WO2007100294A1

WO2007100294A1 - Material system for blood products

Info

Publication number: WO2007100294A1
Application number: PCT/SE2007/000198
Authority: WO
Inventors: Kurt Nilsson
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-02-28
Filing date: 2007-02-28
Publication date: 2007-09-07
Anticipated expiration: 2008-08-28

Abstract

Product characterized by that the product contains at least one binding molecule covalently bound to a matrix, optionally via a spacer, and at least one blood bag or blood bag system, the use of the product on blood and blood plasma and the obtained blood products and blood derived products, proteins and antibodies. The invention is useful for e.g. reducing the amount of blood group specific antibodies in plasma to obtain universal plasma, and also to reduce the amount of blood group determinants on erythrocytes to obtain universal blood.

Description

Material system for blood products

A product for treatment of whole blood and/or blood plasma is described below.

The product according to the invention contains as one part a Material which contains a so-called matrix to which a binding molecule has been bound, optionally via a spacer molecule. The binding molecule is used for binding of a protein, an antibody, a virus or a cell.

The product according to the invention also contains at least one blood bag or a system of blood bags, or part thereof, used for collection of donor blood or for containment of donor blood, parts thereof or of blood plasma or of pooled blood plasma.

The invention also relates to the use of the product according to the invention on collected blood, especially human blood, parts thereof or on blood plasma. The invention also relates to the blood product and also to blood products derived therefrom, such as proteins and antibodies, obtained when using the product according to the invention, and also relates to the uses of the obtained blood products.

The use of the product on whole human blood, parts thereof or on blood plasma results in a blood product with a reduced quantity of protein, antibodies, protein or antibody of specified activity, virus or other components from whole blood, parts thereof or from blood plasma.

When the product is used alone for binding of antibodies specific for blood group A on donor blood or blood plasma from blood group B or O, the product results in a blood product or a blood plasma product which contains a reduced amount of blood group A specific antibodies. When the product is used alone for binding of antibodies specific for blood group B on donor blood or blood plasma from blood group A or O, the product results in a blood product or a blood plasma product which contains a reduced amount of blood group B specific antibodies. When the product is used alone for binding of antibodies specific for blood group A and blood group B on donor blood or blood plasma from blood group O, the combined product results in a blood product or a blood plasma product which contains a reduced amount of blood group A specific antibodies. This type of blood plasma product with reduced amount of blood group specific antibodies is below called universal plasma which is similar to existing blood plasma obtained from blood donors of blood group AB.

This type of blood plasma product may be given to patients of any blood group.

Several proteins and antibodies are purified from human blood. Such proteins and antibodies may contain e.g. antibodies specific for blood group determinants A and/or B, which may lead to side reactions when administered to patients with incompatible blood group. The invention also relates to blood derived human proteins and antibodies from the use of universal plasma obtained using the product, to obtain protein and antibody products with reduced content of blood group specific antibodies.

There are several types of blood bags or blood bag systems on the market for collection of and processing of collected blood and the different types of blood bags or blood bag systems do not limit the scope of the invention. For instance, there are blood bag systems containing three or four connected blood bags, where one is used for collection of human donor blood, and one for containment of human blood plasma produced using the blood bag system. The collected human plasma may be pooled with other plasma bags of the same blood group and stored in a larger blood bag.

One specific type of use of the product according to the invention on donor blood, parts thereof or on donor plasma of blood group A, B or O, results in the binding of the blood group specific antibodies present in the human blood or human plasma, leading to a reduction of blood group antibodies or other specific proteins or components from whole blood, parts thereof or from blood plasma. When the product binds to antibodies specific for blood group A or B or both, thus separating these from the whole blood, parts thereof or from blood plasma, such use of the combined product results in a blood product or blood plasma product which contains a reduced amount of blood group A and B antibodies.

