SADLER CHRIS (GB)
WO2004035199A1 | 2004-04-29 | |||
WO2003080655A1 | 2003-10-02 | |||
WO2004052870A1 | 2004-06-24 |
US6831161B1 | 2004-12-14 | |||
US20140135476A1 | 2014-05-15 | |||
US9162223B2 | 2015-10-20 |
Claims 1. A method of producing an activated substrate, the method comprising: (a) modifying a substrate, wherein the substrate comprises a base matrix, to form a base matrix comprising a leaving group; (b) contacting the base matrix formed in step (a) with an aminating agent, to thereby provide an aminated base matrix; and (c) contacting the aminated base matrix formed in step (b) with a heteroaromatic compound, wherein the heteroaromatic compound is a 5 to 12 membered heteroaromatic ring substituted with at least two halogens and optionally one or more further substituents, to produce an activated substrate. 2. The method of claim 1, wherein the substrate comprises a nucleophilic moiety, which is preferably a hydroxyl moiety. 3. The method of claim 1 or claim 2, wherein step (a) of the method comprises: ai) contacting the substrate with an electrophile, wherein the electrophile comprises an unsaturated hydrocarbon chain, to thereby form a base matrix comprising an unsaturated hydrocarbon chain; and aii) contacting the base matrix formed in step (ai) with a halogenating agent, to thereby provide a halogenated base matrix. 4. The method of claim 3, wherein the electrophile is a compound of formula (I): R1-L1-X1-L2-R2 (I) , wherein R1 is an optionally substituted 3 to 6 membered heterocyclic ring or a leaving group; R2 is an optionally substituted C2-C12 alkenyl or an optionally substituted C2-C12 alkynyl; L1 and L2 are each independently absent, an optionally substituted C1-12 alkylene, an optionally substituted C2-12 alkenylene or an optionally substituted C2-12 alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms; X1 is NR3, O or S; and R3 is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl or an optionally substituted C2-C6 alkynyl. 5. The method of claim 3 or claim 4, wherein the halogenating agent is N- bromosuccinimide, N -chlorosuccinimide, bromine or chlorine. 6. The method of any preceding claim, wherein the aminating agent is NH2R16 or , wherein R16 and R17 are independently H, an optionally substituted C1-24 alkyl, an optionally substituted C2-24 alkenyl or an optionally substituted C2-24 alkynyl and L3 is an optionally substituted C1-12 alkylene, an optionally substituted C2-12 alkenylene or an optionally substituted C2-12 alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms. 7. The method of any preceding claim, wherein the heteroaromatic compound is a 5 or 6 membered heteroaromatic ring which is substituted with at least two halogens, and optionally one or more further substituents independently selected from the group consisting of OH, SH, COOH, NH2, optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl and optionally substituted C2-C24 alkynyl. 8. The method of claim 7, wherein the heteroaromatic compound is dichloro- triazine or cyanuric chloride. 9. The activated substrate obtained or obtainable by the method of any one of claims 1 to 8. , wherein L3 is an optionally substituted C1-24 alkylene, an optionally substituted C2-24 alkenylene or an optionally substituted C2-24 alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms; R16 is H, an optionally substituted C1-24 alkyl, an optionally substituted C2-24 alkenyl or an optionally substituted C2-24 alkynyl; and R18 is a 5 to 12 membered heteroaryl group substituted with at least one halogen, and optionally substituted with one or more further substituents. 11. The activated substrate of claim 10, wherein R18 is wherein R19 is a halogen and R20 is a halogen, H, OH, SH, COOH, NH2, optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl or optionally substituted C2-C24 alkynyl. 12. The activated substrate of claim 10 or 11 wherein L4 is substituted by at least two hydroxyl groups. 13. The activated substrate of any one of claims 10-12 wherein L4 is a C1-24 alkylene, an optionally substituted C2-24 alkenylene or an optionally substituted C2-24 alkynylene interrupted by at least two O atoms. 14. The activated substrate of any one of claims 10-13, wherein the compound of formula (I) is a compound of formula (lIb): (lIb) 15. A method of producing a scaffold for isolation of a biomolecule, the method comprising: conducting the method of any one of claims l to 8 to produce an activated substrate, or providing the activated substrate of any one of claims 9 to 14; and contacting the activated substrate with a molecule comprising a ligand specific for a biomolecule to provide a scaffold for isolation of the biomolecule. 16. The method of claim 15, wherein the biomolecule is selected from the group consisting of an amino acid, an aptamer, a peptide, an affimer, a protein, a glycoprotein, a lipopolysaccharide, an antibody or a fragment thereof, a nucleic acid, an organic polymer, a virus, a bacterium, a cell, and a cell-related structure. 17. The method according to claim 15 or claim 16, wherein the molecule comprising the ligand is a compound of formula (IV): X4-L5-X5 (IV) , wherein X4 is NH2, SH or OH; L5 is absent or is an optionally substituted C1-30 alkylene, an optionally substituted C2-30 alkenylene or an optionally substituted C2-30 alkynylene, where the backbone of the alkylene, alkenylene or alkynlene is optionally interrupted by one or more heteroatoms; and X5 is a ligand specific for a biomolecule. 18. The method according to claim 17, wherein X5 comprises an NH2 group, a boronate group or a naphthol group. 