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Preparation and studies of dyes and dye sorbents for purification of biologicals

Research into specific sorbents to mimic biological
recognition using simple organic molecules. The advantage
is economic: large source of materials, low cost and long
term stability.

1. Antecedents: Protein purification is one of the most important specialities in advanced biotechnology. Scientific articles published lately show that organic dyestuffs have specific reactions with individual proteins, which can be used for their purification. For some years, IBF (a French company specializing in matrices for protein purification) used traditional techniques for the immobilization of several dyes. Two of them are currently available from IBF catalogue and are used in liquid chromotography. These IBF dye-sorbents are their applications have been described in numerous scientific publications in international journals. During 1986-88, IBF and VILMAX (an Argentinian company specializing in organic dyestuffs) did advanced research in this area. Their original findings and discoveries are covered by Argentine patent No. 310.844 of May 1988 which refers to an original way to derivatize dyes and then to copolymerize them into homogeneous three-dimensional polymers. 2. Background and general information In the field of biotechnology, gene expresion and large scale culture of bacteria and cells for specific preparations of proteins are well developed and the knowledge has been applied on a large scale. Today, the limiting factor is the purification method of biologically produced proteins and particularly, the elimination of numerous impurities either derived from the cells or brought in by the culture media. The purification procedures known up to now are very heavy as they involve several steps including precipitation, ion exchange chromotology, hydrophobic chromatography, gel filtration, reverse phase chromatography and affinity chromatography. In view of reducing the number of expensive chromatographic steps, users are looking for very specific sorbents. Unfortunately, these latter are based on biospecific recognition and are built with expensive and relatively unstable biological material. This is the case of, for example, monoclonal antibodies. The objective in the research of specific sorbents is to mimic the biological recognition using simple organic molecules. The advantage of this approval is obviously of economic order: large source of materials, low cost and long term stability. In recent years, it has been decided several times that the use of current dyes can replace advantageously biological ligands. Some of them are already used for the purification of proteins at laboratory scale as well as preparative scale. The difficulties encountered in the development of such an approach are: - present of residual leached dye in the column effluents when the chemical link on the chromotographic support is not stable, - unknown biological effect of dyes in vivo (in case of leakage), - no assay tests available to determine the level of dye leached from a column, - lack of information from the dye manufacturer concerning the presence of impurities and the batch to batch consistency. This situation does not incite potential users to develop a down- stream processing involving immobilized dyes. Many of them are ready to try dyes in their procedures provided the above described problems are solved. Immobilized dyes will have not only a concrete and rapid application in the purification of well known products such as human albumin, transferrin, milk proteins, interferon or insulin, but could be applied to many recombinant proteins of diagnostic and therapeutic interest. 3. Market description: For all biotechnology projects dealing with the production of a particular protein for diagnostic or therapeutic application, the purification step becomes very crucial. Two objectives must be achieved: - obtaining a pure product - use of a simple and cost saving procedure. Downstream processing (extraction and purification) represents in most cases more than 80% of the production cost, a great part of which is dedicated to chromatographic separations. The worldwide market of chromatographic supports for protein isolation (gel filtration, ion exchange, affinity) represents about 450 million FF per year. The growth estimated at 20-25% is dominated by BIO-PHARMA applications. This rate applies particularly to industrial and preparative applications which involve increasingly large columns. In comparison with other chromatographic techniques, affinity chromatography still plays a minor role in preparative purposes because it represents only 15% of the overall, i.e. 65-70 million FF. However, the interest for it keeps growing as long as appropriate and costless chromatographic supports could be marketed. A recent statistic study at academic level showed that 26% of laboratories which isolate proteins by chromotography, make use currently of affinity sorbents. Moreover, when taking into account the type of chromatographic support, 23% of these laboratories use immobilized dyes, 30% used immobilized lectins, the rest use various other supports.
Acronym: 
DYMECHROM
Project ID: 
384
Start date: 
26-11-1989
Project Duration: 
43months
Project costs: 
1 330 000.00€
Technological Area: 
Market Area: 

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