The resulting blood plasma produced by using the product according to the invention on donor blood, parts thereof or on blood plasma, is here called universal plasma, which is similar to existing AB- plasma, and may be used to administer blood plasma to patients of any blood group. The invention also relates to the product according to the invention combined with α-galactosidase and/or N-acetyl-α-galactosaminidase enzymatic activity. The α-galactosidase and/or N-acetyl-α- galactosaminidase enzymatic activity may be obtained from an enzyme product containing the natural enzyme or an enzyme molecule with α-galactosidase and/or N-acetyl-α-galactosaminidase enzymatic activity obtained using recombinant techniques. The enzyme product may according to the invention be used as a soluble molecule or as an immobilized molecule to a solid matrix, such as e.g. agarose or crosslinked agarose, e.g. immobilized to NHS-activated cross-linked agarose. This product of the invention containing the enzyme product, the Material and the blood bag or blood bag system is used for treatment of donor blood, with the enzyme product and the Material used simultaneously e.g. in the first blood bag in a given blood bag system, or separately in different blood bags, e.g. with the enzyme product treatment in the first blood bag or in the blood bag containing erythrocytes, and with the Material in e.g. the plasma blood bag or after the plasma blood bag.

This latter aspect of the invention refers to the blood product obtained from the use of such a combined product to treat donor human blood of e.g. blood group A or B to obtain a blood product which has a reduced amount of blood group determinants A and/or B present on erythrocytes (as obtained with the N-acetyl-α-galactosaminidase activity and/or the α-galactosidase enzymatic activity on the blood group determinants present on erythrocytes) and with a reduced amount of blood group A or blood group B specific antibodies (from the use of the Material containing blood group A and/or blood group B saccharide antigen(s) which specifically binds the blood group specific antibodies A and B respectively).

This type of blood product here called universal blood, can be used to administer to patients of e.g. blood group AB, A, B or O.

The active part of the Material according to the invention, contains at least one binding molecule, such as a protein, or saccharide or glycoprotein, mucin or glycopeptide, obtained e.g from natural sources, synthetically or using recombinant techniques or recombinant cells, and which has been bound via a spacer to a Matrix. This is exemplified for saccharide, glycoprotein and mucin below:

Saccharide-Matrix, or Saccharide-spacer-Matrix, or Glycoprotein-Matrix, or Glycoprotein-spacer-Matrix or Mucin-Matrix, or Mucin-spacer-Matrix

Among the functions of the spacer may be mentioned that the spacer separates the binding molecule, e.g. the Saccharide from the Matrix and thus covalently binds together the Saccharide with the Matrix.

The matrix consists for example of a polymer, a plastic, a polysaccharide, or a cross-linked polysaccharide, and can bind a large number of Saccharide-spacer units. The matrix can be e.g in the form of beads or in the form of a filter.

The Material may according to the invention be present within one or more of the blood bags of the blood bag system before collection of the donor blood, or may be administered after collection.

The matrix can be magnetic or non-magnetic. If the matrix is magnetic, this characteristic can be used to separate the material from the whole blood or the plasma using an externally applied magnet during emptying of the whole blood or blood plasma from the blood bag.

Otherwise the Material may be separated from the whole blood, blood fraction or blood plasma using a filter with a pore size which is lower than the particle size of the material described above, and thus the filter does not allow passage of the material but allow passage of the blood, blood fraction, blood containing erythrocytes but for example not of larger blood cells, or allow passage of blood plasma.

The Material may also be separated by centrifugation of the blood bag containing the material.

The Material may in an alternative mode according to the invention be in the form of or within a filter connected between or after the blood bags in the blood bag system.

The volume of the Material, the treatment time and the treatment temperature are decided by the desired binding capacity of the specific protein, antibody, cell or virus. Non-limiting example is either 0.1, 1, 2, 5, 10, 20 mL volume, or a value between these values, of Material per L of blood or plasma treated. Non-limiting treatment time example is either 1, 5, 20, or 60 minutes or overnight or a value between these values. Non-limiting treatment temperature example is either cool room temperature, room temperature, or 37 oC or a value between these values.

The binding molecule in the Material described above, e.g. a protein, glycoprotein, mucin, glycopeptide and saccharide, has a biological or other affinity to another molecule, e.g. protein, such as antibody, protein, or other blood component or a virus.