19. The method of any one of claim 15 to 17, wherein the ligand comprises an optionally derivatized saccharide molecule, an optionally derivatized amino acid, an optionally derivatized peptide, an optionally derivatized affimer or an optionally derivatized protein, and preferably comprises an optionally derivatized saccharide molecule. 20. The method of claim 19, wherein the compound comprising the ligand is a compound of Formula (IVa) or (IVb): (IVb) 21. The method of any one of claims 15 to 20, wherein the method subsequently comprises contacting the scaffold with an alcohol, a hydroxide, ammonia, an amine or a thiol. 22. A scaffold for isolation of a biomolecule obtained or obtainable by the method of any one of claims 15 to 21. 23· A scaffold for isolation of a biomolecule of formula (III): , wherein L4 and R16 are as defined in any one of claims 10-14; and R21 is a 5 to 12 membered heteroaryl group substituted with at least one group comprising a ligand specific for a biomolecule, and optionally substituted with one or more further substituents. 24. The scaffold of claim 23, wherein R21 is , wherein R22 is a group comprising a ligand specific for a biomolecule and R23 is a group comprising a ligand specific for a biomolecule, a halogen, H, OR24, COOR24, NR24R24, SR24, optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl or optionally substituted C2-C24 alkynyl, wherein R24 and R25 are independently H optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl or optionally substituted C2-C24 alkynyl. 25. The scaffold according to claim 23 or 24 wherein a concentration of the ligand of the scaffold is at least 40 μmol/g. 26. The scaffold of any one of claims 23-25, wherein the or each group comprising the ligand specific for the biomolecule is 27. Use of the scaffold of any one of claims 22 to 26 isolate a biomolecule. 28. A method of isolating a biomolecule on a scaffold, the method comprising contacting a scaffold with a biomolecule, wherein the scaffold is as defined in any one of claims 22 to 26. 29. The method according to claim 28 wherein the biomolecule is selected from isoagglutinins, lipopolysaccharides, albumins, glycosylated proteins and insulin. 30. A method of cleaning a scaffold, the method comprising contacting a scaffold with a caustic substance, wherein the scaffold is as defined in any one of claims to 26. |
More preferably, X 5 is or The compound of Formula (IV) may be an antigen-A trisaccharide ligand, which has Formula (IVa): (IVa) The compound of Formula (IV) may be an antigen-B trisaccharide ligand, which has Formula (IVb): (IVb) The ligand may have an affinity for isoagglutinins. The ligand may, in use, be cationic, preferably protonated. An exemplary ligand which may be cationic in use is a ligand formed from a compound of formula (IVc): H 2 N-L 5 -N(Ak-NH 2 )2 (IVc) wherein Ak in each occurrence is a C 1-12 alkylene where, in the case of a C 2-12 alkylene, the backbone of the alkylene is optionally interrupted by one or more heteroatoms, e.g one or more O atoms. A preferred compound of formula (IVc) is formula (IVd):
(IVd) The ligand formed from the compound of formula (IVd) may have an affinity to lipopolysaccharide and albumin. X 5 of formula (IV) may be a boronate group. A ligand comprising a boronate group may be formed from a compound of formula (IVe): (IVe) The ligand may have an affinity to glycosylated proteins. X 5 of the compound of Formula (IV) may be a naphthol ligand. An exemplary compound of formula (IV) comprising a naphthol ligand has Formula (IVf):
The ligand may have an affinity to insulin. The method may comprise contacting the activated substrate and the molecule comprising the ligand in a solvent. The solvent may be water. The activated substrate and the molecule comprising the ligand may be contacted in a weight ratio of between 1,000,000:1 and 1:1 or between 100,000:1 and 10:1, more preferably between 10,000:1 and 25:1 or between 5,000:1 and 50:1, and most preferably between 2,000:1 and 100:1, between 1,500:1 and 200:1 or between 1,000:1 and 1,000:3. Alternatively, or additionally, the ligand may be present at a concentration of between 1 μg/ml and 750 mg/ml, between 10 μg/ml and 500 mg/ml or between 50 μg/ml and 250 mg/ml, more preferably between 100 μg/ml and 100 mg/ml, between 250 μg/ml and 50 mg/ml, between 500 μg/ml and 10 mg/ml or between 750 μg/ml and 5 mg/ml, most preferably between 1 and 3 mg/ml. Alternatively or additionally, the ligand may be present at a concentration at least 40 µmol per gram of scaffold, optionally a concentration of at least 50 µmol/g, optionally up to 300 or 200 µmol / g. The activated substrate and the molecule comprising the ligand may be contacted under alkaline conditions. The activated substrate and the molecule comprising the ligand may be contacted in solution at a pH between 6 and 14 at 20°C, more preferably at a pH between 7 and 13 at 20°C, and most preferably at a pH of between 8 and 12 at 20°C. The activated substrate and the molecule comprising the ligand may be contacted at a temperature between -20 and 100°C, more preferably between 0 and 75°C, between 5 and 50°C or between 10 and 30°C, and most preferably between 15 and 25°C. The activated substrate and the ligand molecule comprising the may be contacted for at least 1 minute, at least 10 minutes, at least 20 minutes or at least 30 minutes, and more preferably at least 1 hour, at least 2 hours, at least 3 hours or at least 4 hour. The activated substrate and the molecule comprising the ligand may be contacted for between 1 minute and 48 hours, between 30 minutes and 24 hours or between 1 and 12 hours, more preferably between 2 and 10 hours, between 3 and 8 hours or between 4 to 6 hours. The method may subsequently comprise contacting the scaffold with an