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Press Info item.
11/12/2006
Improving the freeze-drying process for pharmaceutical proteins
The lyophilisation, or freeze-drying, of biological materials, such as proteins, vaccines or microorganisms, helps keep them physically and chemically intact for several months or even several years. As part of the European project known as Lyopro, INRA researchers from Versailles-Grignon have been studying the process for freeze-drying proteins and looking for ways to improve the long-term stability of freeze-dried proteins, especially in pharmaceuticals. After studying various molecule combinations designed to protect the biological activity of proteins, three formulations were chosen for their effectiveness and a patent has been filed.
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Freeze-drying biological materials, such as proteins or vaccines, is a costly process with varying results; it is currently, however, the only option for temperature-sensitive materials.
Water molecules are essential for maintaining a protein’s native conformation, but they also stimulate chemical breakdown reactions. Because it slows or stops these chemical reactions, the removal of water during the freeze-drying process has a positive effect on long-term storage. On the other hand, it can also destabilise the native conformation, making proteins more likely to become biologically inactive.
Active search for protective molecules
How might scientists ensure the long-term stability of proteins and make the freeze-drying process more effective? This was the task set before INRA researchers, in order to prevent the loss of biological activity of proteins. One option was to look for a molecule to replace water and yet play the same role, in other words, a protective molecule. However, the most effective molecules generally limited the productivity and industrial application of the process. Other solutions had to be found to meet the stability and productivity challenges facing the pharmaceutical industry.
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The Freeze-Drying Process
It was in the 12th century that the ancient Incas first discovered the freeze-drying process to preserve food, such as meat.
In 1906, Arsène d’Arsonval, then head of the biophysics laboratory at the Collège de France in Paris, together with colleague F. Bordas, invented the freeze-drying process. This now one hundred year old system involves eliminating the water contained in food or other products in order to keep it stable at room temperature and facilitate its storage.
Freeze-drying a product consists of three stages. First, the product is frozen so that the water contained in it becomes ice. Second, the ice in the product is made to vaporize without passing through the liquid phase; this process is known as sublimation. This vapour is collected by use of condensers, and, once removed from the freeze-drying chamber, becomes ice again. Finally, secondary desiccation involves eliminating the water that was not converted into ice during the first stage.
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© INRA / S. Passot
Close-up of a freeze-dryer
Tested and patented answers to the problem
The researchers worked closely with several industrial partners for over a year and tested 32 molecule combinations using two model proteins, the A and B toxins secreted by Clostridium difficile. These toxins are used as positive controls in Clostridium difficile diagnosis kits.
The formulations were studied for their impact on the long-term stability of the proteins and their physical behaviour during the freeze-drying process.
Adding one molecule with a specific protective action, such as the surfactants Tween 80 and polyvinylpyrrolidone, proved beneficial for the stability of both proteins, in that it limited their absorption at interfaces and/or stimulated their folding during rehydration.
The combination of these protective molecules with a molecule that provided mechanical resistance to the product during the process, such as glycine, led to shorter freeze-drying cycles with easier industrial application, without compromising the proteins’ long-term stability. Nevertheless, each protein has a unique structure and it is difficult to ensure the absolute effectiveness of these formulations using other proteins.
Three of the formulations tested were chosen for their effectiveness and a patent was then filed.
The researchers’ goal was to make the freeze-drying process safer and easier for use by the pharmaceutical industry. Freeze-dried products have distinct advantages over frozen products: lower storage and transport costs and immediate rehydration of the products.
Other improvements may yet be found in terms of homogeneity between products. For instance, proteins do not react the same way depending on their location in the freeze-dryer. Researchers are now studying the impact of the freezing stage on the freeze-drying process.
Patent Reference
S. Passot, F. Fonseca, M. Marin (2005). Compositions pour la lyophilisation de protéines. France (patent no. 05 00791).
European Project Reference
Competitive and Sustainable Growth Programme “LYOPRO” (Optimisation and control of the freeze-drying of pharmaceutical proteins – Contract No. GIRD-CT2002-00736 – Project No. GRD1-2001-40259).
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Written by :
INRA press service, phone: +33 (0)1 42 75 91 69
Contacts :
Stéphanie PASSOT
tel: 01 30 81 59 40
spassot@grignon.inra.fr
Joint Research Unit for Food Process Engineering and Microbiology, Science and Process Engineering of Agricultural Products Department, INRA Versailles-Grignon Research Centre.
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