The saccharide can consist of a glycoprotein, a mucin, neoglycoprotein, a glycopeptide or a glycosylated amino acid, a glycolipid, or a part, a fragment or a modified variant thereof, or another di-, tri-, tetra-, or pentasaccharide or higher oligosaccharide substance, obtained from natural sources, by synthetic methods, or by recombinant techniques and this do not limit the scope of the invention.

The binding molecule as exemplified above can be produced chemically or with biological methods, e.g. involving recombinant cells or bacteria, or by a combination of these methods.

The binding molecule may contain one or more of a di-, tri, or higher oligosaccharide linked together synthetically, e.g. via a di-, tri or oligomeric molecule, a peptide or a protein, thus creating a di-, tri-, oligomeric binding molecule. This may then be linked to the Matrix directly or via a spacer.

A few non-limiting examples of biologically active Saccharide, spacer and Matrix which can be used according to the invention, are further exemplified below.

The function of the binding molecule as exemplified above, is to bind to one or more of the molecules mentioned above, for example antibodies specific for blood group determinants A or B, other antibodies, proteins or components of whole blood or blood plasma.

The binding molecule as exemplified above may consist of blood group determinants, e.g. blood group A, blood group B, other blood group determinants, or other carbohydrate structures found on gangliosides, e.g. of the type GQIb, GTIa, GD3, GMl or GM2, e.g. containing the saccharide structure NeuNAc(α2-8)NeuNAc(α2-3), the trisaccharide structure NeuNAc(α2-8)NeuNAc(α2-3)Gal, or tetra-, penta- or higher oligosaccharide structures containing one or more of these di- or trisaccharide structures, glycolipids, or glycoproteins. The saccharide or the binding molecule may contain the terminal or internal di-, tri-, tetra, penta- or higher oligosaccharide structures, branched or unbranched, found in gangliosides, glycolipids, mucins or glycoproteins in Nature.

The product is further characterised by that the Material as described above, has been sterilized, e.g. preferentially autoclaved, e.g. at least at 121 oC for a time which allows validated sterilization, or alternatively treated with heat and or steam, or treated with other sterilization method such as UV-light or ethylene oxide. The product is further characterized by that it has been optionally produced under validated clean room conditions under GMP to allow for function and safety for its clinical purpose.

The Material according to the invention may consist, as a non-limiting example, of for example either:

1. Blood group A determinant-spacer -Matrix or:

2. Blood group B determinant-spacer-Matrix or

3. A combination of the above type of Saccharides (blood group A and blood group B determinants) each bound via a spacer to Matrix.

Specific example of spacer:

1. Blood group A-O(CH2)_nPhNH-CO-(CH2)_mNH-CH₂-CH(OH)-CH2-O-Matrix or:

2. Blood group B-O(CH2)_nPhNH-CO-(CH2)_mNH-CH2-CH(OH)-CH2-O-Matrix or

3. A combination of the above two Saccharide structures (blood group A and blood group B) each bound via a spacer to Matrix

Matrix denotes e.g. a polymer, a plastic or a polysaccharide, for example cross-linked agarose.

As a specific example of cross-linked agarose may be mentioned Sepharose^- Fast Flow, where -O(CH2)_nPhNH-CO-(CH2)_mNH-CH2-CH(OH)-CH2- is spacer, according to the invention, to separate the Saccharide, in above examples blood group determinant A- and B-, respectively, from the Matrix, where n and m, respectively, is an integer, n is for example one of 0, 1, 2, 3 or 4, and m is for example 1, 2, 3, 4, 5, 6 or 7, and where the linkage between -O- and Matrix is formed between -O- and for example a carbon atom in the Matrix.

Saccharide-spacer, for example Blood group A-O(CH2)_nPhNH-CO-(CH2)_mNH- and Blood group B-O(CH2)_nPhNH-CO-(CH2)_mNH-, respectively, is below called (the) ligand.