alcohol, a hydroxide, ammonia, an amine or a thiol. The alcohol, hydroxide, ammonia, amine or thiol may be HOR 24 , -OH, HNR 24 R 25 or HSR 24 , wherein R 24 and R 25 are independently H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl or optionally substituted C 2 -C 12 alkynyl. More preferably, R 24 and R 25 are independently H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkynyl. Most preferably, R 24 and R 25 are independently H, optionally substituted C 1 -C 3 alkyl, optionally substituted C 2 -C 3 alkenyl or optionally substituted C 2 -C 3 alkynyl. The alkyl, alkenyl or alkynyl may be optionally substituted with –OH, NH 2 or SH. The alcohol, hydroxide, ammonia, amine or thiol may be selected from the group consisting of 2-aminoethanol, methylamine, ammonia, sodium hydroxide, glycine, alanine, dimethylamine, tris(hydroxymethyl)aminomethane or 2-mercaptoethanol. Advantageously, this step removes residual halogen sites after ligand coupling. The scaffold and the alcohol, hydroxide, ammonia, amine or thiol may be contacted in a weight ratio of between 1:10 and 1,000:1 or between 1:1 and 500:1, more preferably between 2:1 and 250:1 or between 4:1 and 100:1, and most preferably between 10:1 and 50:1, between 25:2 and 25:1 or between 100:6 and 100:5. The scaffold may be washed with a solvent. The solvent may be water. In accordance with a fifth aspect, there is provided the scaffold for isolation of a biomolecule obtained or obtainable by the method of the fourth aspect. In accordance with a sixth aspect, there is provided a scaffold for isolation of a biomolecule, wherein the scaffold is represented by formula (III): (III) , wherein L 4 and R 16 are as defined in relation to the third aspect; and R 21 is a 5 to 12 membered heteroaryl group substituted with at least one group comprising a ligand specific for a biomolecule, and optionally substituted with one or more further substituents. The group comprising the ligand and biomolecule may be as defined in relation to the fourth aspect. In particular, the group comprising the ligand may have formula –L 6 -L 5 -X 5 , wherein L 5 and X 5 are as defined in the fourth aspect and L 6 is O, S or NH. R 21 may be a 5 to 10 membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, more preferably a 5 or 6 membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, and most preferably a 6 membered heteroaryl group substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents. The heteroaryl group which is substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents, may be a pyridinyl, a pyridazinyl, a pyrimidinyl, a pyrazinyl, a 1,2,4- triazinyl or a 1,3,5-triazinyl. Preferably, the heteroaryl group is a 1,2,4-triazinyl or a 1,3,5-triazinyl substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents and most preferably a 1,3,5-triazinyl substituted with at least one group comprising the ligand specific for a biomolecule, and optionally substituted with one or more further substituents. The heteroaryl group is substituted with at least one group comprising the ligand, and may be substituted with 1, 2, 3 or 4 groups comprising the ligand. Preferably, the heteroaryl group is substituted with 1 or 2 group comprising the ligand, most preferably 2 group comprising the ligand. The heteroaryl group may be substituted with one or more further substituents. The one or more further substituents may be a halogen, OR 24 , SR 24 , COOR 24 , NR 24 R 25 , optionally substituted C 1 -C 24 alkyl, optionally substituted C 2 -C 24 alkenyl or optionally substituted C 2 -C 24 alkynyl, wherein R 24 and R 25 are independently H optionally substituted C 1 -C 24 alkyl, optionally substituted C 2 -C 24 alkenyl or optionally substituted C 2 -C 24 alkynyl. Accordingly, R 21 may be , wherein R 22 is a group comprising a ligand specific for a biomolecule and R 23 is a group comprising a ligand specific for a biomolecule, a halogen, H, OR 24 , COOR 24 , NR 24 R 25 , SR 24 , optionally substituted C 1 -C 24 alkyl, optionally substituted C 2 -C 24 alkenyl or optionally substituted C 2 -C 24 alkynyl, wherein R 24 and R 25 are independently H optionally substituted C 1 -C 24 alkyl, optionally substituted C 2 -C 24 alkenyl or optionally substituted C 2 -C 24 alkynyl. Preferably, R 24 and R 25 are independently H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 - C 12 alkenyl or optionally substituted C 2 -C 12 alkynyl. More preferably, R 24 and R 25 are independently H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkynyl. Most preferably, R 24 and R 25 are independently H, optionally substituted C 1 -C 3 alkyl, optionally substituted C 2 -C3 6 alkenyl or optionally substituted C 2 -C 3 alkynyl. The alkyl, alkenyl or alkynyl may be optionally substituted with –OH, NH 2 or SH. In a most preferred embodiment, the scaffold is represented by formula (IIIa): (IIIa)
