ALKALI RESISTANT PROTEIN ADSORBENT

Abstract: An adsorbent preferably for proteins and peptides, but also for e.g. certain metals, with hydrophilic ligands covalently bonded to a polymer net around a carrier, and having the general structure: -X-S-(CH2)n-R, wherein X is a spacer arm, n is an integer 1 or 2, and R is a pi -electron-rich ring system with at least one nitrogen atom in the ring structure. Thanks to its resistance to 1 M caustic, a concentration which eliminates bacteria, viruses and pyrogenes, the adsorbent can be re-used for medical purposes in large scale work.

Description: Alkali Resistant Protein Adsorbent DESCRIPTION The invention relates to an adsorbent for the separation and immobilization of proteins, comprising a carrier material with covalently bound ligands, and a method of manufacturing the adsorbent and the use thereof. More specifically, the adsorbent is alkali resistant, and thereby suitable for the use together with proteins which, during certain treatment steps, can be subjected to alkaline solutions. At the chromatographic process the adsorption of proteins increases through the addition of different types of salts in different salt concentrations, and after the removal of salt from the eluation buffer the proteins will desorb from the adsorbent, and can be purified in this way or be divided into different fractions. PRIOR ART At present, for the purification of proteins adsorbents with ligands giving large protein losses at the eluation of the protein are used. Even after washing of the adsorbent with 1 M caustic solution, protein remains on the adsorbent and alters its characteristics. Re-use thereof may thus cause risks of contamination of further test mixtures with the proteins that remain on the adsorbent. As an alternative to these adsor¬ bents more specific adsorbents have been developed for the isolation of a.o. IgG. These adsorbents are characterized by that a protein is covalently bound to the adsorbent and binds specifically to IgG. The disadvantages are that the covalent¬ ly bound protein is released and leaks out and can cause medical complications thereby. As an alternative to such biospecific adsorbents (Protein A and Protein G, resp.) so called tiophilic adsorbents have been developed (Porath, Belew - SE-PS 8405431-1) . The tio¬ philic adsorbents of the kind having been developed hitherto, however, are not alkali stable, wherefore purification and. washing with 1 M caustic solution can not be performed without risk of the undesirable effect, which is a condition for preventing bacterial and virus infections and risk for pyrogenes, when the adsorbent is re-used in large scale processes. An adsorbent with the structure Sepharose - 0 - CH 2 - CH - CH 2 - S * ^δ OH ^ " -5 is previously described in EP-A-0 180 563 and SE-B-462 165 (Porath and Oscarsson) . The disadvantage with this adsorbent is that it is not alkali stable, because of the unfavourable electron density distribution within the molecule, which is determined by the positions therein of the sulphur and nitrogen, respectively. The electron density distribution within the pyridyl sulphide molecule in formula I is shown in formula II below H 4 0.91 When the electron density distribution within the mole¬ cule is calculated carbon atom C- ^ is shown to have an elec¬ tron deficit in its position, which means that the nucleophi- lie hydroxyi group can react at position C^_ and result in elimination of hydroxipyridin. If the sulphur is moved from the pyridine ring within the above describe molecule by the introduction of a methylene group between the sulphur and the pyridine ring the undesired effect of an electron deficit on C-, does not occur. This has also been verified experimental¬ ly. The adsorbent will thereby remain stable against alkali, and has a high desorption effectivity towards proteins having been into contact with the adsorbent, and further very low protein residues. The object of the invention is now to provide an adsor¬ bent which can be subjected to 1 M alkali during a prolonged time without being broken down, and thereby can be effective¬ ly cleaned of bacteria, viruses and pyrogenes. A further object of the invention is to provide an adsorbent which can be eluated without noticeable losses. Further, the invention aims at providing an adsorbent which can be re-used after eluation with 1 M caustic solu¬ tion, without causing a risk of passing on infection. The adsorbent according to the invention comprises a polymeric network in the form of finely divided particles with a diameter less than 1 mm or diaphragms with a thickness not exceeding 1 mm. The polymeric network is comprised