Other spacers may be used according to invention and a few examples with binding molecule (BM below) linked to Matrix, are:

BM-N(Ac)-(CH2)n-PhNH-CO-CH2- linked directly or via a further molecule to Matrix, eg.

BM-N(Ac)-(CH2)_nPhNH-CO-(CH2)_mNH-CH2-CH(OH)-CH2-,

-N(Ac)-CH₂-CH(OH)-CH2-O-.

The binding molecule of the Material consists, as another non-limiting example, of for example a mucin or a glycoprotein or a glycopeptide linked to for example cross-linked agarose. When producing the material, for example, these molecules, or a derivative thereof, may be reacted with cross-linked agarose which has been activated with N-hydroxysuccinimide (NHS) groups. NHS-activated Sepharose is an example of such an NHS-activated cross-linked agarose.

The Matrix has a large number of bound molecules of ligand. Examples of bound amount of ligand is 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 rnnole per liter of Matrix, or an amount of mmole which is between two of the above given values per liter of Matrix. Per liter Matrix here means the volume occupied by the ready-to-use Matrix product.

A combination of two or more different saccharides can be used according to the invention, for example as non-limiting example, a combination of Blood group A-O(CH2)_nPhNH-CO-(CH2)_mNH-, and Blood group B-O(CH2)_nPhNH-CO-(CH2)_mNH-, where both ligands in this example are bound to Matrix.

A non-limiting example of a preferred variant of blood group A containing Material 1 above is:

l.a. GalNAcαl-3(Fucαl-2)Galβ-0(CH2)2PhNH-CO-(CH₂)5NH-CH2-CH(OH)-CH₂-0-Matrix This type of Material can be produced by reaction between for example GalNAcαl-3(Fucαl-2)Galβ-O(CH2)2PhNH₂ and for example NHS-activated Sepharose^R 4FF, where the latter is commercially available, or cross-linked agarose or other Matrix with corresponding properties.

Similarly the Material according to the invention may contain GalNAcαl-3Galβl-4GlcNAcβl-3Lactoseβ-O O(CH₂)2PnNH-CO-(CH2)5NH-CH2-CH(OH)-CH₂-O-

Matrix, And or:

GalNAcαl-3Galβl-4GlcNAcβ-O O(CH2)2PhNH-CO-(CH₂)5NH-CH2-CH(OH)-CH2-O-Matrix These are produced in a similar manner as above.

The reaction conditions are chosen by the expert and does not limit the scope of the invention. Other examples are for example Material containing a in the same manner bound, higher oligosaccharide than the A-trisaccharide in the above example, which contains the A-determinant terminally, for example A-determinant of type 1, 2, 3 or 4. Similarly structures 2, 3, 4 and 5 above can be prepared via reaction with NHS-activated Sepharose and the corresponding Saccharide- O(CH2)2PhNH_2.

Material above can be prepared as another example, via reaction of e.g. amino group containing matrix such as cross-linked agarose or amino group containing Sepharose, with the corresponding biomolecule, such as a Saccharide followed by acetylation of the amino group of the formed aminoglycoside.

The trisaccharide derivative GalNAcαl-3(Fucαl-2)Galβ-O(CH2)2PhNH₂ and other saccharide derivatives, mentioned in this application can be produced with different chemical and/or biochemical methods and this do not limit the scope of the invention.

Further examples of product 1 is Product where a combination of the trisaccharide and one or more of mentioned blood group A variants, are bound via the same type of spacer as shown above to Matrix, or via a different type of spacer. A non-limiting example of a preferred variant of the Material 2 above is:

2.b. Galαl-3(Fucαl-2)Galβ-O(CH2)2PhNH-CO-(CH2)5NH-CH2-CH(OH)-CH2-O-Matrix

This type of Material can be produced by reaction between for example Galαl-3(Fucαl-2)Galβ-O(CH2)2PhNH₂ and for example NHS-activated Sepharose^R 4FF₅ where the latter is commercially available. The reaction conditions are chosen by the expert and does not limit the scope of the invention. Other examples are for example Material containing a in the same manner bound higher oligosaccharide, which contains the B-determinant terminally, for example B- determinant of type 1, 2, 3 or 4. Further examples of Material 2 are Material where a combination of 2.b. and one or more of mentioned blood group B variants, are bound via the same type of spacer as above to Matrix, or via a different type of spacer.