R 22 may be H or Preferably, R 23 is a group comprising a ligand specific for a biomolecule. Preferably, R 23 is the same as R 22 . In accordance with a seventh aspect, there is provided use of the scaffold of the fifth or sixth aspect to isolate a biomolecule. In accordance with an eighth aspect, there is provided a method of isolating a biomolecule on a scaffold, the method comprising contacting a scaffold with a biomolecule, wherein the scaffold is as defined in either the fifth or sixth aspect. It may be appreciated that the use of the seventh aspect is in affinity chromatography. The method of the eighth aspect is preferably a method of conducting affinity chromatography. The biomolecule may be as defined in relation to the fourth aspect. The method may comprise contacting the scaffold for isolation of a biomolecule with a solution comprising the biomolecule. The biomolecule may be present in human intravenous immunoglobulin (IVIG) intermediate product. Accordingly, the method may comprise contacting the scaffold for isolation of a biomolecule with human IVIG intermediate product. In accordance with a ninth aspect, there is provided a method of cleaning a scaffold, the method comprising contacting a scaffold with a caustic substance, wherein the scaffold is as defined in either the fifth or sixth aspect. Advantageously, the scaffold may be cleaned to avoid contamination from bacteria, viruses, and endotoxin without loss of performance. The caustic substance may be or comprise an alkaline solution. The alkaline solution may have a pH of at least 7.5, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 at 20°C. The alkaline solution may have a pH of between 8 and 14.5, between 9 and 14, between 10 and 13.75, between 11 and 13.5, between 12 and 13.25 or between 12.5 and 13 at 20°C. The alkaline solution may comprise an alkali metal hydroxide or an alkaline earth metal hydroxide. Preferably, the alkaline solution comprises an alkali metal hydroxide. The alkaline solution may comprise lithium hydroxide, sodium hydroxide or potassium hydroxide. In some embodiments, the alkaline solution comprises sodium hydroxide. The alkaline solution may comprise the alkali metal hydroxide or the alkaline earth metal hydroxide at a concentration of between 0.01 and 10 M, between 0.05 and 5 M, between 0.1 and 2.5 M, between 0.2 and 1 M, between 0.3 and 0.75 M or between, between 0.4 and 0.6 M. The method of the ninth aspect may be performed subsequent to the method of the eighth aspect. The scaffold may then be used in a further method of isolating a biomolecule. Accordingly, the method of the eighth aspect may be repeated after the method of the ninth aspect. The methods of the eighth and ninth aspect may be cycled, one after the other. The methods of the eighth and ninth aspect may be repeated at least 2 times, at least 3 times at least 4 times or at least 5 times, more preferably at least 10 times, at least 20 times or at least 30 times, and most preferably at least 40 or 50 times. All of the features described herein (including accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:- Figure 1 shows a synthesis route and structure of dihalogentriazine activated base matrix represented by dichlorotriazine; Figure 2 shows an agglutination assay plate for A-antigen adsorbent. Two replicate load samples were applied to the top two rows and four non-bound samples (labelled NB rep.1 and 2), recovered from the A-isoagglutinin column in two separate experiments (Exp.1 and 2), are shown in the bottom four rows. The dilutions of the loads are indicated above the top row, while the figures below the bottom row indicate the dilutions applied to the non-bound samples; Figure 3 shows as agglutination assay plate for B-antigen adsorbent. Two replicate load samples were applied to the top two rows and two replicate non-bound samples (labelled NB Rep.1 and 2), recovered from the B-isoagglutinin column, are shown in the bottom two rows. The dilutions of the loads are indicated above the top row, while the figures below the bottom row indicate the dilutions applied to the non-bound samples; Figure 4 is a graph of A-antigen trisaccharide ligand concentration vs. time in the presence of dihalogentriazine activated base matrices having differing activation densities; Figure 5 is a graph of BSA binding capacities across 21 caustic cleaning cycles for a dihalogentriazine scaffold and comparative scaffolds having a Tren ligand; Figure 6 is a graph of HSA binding capacities across 21 caustic cleaning cycles for a dihalogentriazine scaffold and comparative scaffolds having a Blue ligand; Figure 7 is a graph of Blue ligand leachate over time for a dihalogentriazine scaffold and a comparative scaffold in NaOH; and Figure 8 is a graph of binding capacity over time for a dihalogentriazine scaffold and a comparative scaffold having a Blue ligand in NaOH; Examples EXAMPLE 1 A synthetic route for producing a dihalogentriazine scaffold is shown in Figure 1. The steps are described in more detail below. Solid phase activation of a solid support 1.1 Allyl activation Approximately 1 kg of a solid support, such as beaded agarose, is slurried in water with up to 380 g, optionally approximately 250g of sodium sulphate, 10 M NaOH, and 2.5 g of sodium borohydride. The slurry is heated to 45 to 55 °C before adding allyl glycidyl ether (up to 1800 mL, optionally 250 mL). The slurry is left to react for no more than 18 hours, after which the drained gel is washed with ethanol and water. The resulting material is an activated base matrix that contains up to about 200 µmol allyl groups per g adsorbent. 1.2 Bromination of allyl-activated base matrix The allyl-activated base matrix is slurried at room temperature with an acidic solution of pH around 4, followed by the addition of N-bromosuccinimide, and incubation for at least one hour. The resulting brominated base matrix is then washed with water and left to settle. 1.3 Amination of brominated base matrix The brominated base matrix is resuspended in water followed by the addition of 600 mL of an ammonia solution, and heating to no more than 65 °C under stirring for less than 24 hours. At the end of the reaction period, the reaction is drained and washed with water, then a pH-neutral solution, followed by settling. 