of a hydrophilic polymer such as a polysaccharide, e.g. cross-linked cellulose or agarose, a cross-linked polyhydric polymer such as poly¬ vinyl alcohol, or can also be a cross-linked polyacryl amide. To the polymeric network ligands with the general structure - X - S - (CH 2 ) n - R are covalently bonded, and in which X is a spacer arm, n is an integer 1 or 2, and R is a π - electronrich ring system with at least one nitrogen atom in the ring structure such as a nitro or cyano group. The adsorbent according to the present invention show particular adsorption features, which are shown especially in contacts with protein solutions, especially in the presence of high concentrations of lyotropic salts, such as sulfate and phosphate of alkali metals, magnesium and ammonium, where the proteins are distinguished in classes having different adsorptive strength depending on their preferential affinity proportion to the adsorbent. The adsorbent according to the invention can also be used for the adsorption of certain soft metal ions such as Ag + , Hg 2+ , Pd 2+ and Pt 2+ . The invention will now be described more in detail below together with examples of the manufacture of the adsorbent. Example 1 Pyridy1-2-meth Agarose - 0 - CH 2 - Firstly, an epoxy active gel is manufactured by further washing 50 g washed, 6 % agarose gel (Sepharose 6B) with distilled water and thereafter adding 0.34 g NaBH 4 and 35 ml 2 M NaOH and drop by drop 25 ml epichlorohydrin. The reaction is then continued for about 20 hours at room temperature. To 50 g of this gel is added 10 ml 2 M Na 2 S 2 0 3 and the reaction is allowed to continue for 10 hours at room tempe¬ rature. Activation is performed adding 1.5 g dithio-threitol (DTT) in 100 ml 0.1 M NaHC0 3 to 25 g gel and reacting for 30 minutes at room temperature. The activated gel is washed on a glass filter with distilled water, whereupon 2.5 g pikolylchloride (2-chloro- methyl-pyridine) in 0.1 M NaHC0 3 is added, and pH is adjusted to 8.0. The reaction is performed at room temperature for 3 hours. Stability studies of the gel is made by elementary analysis of the nitrogen content before and after keeping the gel in I H NaOH at 40°C for 13 days. TABLE % by weight of nitrogen Testing of new gel according to the invention 1.4 After 13 days and nights in 40°C caustic 1.3 Comparative testing of gel with sulphur direct¬ ly bonded to the pyridine nucleus 1.4 After washing of comparative specimen with cold caustic 0.5 The gel according to the invention can thus be washed with 1 M caustic with any risk of degradation while pre¬ viously used gels for purification of e.g. IgG are destroyed already when washing with 1 M caustic at room temperature. Purification (fractionation) of IgG with a gel according to the invention before and after washing did not show any significant changes regarding capacity and specificity. In re-use of the gel for fractionation of IgG no differ- ence could be noticed. Correspondingly good results were obtained also with, among others, gels with the following ligands - 0 - CH 2 - CHOH - CH 2 - S - CH 2 - (Q/ -N0 2 - CH 2 - CHOH - CH 2 - S - CH 2 - CH 2 -- Example 2 Pyridyl-4-methyl thioether agarose In the same way as in Example 1 pyridyl-4-methyl thio¬ ether agarose is manufactured but instead of 2-pikolyl- chloride the same amount of 4-pikolylchloride (4-chloro methyl pyridine) is used. Example 3 Quinoline-2-methyl thioether silica. In a round flask 100 ml dried toluene and 24 g silica are added, then 3 ml dried triethylamine is added followed by 30 ml g-glycidoxypropyl-trimethoxysilan. The mixture is refluxed for 3 hours and is thereafter washed on a glass filter with toluene and activated with 10 ml 2 M Na 2 S 2 0 3 for 10 hours at room temperature. To the washed gel 1 g dithioerythritol dis¬ solved in 0.1 M NaHC0 3 is added, and pH is adjusted to 7.5. After 30 minutes reaction the gel is washed on a glass filter with distilled water followed by toluene. 2 g quinoline-2- ethylchloride dissolved in toluene is added to the toluene- washed silicate gel and is allowed to react for 6 hours at 60°C, whereafter the gel is washed with toluene on a glass filter. Example 4 Imidazolylethyl thioether agarose. Manufacture in the same way as in Example 1, with the ex- ception that instead of 2-pikolylchloride the same amount of vinylimidazol is used. Example 5 Uracilyl-6-methyl thioether agarose. Manufacture in the same way as in Example 1 with the ex¬ ception that instead of 2-pikolylchloride, 6-chloromethyl- uracil is used. Use of the σel according to the invention In the chromatographic process the adsorption of the protein is amplified by the addition of different