Another non-limiting example of a preferred variant of the Material 2 above is:

Galαl-3Galβ-O(CH2)2PhNH-CO-(CH₂)5NH-CH2-CH(OH)-CH2-O-Matrix

Instead of the -O(CH2)2PhNH- group in the formulas above, another suitable Spacer or part of Spacer can be used, as for example the type of spacers mentioned above, or -O(CH2)_nNH- or for example N(Ac)-(CH^)_nNH- (Ac = Acetyl group; n is an integer, for example 1, 2, 3, 4, 5, 6, or 7 or higher), or another aliphatic compound, or another aromatic compound.

The blood group A- and B-saccharides exemplified above may also be parts of mucins, glycoproteins or glycopeptides and as such linked to Matrix, e.g. via reaction with NHS-activated cross-linked agarose as exemplified above.

The saccharide, as for example the blood group A- or B- determinant mentioned above, can also be bound, directly or indirectly, to an oligomeric molecule acting as Spacer, or part of Spacer, as for example an aliphatic or aromatic molecule, a mono-, di-, or higher oligosaccharide or polysaccharide, peptide, for example a peptide consisting of amide bound glycine and glutamic acid residues, for example Gly-(Glu-Gly)n-Glu, where n is an integer between for example 1 and 20. In this manner the Saccharide-spacer consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more saccharide units bound to each oligomeric substance or peptide.

In this latter example, the linkage between the saccharide and the peptide can for example be formed via O-glycosidically bound -0(CH^PhNH- group (see for example formulas 2. a. and 2.b. above), or via for example O-glycosidically bound -0(CH^)_nNH-, (where n is an integer for example 1,2,3,4,5,6,7 or higher), where NH- is bound via an amide linkage (NH-CO) to the carboxyl group on the side-chain of the GIu- residues in the peptide. -O in -O(CH2)2PhNH- and in -0(CH2)_nNH-, respectively, is then bound glycosidically to the Saccharide.

The peptide can first have been coupled to Matrix, for example NHS-activated Sepharose^R 4FF via the α-amino-group on the peptide, and thereafter can the saccharide be bound to the peptide via -O(CH2)2PhNH-, or for example -O(CH2)_nNH-, to the carboxyl group on the Glu-residues in the peptide. This linkage between saccharide and Glu-residues can be achieved by for example carbodiimide-(for example EDC-) mediated coupling, or by for example succinimide-mediated coupling. The saccharide-spacer can herewith be added to the reaction mixture in for example a desired molar excess in relation to the amount of moles of peptide, e.g. in a molar excess of 2, 3, 4, 5, 6, 7, 8, 9 or 10 times excess or more. These and other reaction conditions are chosen by the expert and do not limit the scope of the invention. Non-limiting examples of in this manner bound amount of Saccharide is 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 irmole of ligand per liter of Matrix. Per liter of Matrix means here the volume occupied by the ready to use Matrix product.

Another example of peptide is as above, but instead of GIu, containing at least one Lysine residue, where for example the alfa-amino group of the peptide or the ε-amino group in the Lysine residue of the peptide is used for covalent coupling to Matrix, for example NHS-activated Sepharose^R 4FF or NHS-activated cross-linked agarose. Such a peptide may contain intervening amino acid residues, e.g. Glycine residues. An example of such a peptide is thus (Glyn-Lys-Glym-Lys)z, where n, m and z are integers as exemplified above. An example using a peptide or protein containing at least one Lysine residue, is to couple the peptide or protein to for example NHS-activated cross-linked agarose or Sepharose, followed by reaction of amino-groups, of the peptide or protein, with the reducing end of the Sacccharide, followed by acetylation of amino groups, thus forming Saccharide-N(Ac)- linkages between the Saccharide and amino group on the peptide or protein.