1.4 Dichlorotriazine (DCT) activation of the aminated base matrix The aminated base matrix resulting from the previous step is then slurried in an aqueous solution of potassium phosphate (1M), followed by settling and re-suspension in the same solution, with the addition of 160 mL of acetone, under stirring at around 2 °C. Approximately 1.4 molar equivalents of cyanuric chloride with respect to the precursor activation density dissolved in acetone is added to the slurried aminated base matrix, followed by incubation under refrigeration for approximately one hour, after which it is drained, washed with aqueous acetone solutions of decreasing concentration, then a final wash in water, after which the slurry is settled under gravity. The final product of this reaction is the DCT-activated base matrix. EXAMPLE 2 Functionalisation of an activated solid support 2.1 Coupling an antigen-A trisaccharide ligand to DCT-activated base matrix A DCT-activated base matrix is suspended in water and set to stir at room temperature. A solution of A-antigen trisaccharide ligand comprising a flexible linker in water is added to the DCT gel slurry maintained under high pH at room temperature for 4 to 6 hours. The A-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVa), as described herein. After reaction, the derivatised base matrix is blocked using mercaptoethanol and thoroughly washed with water before being allowed to settle under gravity. This reaction results in a chromatographic scaffold material containing an affinity ligand which binds A-isoagglutinins (Scaffold Example 1). 2.2 Coupling antigen-B trisaccharide ligand to DCT-activated base matrix A DCT-activated base matrix is suspended in water and set to stir at room temperature. A solution of B-antigen trisaccharide ligand comprising a flexible linker in water is added to the DCT gel slurry maintained under high pH conditions at room temperature for 4 to 6 hours. The B-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVb), as described herein. After reaction, the derivatised base matrix is blocked using mercaptoethanol, thoroughly washed with water then settled under gravity. This reaction results in a chromatographic scaffold material containing an affinity ligand which binds B-isoagglutinins (Scaffold Example 2). EXAMPLE 3 3.1 Removal of A-isoagglutinin from human IVIG intermediate product feedstock using a product of this invention A column containing the antigen A ligand (Scaffold Example 1) is packed, followed by the application of an IVIG solution containing isoagglutinins at a flow rate of 1 mL/min. The flow-through from the loading process is collected. The load and flow-through samples are analysed for of A-isoagglutinins using a standard agglutination assay. 3.2 Agglutination assay 3.2.1 Preparation of red blood cells Human red blood cells are washed with PBS buffer by adding 2mL red blood cells into a 5 mL centrifuge tube and centrifuging at 1400 rpm for 3 minutes. The supernatant from the centrifuged cells is removed before adding PBS buffer to the cell pellet to make the suspension up to a 2 mL volume and the cells mixed by inverting the tube. This PBS wash is repeated three times. Freeze dried papain is reconstituted with PBS buffer (2 mL) before adding 200 µL to the cells after centrifugation and removal of the supernatant. These suspensions are then made up to 2 mL volume with PBS buffer before incubation with mixing for 10 minutes at 37 °C. At the end of this incubation period, the suspension is centrifuged for 3 minutes at 1400 rpm. The supernatant is removed from the centrifuged cells, then PBS buffer added to make the suspension up to a 2 mL volume. The cells are mixed by inverting the tube. This PBS wash is repeated three times. The spun cells are mixed with inversion made up to 2 mL volume with 2 mg/mL BSA solution. 3.2.2 Performing the agglutination test A sample of the feedstock from step 3.1 is diluted 1/2, 1/4, 1/6, 1/8, 1/10, 1/12, 1/14 and 1/16 with a solution of 2 mg/mL BSA solution. A sample of the non-bound from step 3.1 is diluted 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8 and 1/9 with a solution of 2 mg/mL BSA solution. The feedstock and non-bound dilutions described above (20 µL) are transferred to a 96 well v-bottomed plate along with the blank 2mg/mL BSA solution. The red blood cells prepared in stage 3.2.1 are mixed by inversion before transferring to a pipette reservoir. The blood cell suspension (20 µL) is transferred to the plate using a multichannel pipette. The plate is agitated to mix the solutions for 30 seconds before centrifuging the plate at 1400 rpm for 3 minutes. The plate is placed onto a stand at a 70° angle for 15 minutes. Each well of the plate is studied to determine the level of clearance of isoagglutinin from the red blood cells. This is achieved by assessing the sample dilution required to prevent agglutination, indicated by streaming of the red blood cells in the plate wells (Figure 2). As shown in Figure 2, a dilution of greater than 1/8 for the load sample causes the red blood cells to stream. For the non-bound solution sample, a dilution of greater than 1/3 gives streaming of the blood cells. It can be concluded from this assay that the A- antigen adsorbent (Scaffold Example 1) gives a 1/8 to 1/3 clearance of isoagglutinin from the feed. EXAMPLE 4 Removal of B-isoagglutinin from human IVIG intermediate product feedstock using a product of this invention 4.1 Column chromatography purification of IVIG feedstock A column containing the antigen B ligand (Scaffold Example 2) is packed followed by the application of an IVIG solution containing isoagglutinins at a flow rate of 1 mL/min. The flow-through from the loading process is collected. The load and flow-through samples are analysed for the content of B-isoagglutinins using a standard agglutination assay. 