat 33076 Bordeaux cedex France
(2) IHP (Interface Hautes Pressions), ENSCPB, Avenue Pey Berland, BP 108 33402 Talence cedex France
(3) Bertin Technologies, 10 av. Ampere, Parc d'Activités du Pas du Lac, 78180 Montigny-le-Bretonneux, France
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Ionizing radiations or temperature are the most commonly ways to sterilize drugs. In the case of fragile drugs, such as molecules issued from biotechnologies, ultrafiltration or aseptic processes are the only conventional alternative methods which can be used in industrial pharmacy in order to sterilize without altering them. High Hydrostatic Pressure (HHP) could represent another soft alternative process.

The aim of this study was to determinate the parameters of HHP treatment in order to inactivate various strains of microorganisms described in European Pharmacopoeia and to preserve liquid and semi-solid pharmaceutical drugs integrity.

These essays have been performed with a high hydrostatic press designed by NFM-FRAMATOME. The range of temperature and pressure was respectively -20C up to 80C and 0,1 up to 800 MPa (100 MPa = 1000 bar).

Several molecules chosen with molecular weight from 1200 g/mol up to 150 000 g/mol have been tested. According to HPLC control methods, HHP appears to be a safe treatment for biomolecules with low molecular weight (two peptides around 1200 g/mol have been treated : 600 MPa, room temperature). In the same way insulin (5800 g/mol) did not show significant modifications (600 MPa, -20 C and 25C). But, according to ELISA tests, monoclonal antibodies anti-influenza A (150 000 g/mol), have shown a reversible loss of their specificity from 500 MPa.

10 min at 400 MPa are necessary to inactivate totally pure suspensions of P aeruginosa, C albicans and spores of A niger.
The association of HHP with low temperature (-17C) on P aeruginosa and C. albicans has shown a an improvement of the inactivation than at room temperature, using the same pressure. Such an effect had been observed previously for S. cerevisiae.
Because of the difference in the structure of the membrane between Gram - and Gram +, Gram + are less barosensitive. The increase of processing time and pressure has been tested in order to inactivate S. aureus, a Gram + bacteria. A multi-step process at 25C, 500 MPa and during a total time of 150 min, inactivate totally S. aureus. At fixed conditions of pressure, temperature and time processing, best results were achieved by using a multi-step process compares to a single high pressure application.

Nevertheless, it appears that the same tests (i.d. cycling, time processing, temperature) applied on spores of B. subtilis did not lead to a total inactivation as for vegetative strains of the European Pharmacopoeia.
Further processes are now in progress in order to obtain a significant inactivation of this form.

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