In this way each spacer can be made to be linked to two or more Saccharide molecules, thus creating a multivalent spacer concerning the number of Saccharide molecules linked to each Spacer. By adjusting the distance (by for example choosing different number of GIy residues) between each Lysine residue in the peptide, the distance between each Saccharide molecule (linked as above to each Lysine residue) may also be varied, thus creating an optimal distance for binding of the protein, virus or cell desired to be separated. TWs distance may also be varied by choosing different starting concentrations of the Saccharide and/or of the peptide or protein, when reacting the saccharide with the peptide or protein.

An advantage of using mucins, glycopeptide, glycoprotein which contain several units of the binding molecule per mucin, glycopeptide or glycoprotein molecule, is that a stronger binding often can be achieved, due to the plurality of Saccharide molecules on each spacer molecule, to the molecule which is desired to separated by the product, for example of antibodies towards blood group determinant or of other antibodies, proteins, viruses or cells, and that therewith a more efficient product can be obtained as compared with non-oligomeric ligand.

As a non-limiting example of one preferred variant of Material according to the invention and as a non-limiting example of its production may be mentioned the coupling of ca 3, 1.5 and 1 μmol, respectively, of the peptide

Ac-Lys-(ε-amino)-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-Glu-Gly-amide via its ε-amino group, to 2 ml of NHS-activated Sepharose 4 FF, from Pharmacia-Biotech, at pH 7.5 (0.2 M sodium fosfate buffer + 0.5 M NaCl) under 4 h at room temperature followed by 0.3 M Tris-HCl, pH 8, at room temp., over night. The gel is washed with Tris-buffer and 0.1. M MES, pH 4.7, which gave Peptide-Sepharose 4FF. Saccharide-spacer, as for example Gala 1-3 GaIa-OPhNH₂, ca 25 mikromole, dissolved in 0.1 M MES-buffert, pH 4.7, was added to a solution of 48 mg EDC ((l-Ethyl-3-(3- Dimethylaminopropyl)-Carbodiimide)), to which was added 2 ml of Peptide-Sepharose 4FF, the mixture was incubated under 4 h at room temperature, after which it was washed with Tris-HCl, pH 8, 0.1 M acetate- and 0.1 M sodium fosfate buffer, respectively. The gels were tested for binding of antibodies, in the example of anti-Galαl-3Gal antibodies and showed a better binding of IgM antibodies as compared with the same amount of Saccharide-spacer-Sepharose 4FF, obtained by coupling of the corresponding amount of Gala 1-3GaIa-OPhNH- , but without peptide, directly to NHS-activated Sepharose 4FF.

Other saccharide derivatives than Gala 1-3 GaIa-OPhNH₂ can be used according to the invention, such as for example

GalNAcαl-3(Fucαl-2)Galβ-O(CH2)2PhNH2 or Galαl-3(Fucαl-2)Galβ-O(CH₂)2PhNH2.

The saccharide-peptide conjugate can for example first be prepared by using for example the, ε-BOC- derivative (BOC = tert-butyloxycarbonyl group, situated on the ε-amino group of the Lysine residue) of for example the peptide in the above mentioned example. The Saccharide-spacer-peptide konjugat is then first formed by for example EDC-mediated reaction between amino groups on the saccharide- spacer and the carboxyl groups on the peptide, the resulting conjugate can be purified by for example Sephadex chromatography, the BOC group can be eliminated by for example trifluoroacetic acid reaction according to standard conditions for peptide chemistry, and the conjugate can be coupled for example in the same manner as describe above via the e-amino group of the lysine residue to the Matrix, for example NHS-activated Sepharose 4 FF.