4.2 Agglutination assay on flow through from column chromatography purification of IVIG feedstock with B-antigen beaded agarose adsorbent 4.2.1 Preparation of red blood cells To make the type-B red blood cells up in PBS buffer, the cells are washed with PBS buffer by adding 2mL red blood cells into a 5 mL centrifuge tube and centrifuging at 1400 rpm for 3 minutes. The supernatant from the centrifuged cells is removed before adding PBS buffer to the cell pellet to make the suspension up to a 2 mL volume, and the cells are mixed by inverting the tube. This PBS wash is repeated three times. Freeze dried papain was reconstituted with PBS buffer (2 mL) before adding 200 µL to the cells after centrifugation and removal of the supernatant. These suspensions are then made up to 2 mL volume with PBS buffer before incubation with mixing for 10 minutes at 37 °C. At the end of this incubation period, the suspension is centrifuged for 3 minutes at 1400 rpm. The supernatant is removed from the centrifuged cells, then PBS buffer added to make the suspension up to a 2 mL volume. The cells are mixed by inverting the tube. This PBS wash is repeated three times. The spun cells are mixed with inversion made up to 2 mL volume with 2 mg/mL BSA solution. 4.2.2 Performing the agglutination test A sample of the feedstock from step 4.1 is diluted 1/2, 1/4, 1/6, 1/8, 1/10, 1/12, 1/14 and 1/16 with a solution of 2 mg/mL BSA solution. A sample of the non-bound from step 4.1 is diluted 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8 and 1/9 with a solution of 2 mg/mL BSA solution. The feedstock and non-bound dilutions described above (20 µL) are transferred to a 96 well v-bottomed plate along with the blank 2mg/mL BSA solution. The red blood cells prepared in stage 4.2.1 are mixed by inversion before transferring to a pipette reservoir. The blood cell suspension (20 µL) is transferred to the plate using a multichannel pipette. The plate is agitated to mix the solutions for 30 seconds before centrifuging the plate at 1400 rpm for 3 minutes. The plate is placed onto a stand at a 70° angle for 15 minutes. Each well of the plate is studied to determine the level of clearance of isoagglutinin from the red blood cells. This is achieved by assessing the sample dilution required to prevent agglutination, indicated by streaming of the red blood cells in the plate wells (Figure 3). As shown in Figure 3, a dilution of greater than 1/4 for the load sample causes the red blood cells to stream. For the non-bound solution sample, any dilution gives streaming of the blood cells. It can be concluded from this assay that the B-antigen adsorbent (Scaffold Example 2) gives a 1/4 to neat clearance of isoagglutinin from the feed. EXAMPLE 5 Caustic stability of products of the invention 5.1 Stability of an A-Antigen adsorbent Two samples of the A-Antigen adsorbent were incubated in 0.5 M NaOH at 40 o C for 1 week, and subsequently tested for performance using a standard agglutination assay. The samples both showed reduction in agglutinin titres of 1/10 to 1/4 before and after incubation under caustic conditions, indicating the stability of the adsorbent under those conditions. In another experiment, a batch of the A-Antigen adsorbent was subjected to a cycling study, where a packed column of the adsorbent went through 51 cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using 0.5 M NaOH. The isoagglutinin titre of samples taken before and after the column run both showed a reduction of agglutinin titre from 1/8 to 1/2 after the first and 51 st runs, demonstrating the stability of the material to caustic conditions. 5.2 Stability of a B-Antigen adsorbent A batch of the B-Antigen adsorbent was subjected to a cycling study, where a packed column of the adsorbent went through 51 cycles of a procedure containing a caustic- based cleaning-in-place (CIP) step using 0.5 M NaOH. The isoagglutinin titre of samples taken before and after the column run both showed a reduction of agglutinin titre from 1/4 to 1/1 (neat) after the first and 51 st runs, demonstrating the stability of the material to caustic conditions. EXAMPLE 6 Functionalisation of an activated base matrix 6.1 Coupling an antigen-A trisaccharide ligand to DCT-activated base matrix at varying activation densities A range of DCT activated substrates with activation densities ranging from 30 µmol/g to 140 µmol/g were generated using the method described in Example 1. A solution of A-antigen trisaccharide ligand comprising a flexible linker in water was added to the DCT gel slurries maintained under high pH at room temperature for 24 hours. The A-antigen trisaccharide ligand comprising the flexible linker is a compound of formula (IVa), as described herein. After 0, 0.5, 1, 2, 3, 5 and 24 hours, samples of the reaction supernatants were collected for quantitation of unreacted ligand. As shown in Figure 4, higher activation densities afforded by the invention allow coupling of the ligand to the target concentration within 5-hours reaction time. It can be concluded that activation densities equal to or lower than 40 µmol/g would fail to meet the target immobilisation level within 24 hours. Other activation methods such as epichlorohydrin activation (Example 12) are not capable of achieving activation densities higher than 30 µmol/g settled on a beaded agarose support. EXAMPLE 7 (Comparative) A synthetic route for producing a n-hydroxy succinimide scaffold is shown in Scheme 1. The steps are described in more detail below. Solid phase activation of a solid support 7.1 Carboxy activation Scheme 1 Approximately 1 kg of a solid support, such as beaded agarose, is suspended in water before heating to 40 to 50 °C. Sodium chloroacetate (approximately 235 g) is added to the reaction slurry and left to react for no more than 18 hours, after which the drained gel is washed with water. The resulting material is an activated matrix that contains 20 to 30 µmol carboxy groups per g adsorbent. 7.2 N-hydroxy succinimide esterification
