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Preparation of efficient chromatography media for peptide and protein downstream processing/fast in-process control

Development of new media for fast downstream processing and
in-process control as well as development of specific
peptides for specific ligands in the purification of
plasma-derived and recombinant proteins.

Production and downstream processing in biotechnology requires, besides a fast and efficient isolation process, the fast and accurate control of each step in the process. Improved techniques which can be validated are required in order to meet these demands. Analytical methods such as gas chromatography (GC), capillary electrophoresis (CE) and especially high performance liquid chromatography (HPLC) allow monitoring of one more key substances without interference in the process itself. Recently, progress in the HPLC of biopolymers, especially as regards reduced time of separation and improved separation performance has been observed. It is to be anticipated that this analytical method will be among the methods of choice where on-line monitoring is concerned. The project is based on the application and optimization of newly developed, fast separation media, on analytical as well as preparatory scale, for the production of plasma proteins. The main advantage of these media is that they enable the different proteins to be separated within a few minutes. This fast separation is possible because the mass transfer of proteins between the mobile and stationary phase is not based on the diffusion as is the case with traditional porous media but rather on the convective flow. This feature means not only economic benefits due to a more efficient production process in a defined period of time but also an increase in the quality and yield of unstable products. The work will be oriented in three main areas: 1. Development of peptides for special binding of plasma proteins and their immobilisation on fast media 2. Application and optimisation of fast media for the downstream processing of plasma proteins 3. Application of fast media for fast in-process control. Peptides can be used for the specific binding of proteins. They have many advantages: they can be synthesized completely artificially and in higher quantities, the production is well controlled in comparison with isolation of proteins as they are more stable and can be heated well above 120 deg.C, which give them the opportunity to be used as in-line sterilisable separation modules. Octa and nona peptides will be used as ligands for affinity chromatography. They will be immobilised on the matrix in two different ways: * by adding the peptide into the monomer mixture, and * immobilising the peptide afterwards. In the first case, the peptide's influence on the characteristics of the polymer material should be checked and the optimal peptide concentration to be added should be determined. If the peptide is bound after immobilisation, the most suitable active group and immobilisation conditions should be examined. The result of this study should be a product with well defined characteristics as long-term stability of binding capacity. Depending on the peptide, many applications in research as well as production can be found. The second area of research will be directed to the production of plasma proteins using fast media. Some very encouraging preliminary studies have indicated that such media can be successfully used, giving high yields within a short time. However, to optimise the production, separation media with high binding capacity and mechanical stability, allowing easy scale-up, are required. The study will therefore be directed towards scaling up and optimisation of the separation units packed with fast media. Besides low dead volume and good flow characteristics, such units should also guarantee proper mechanical stability of the material. On the other hand, different active groups (such as ion exchange, metal- chelate, affinity, hydrophobia) will be introduced in order to improve selectivity and binding capacity. These tests will be run in parallel on small units which will be prepared for fast in-process control. Such units will be applied on different production processes, if possible, on-line. Keywords: virus removal, lymphocite, plasma proteins.
Acronym: 
FAST1
Project ID: 
1 766
Start date: 
01-07-1997
Project Duration: 
48months
Project costs: 
1 110 000.00€
Technological Area: 
Market Area: 

Raising the productivity and competitiveness of European businesses through technology. Boosting national economies on the international market, and strengthening the basis for sustainable prosperity and employment.