As another non-limiting example, can be mentioned peptide consisting of amide bound GIy and Lys units, for example Gly-(Lys-Gly)n-Gly, where n is an integer between for example 1 and 20. In this case can for example the peptide be bound to the Saccharide via amino groups on the peptide, a N- glycosidic linkage is formed between the reducing end on the Saccharide and the ε-amino group on the Lysine residue(s), and the Saccharide-peptide can be coupled to the Matrix via for example either the remaining amino group(s) on the peptide to for example NHS-activated Sepharose as described above, or via for example the terminal COO-group on the peptide and amino groups on amino group containing Matrix, for example aminohexyl-Sepharose (by for example carbodiimide or succinimide coupling according to examples given above). The N-glycosidic linkage can be stabilised by acetylation under standard conditions, for example before coupling to the Matrix, e.g. NHS- activated Sepharose 4 FF or NHS-activated cross-linked agarose. In the same manner as for the GIy- Glu-peptide above can also an aliphatic or aromatic spacer be used to bind the Saccharide to the Lysine residues of the peptide, but in this case is, for example, glycosidically bound groups of the type -0(CH2)2PhCOO, or for example -O(CH2)_nCOO-, used for carbodiimide- or succinimide-mediated coupling between Saccharide and Lysine residues in the peptide.

The coupling to the peptide can also be carried out by first coupling the Saccharide part to the amino acid and thereafter form the peptide linkages.

Further examples of Ligand according to the invention, is to use a protein or a polysaccharide as Spacer, or part of Spacer, between Saccharide and Matrix. Here for example a protein, such as serum albumin, or a polysaccharide, such as dextran, is used. The Saccharide can then first be coupled to the protein, or to the polysaccharide, which then is coupled to the Matrix. The same type of chemistry as exemplified above can, as non-limiting examples, be used to achieve the linkages between Saccharide, protein, or polysaccharide, and Matrix. This do not limit the scope of the invention, and the conditions are chosen by the expert.

To use a glycopeptide, glycoprotein, mucin with branched saccharide determinants or polysaccharide according to what have exemplified above, can in some cases be an advantage to increase the ability of the Material to bind protein, and thereby increase the efficiency of the product according to the invention.

As another example of Matrix can be mentioned the filters which are used for plasma separation or separation of white blood cells in blood bag systems. These can be chemically modified with standard technique and be used for coupling of biomolecules mentioned in this description.

In this manner is achieved a product which can be used for specific removal of proteins in connection with blood plasma separation.

In a variant of the invention, the product in addition contains a Tris structure according to the following non-limiting example: (HOCH₂)3C-NH-CO-(CH2)5NH-CH₂-CH(OH)-CH2-O-Matrix where (HOCH2)3 C-NH- is a so called Tris-group. This product can be made by reaction between Tris- HCl and for example NHS-activated Sepharose 4 FF in which case Matrix above is Sepharose 4 FF. In the production of the product according to the invention can for example be used commercially available activated Matrix, for example so called NHS-activated Sepharose^- 4 Fast Flow (NHS- is an abbreviation of N-hydroxysuccinimide; this variant of agarose is relatively strongly cross-linked, commercially available), which is present in the form of practically spherical particles. The particle size is chosen in, for example, the interval 45 - 165 μm.

This activated Matrix can be used for covalent binding of for example, Blood group A-

O(CH2)_nPhNH2-₅ to give Material 1.a. above, and of

Blood group B-O(CH2)_nPhNH2 , which give Material 2.b. above, respectively, at, as non-limiting and typical example, pH 7.5 or pH 8.0, in buffer, for example 0.1 M sodium phosphate as non-limiting example, under for example 1, or 2 hours or for 20 hours, or at a pH and for a reaction time between above values, and in the example at room temperature or at other desired temperature.

The material is washed for example on a glass filter or under other conditions, for example sterile conditions, with for example buffer and is subsequently treated with for example

Tris-HCl buffer to react any remaining reactive groups. The expert chooses the conditions for the reactions and this do not limit the scope of the invention.

In the production of the Material according to the invention can, as another example, be used so-called epoxy-activated Sepharose^R 4 Fast Flow, to which is covalently bound, for example Blood group A-O(CH2)_nPhNH-CO-(CH2)_mNH-, or to which is covalently bound

Blood group B-O(CH2)nPhNH-CO-(CH2)_mNH-, where n och m are specified above as are Blood group A and Blood group B, respectively.