Scheme 2 The carboxylated support is acidified by washing with 0.1 M HCl and then washed free of water with acetone. The gel is then suspended in acetone before reacting with N- hydroxysuccinimide (NHS) (16 g per kg of support) and N-ethyl-N′-(3- (dimethylamino)propyl)carbodiimide (EDC) (10 g per kg of support) at ambient temperature for at least 16 hours. After the reaction, the gel is drained and washed with N,N-dimethylformamide. The resulting material is an activated matrix that contains 20 to 30 µmol NHS groups per g adsorbent. EXAMPLE 8 Functionalisation of a base matrix with tris 2-aminoethyl amine ligand 8.1 Coupling tris 2-aminoethyl amine ligand to brominated base matrix (Comparative Scaffold 1)
Scheme 3 The bromo activated base matrix produced as described in Example 1 is suspended in water before adding tris 2-aminoethyl amine ligand. The charge of tris 2-aminoethyl ligand is calculated with respect to the precursor allyl activation density to give an excess of 30 molar equivalents. After addition of the amine, the reaction is left to react at 60 °C for at least 16 hours. At the end of the reaction period, the gel is drained and washed with water, 0.1 M HCl and 0.1 M NaCl before settling under gravity. This reaction results in a chromatographic material containing an affinity ligand which binds bovine serum albumin. 8.2 Coupling tris 2-aminoethyl) amine ligand to NHS activated base matrix (Comparative Scaffold 2)
Scheme 4 The NHS activated base matrix produced as described in Example 7 is suspended in N,N-dimethylformamide before adding tris 2-aminoethyl ligand . The charge of tris 2- aminoethyl ligand is calculated with respect to the precursor NHS activation density to give an excess of 30 molar equivalents. After addition of the amine, the reaction is left to react at ambient temperature for at least 16 hours. At the end of the reaction period, the gel is drained and washed with water, 0.1 M HCl and 0.1 M NaCl before settling under gravity. This reaction results in a chromatographic material containing an affinity ligand which binds bovine serum albumin. 8.3 Coupling tris 2-aminoethyl) amine ligand to DCT activated base matrix (Scaffold Example 3)
Scheme 5 The DCT activated base matrix produced as described in Example 1 is suspended in water before adding tris 2-aminoethyl ligand. The amount of tris 2-aminoethyl ligand is calculated with respect to the precursor activation density to give an excess of 10 molar equivalents. After addition of the amine, the reaction is left to react at 45 °C temperature for at least 16 hours. At the end of the reaction period, the gel is drained and washed with water, 0.1 M HCl and 0.1 M NaCl before settling under gravity. This reaction results in a chromatographic material containing an affinity ligand which binds bovine serum albumin. EXAMPLE 9 Caustic stability of tris 2-aminoethyl ligand adsorbent 9.1 Stability of tris 2-aminoethyl amine (tren) adsorbents A sample of the tren adsorbent produced as described in Example 8.1 (bromo attachment, Comparative Scaffold 1), 8.2 (NHS attachment, Comparative Scaffold 2) and 8.3 (DCT attached product of the invention, Scaffold Example 3) were subjected to a cycling study, where packed columns of the adsorbents went through 21 cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using 0.5 M NaOH. On the first, 11 th and 21 st cycle, the column was loaded to 10% breakthrough with bovine serum albumin (BSA) and the binding capacity was calculated. As shown in Figure 5, the BSA binding capacity of the Scaffold Example 3 adsorbent at 10% break through (approximately 41 mg/mL) did not change significantly from cycle 1 to 21, demonstrating stability of the Scaffold Example 3 to caustic conditions. In contrast, binding capacity of Scaffold Example 2 fell significantly under the same conditions. The bromo attached Tren adsorbent of Comparative Scaffold 1 also showed good caustic stability however, as described in Example 8.1 this required much stronger reaction conditions compared to the triazine attachment process for Scaffold Example 3, including higher temperature and a three-fold excess of amine (Tren ligand). EXAMPLE 10 Functionalisation of activated scaffolds with Blue chromophore ligand 10.1 Coupling Blue chromophore ligand to brominated base matrix (Comparative Scaffold 3)
Scheme 6 A solution of 4.5 g of Mimetic Blue SA ligand available from Astrea Bioseparations (referred to herein as “Blue ligand”) per kg of adsorbent is prepared in water with adjustment to pH 12 with NaOH. The bromo activated base matrix produced as described in Example 1 is suspended in the Blue ligand solution and the reaction is left to react at 60 °C for at least 16 hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at 60 °C for at least 16 hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately 3.0 µmol ligand per g of adsorbent which binds human serum albumin. 10.2 Coupling Blue chromophore ligand to NHS activated base matrix (Comparative Scaffold 4)
Scheme 7 A solution of 4.5 g of Blue ligand per kg of adsorbent is prepared in water with adjustment to pH 12 with NaOH. The NHS activated base matrix produced as described in Example 7 is suspended in the Blue ligand solution and the reaction is left to react at ambient temperature for at least 16 hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at room temperature for at least 16 hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately 3.4 µmol ligand per g of adsorbent which binds human serum albumin. 10.3 Coupling Blue chromophore ligand to DCT activated base matrix (Scaffold Example 4)