As has been mentioned above, a combination of ligands can also be covalently bound the Matrix.

When using Products according to the invention for treatment of plasma, can for example be used membranes which have a lower porosity and Material particles which have lower particle size as compared with the case when the Product is applied for treatment of whole blood.

Thus, for example, in the case of treatment of plasma, membrane with porosity of for example 30 micrometer, or membrane with a porosity in the interval 10 till 40 micrometer, and particle size of Matrix of for example 90 micrometer, or Matrix of for example particle size in the interval 40 - 200 micrometer, can be used. The combination of particle size and filter pore size is chosen by the expert. When using Products according to the invention for treatment of whole blood, for example membrane with porosity of for example 30 micrometer or 70 micrometer, or membrane with a porosity in the interval 20 to 100 micrometer, can be used, and the particle size of the Matrix can be for example 150 micrometer, or the Matrix particle size can be for example in the interval 100 - 250 micrometer. The porosity is chosen by the expert and does not limit the scope of the invention.

Non-limiting example of autoclaving the material as described above, is treatment of the material in an autoclave of for example counter-pressure type, which involves treatment under for example at least 20 minutes at 121 ⁰C or higher and with for example water steam. Other conditions can be chosen by the expert from what is suitable, e.g. sterility and stability of the product. As an example can be mentioned that Saccharide-spacer-Matrix according to examples l.a. and 2.b., obtained via coupling of the respective ligand to NHS-activated Sepharose 4FF, exhibits the same properties after autoclaving as before autoclaving concerning tested parameters such as antibody binding properties and other properties.

As background it may be mentioned that there are different blood bags and blood bag systems for collecting and processing of donor blood. These different systems do not limit the scope of the invention, in which the product can be adapted to the different blood bags and blood bag systems available. The blood bag systems often contain three or four blood bags connected with tubings integrated in the system. Non-limiting commercial examples are Baxter R6488 or 7544.

Some of these systems have a filter which allows passage of blood plasma and erythrocytes, but not of larger components. In the latter type of system collected blood is passed from the first bag, bag number one, via a plastic tubing through a filter and through a tubing to bag number two, which thus is filled with blood plasma containing erythrocytes. This bag may then be centrifuged separating erythrocytes from the blood plasma. The blood plasma is then transferred to bag number three via tubings.

The Material according to the invention may for example be administered to the donor blood bag before or after blood collection, or may be included in a filter which is connected before the blood bags, between two of the blood bags, or after the blood bags which are used in the blood collecting blood bag system. When used in conjunction with blood collection the material may optionally be present in the blood collecting blood bag and the material can be separated e.g. by a filter between blood bag number one and blood bag number two in the blood bag system, where the filter allows passage of blood plasma and erythrocytes, but not of the material and optionally also do not allow passage of white blood cells.

The material may also be separated using a filter, or by centrifugation of the blood plasma bag, from the blood plasma in the product according to the invention.

The Material according to the invention may also be used on pooled plasma from different blood bags and may be administered to the pooled plasma and separated after treatment.

If the material contains a magnetic matrix, as exemplified above, separation is achieved by magnetic separation of the material from the blood, parts thereof or from blood plasma.

The amount of the material used in the examples above and also the contact time between the material and the blood, or parts thereof or the blood plasma, is determined by the expert depending on the configuration used and this do not limit the scope of the invention.

Claims

Claims.

1. Product characterized by that the product contains at least one material which contains at least one binding molecule covalently bound to a matrix, optionally via a spacer, and at least one blood bag or blood bag system.

2. Material according to claim 1, which has been treated by autoclaving or sterilized using chemical or UV-light.

3. Material according to claim 1, which has been produced in validated clean room area.

4. Use of product according to claim 1 on blood, parts thereof or on blood plasma.

5. Blood product or blood derived product obtained after use according to claim 4.

6. Blood product and blood derived proteins and antibodies obtained by use of product according to claim 1 on whole blood, parts thereof or on blood plasma.