Scheme 8 A solution of 4.5 g of Blue ligand per kg of adsorbent is prepared in water with adjustment to pH 12 with NaOH. The DCT activated base matrix produced as described in Example 1 is suspended in the Blue ligand solution and the reaction is left to react at ambient temperature for at least 16 hours. At the end of the reaction period, any residual reactive sites on the gel are blocked by addition of ethanolamine followed by reaction at 45 °C for at least 16 hours. After the blocking reaction, the gel is drained and washed with water. This reaction results in a chromatographic material containing an affinity ligand at approximately 6 µmol ligand per g of adsorbent which binds human serum albumin. EXAMPLE 11 Caustic stability of Blue ligand scaffolds A sample of the Blue chromophore ligand scaffolds produced as described in Example 10.1 (bromo attachment, Comparative Scaffold 3), 10.2 (NHS attachment, Comparative Scaffold 4) and 10.3 (DCT attached product of the invention, Scaffold Example 4) were subjected to a cycling study, where a packed column of the adsorbents went through 21 cycles of a procedure containing a caustic-based cleaning-in-place (CIP) step using 0.5 M NaOH. On the first, 11 th and 21 st cycle, the columns were loaded to 10% breakthrough with human serum albumin (HSA) and the binding capacity was calculated. As shown in Figure 6, the HSA binding capacity of the DCT coupled adsorbent (Scaffold Example 4) and bromo coupled adsorbent (Comparative Scaffold 3) at 10% break through did not change significantly from cycle 1 to 21, demonstrating stability of the adsorbents to caustic conditions. The NHS coupled comparative example (Comparative Scaffold 4) showed slight decrease in binding capacity by the 21 st cycle. Despite being activated to the same density and having the same excess of ligand in the immobilisation reaction, the triazine adsorbent showed higher binding capacity than the bromo attached adsorbent. Without wishing to be bound by any theory, this is afforded by the higher reactivity of the chlorotriazine reactive group leading to increased ligand density of the adsorbent and demonstrating an advantage in efficiency of the synthesis process. In another experiment, a sample of the Blue chromophore ligand adsorbents produced as described in Example 10.2 (NHS coupled Comparative Scaffold 4) and 10.3 (DCT attached product of the invention Scaffold Example 4), were incubated in 0.5 M NaOH at 40 °C for three days. Approximately every 24 hours, a supernatant sample was collected for quantitation of ligand leachate concentration by HPLC. As shown in Figure 7, this analysis showed negligible levels of ligand leachate in the incubation supernatant of the triazine coupled Scaffold Example 4 demonstrating the stability of the product to caustic conditions. Conversely, the NHS coupled Comparative Scaffold 4 showed significant levels of Blue ligand leachate increasing over the course of the incubation, indicating a lack of caustic stability. The two adsorbents were tested for HSA binding capacity at 10% breakthrough before the stability study and after incubation in 0.5 M NaOH at 40 °C for 66 hours. As shown in Figure 8, the triazine coupled Scaffold Example 4 showed negligible change in binding capacity after exposure to 0.5 M NaOH at 40 °C for 66 hours. This demonstrates the caustic stability of the product. For the NHS coupled Comparative Scaffold 4, binding capacity significantly dropped over the 66-hour incubation period demonstrating the poor caustic stability of this attachment method. EXAMPLE 12 (Comparative) 12.1 Activation of beaded agarose with epichlorohydrin Approximately 1 kg of a beaded agarose solid support, is suspended in 0.9 L of water before addition of 166 mL of 10 M NaOH. Epichlorohydrin is added to the reaction slurry at 250 mL per kg of base matrix. The reaction is left to stir at 16 °C for at least 16 hours before adding an additional 125 mL portion of epichlorohydrin and 84 mL of 10 M NaOH. The reaction mixture is left to react for 3 hours before draining and washing with water. The resulting material is an activated matrix that contains up to 30 µmol epoxide groups per g adsorbent, demonstrating the limited activation density of epichlorohydrin activation method on beaded agarose support. Summary The inventors have shown that their functionalised solid support can be used to selectively isolate isoagglutinin. However, it will be appreciated that the scaffold could be functionalised with alternative ligands to enable the isolation of other biomolecules. The inventors have also shown that the functionalised solid support is compatible with cleaning and sanitisation using sodium hydroxide, thereby preventing contamination thereof. The functionalised solid support is stable under these conditions, allowing repeated use thereof. This will significantly reduce the cost of isolating biomolecules, and in turn any products manufactured therewith. In addition, the inventors have shown that their activated solid support can be functionalised under mild reaction conditions (low temperature, low molar excess of amine and short reaction times) by virtue of the high activation densities achievable by this invention and the high reactivity of the dichlorotriazine attachment chemistry. It will also be appreciated by those skilled in the art that this high potential activation density enables higher ligand densities which in turn can provide increased binding capacity of target biomolecules.