types of salts to different salt concentrations. When the salt is removed from the eluation buffer the proteins are desorbed. Under optimal conditions the adsorbent according to the invention has shown a retainment of 2.7 % of the totally applied protein after desorption with a buffer free from salt. The corresponding commercial preparation, octyl-Sepharose and phenyl-Sepharose showed under optimal conditions a retainment of 29.3 and 8.1 % by weight, respectively, of the totally applied protein after desorption with a buffer free from salt. Since every percent retained protein is a corre¬ spondingly large loss every reduction of the retainment is of great economic importance. As noted above both retained protein and adsorbent may carry infections, e.g. viruses, bacteria, pyrogenes, but these are effectively rendered harmless by washing with 1 M caustic. The total amount of remaining protein on the adsor¬ bent after washing with 1 M soda-caustic has been determined with aminoacid analysis and has been compared with the values of the presently used adsorbents after a considerably milder treatment, i.e. not sufficient for a safe reuse. As is evident from the table the nitrogen value was kept at 1.4 % by weight on the adsorbent according to the invention upon treatment for 13 days and nights at 40°C, while the nitrogen value already after a washing with short duration with cold caustic was reduced from 1.4 to 0.5 % by weight for pyridine thioether adsorbent where sulphur is directly bonded to the pyridine nucleus. The reason for the enhanced characteristics of the gel according to the invention is assumed to depend on the effect of the nitrogen atom and the sulphur on the electron distri¬ bution within the ligand. With an unfavourable placement of these within the ligand, electron deficit especially on the carbon atoms will make possible nucleophilic attack of the hydroxyi groups (from the alkali solution) on those carbon atoms having an electron deficit, which means a destabilisa- tion of the ligand. Investigations of the electron distribu¬ tion have been made on the adsorbent according to the in¬ vention and on corresponding, presently used, commercial preparations, and confirm this assumption.

Claims: CLAIMS 1. An adsorbent for the separation and immobilisation of biopolymers such as proteins, peptides, etc., comprising a carrier material with covalently bonded ligands, charac- terized in that the carrier material is comprised of a solid phase which is completely or partially penetrated or surface- coated with a polymeric network, and the ligands having the structure X - S - (CH 2 ) n - R, wherein X is a spacer arm, n is l or 2, and R is a π-elec- tron-rich ring system with at least one nitrogen atom in the ring structure. 2. An adsorbent according to claim 1, characterized in that the solid phase is comprised of particles with a dia- meter less than 1 mm or of a diaphragm with a thickness not exceeding 1 mm. 3. An adsorbent according to any of claims 1 or 2, characterized in that the polymer network is a polyhydroxy polymer. 4. An adsorbent according to any of claims 1 - 3, characterized in that the polyhydroxy polymer is a poly- galaktan, such as agar or agarose. 5. An adsorbent according to claim 1 or 2, characterized in that the polymer network is comprised of a polyamid, such as cross-linked polyakrylamid or derivative thereof. 6. An adsorbent according to any of claims 1 - 5, characterized in that the solid phase is comprised of an inorganic polymer, such as silica or derivative, to which the hydrophilic polymer component has been bonded, included or surface-coated. 7. An adsorbent according to any of claims 1 - 5, characterized in that the solid phase is comprised of a hydrophobic, organic polymer such as polystyrene, to which the hydrophilic polymer component has been bonded, included or surface coated. 8. An adsorbent according to any of claims 1 - 7, characterized in that R is comprised of wherein Z is N or CH. 9. An adsorbent according to any of claims 1 - 8, characterized in that the ligand comprises or is comprised of a pikolyl thioether group. 10. An adsorbent according to any of claims 1 - 9, characterized in that the spacer arm contains the structure -CH 2 -CHOH-CH 2 -. 11. A method, of manufacturing an adsorbent according to the above claims, characterized in that first a thiol group is introduced into a polymer network, and in that then, a heteroaromatic group with an electrophilic side chain is introduced. 12. A method of manufacturing the adsorbent according to claim 11, characterized in that the heteroaromatic group has a side-group substituent with the structure -(CH 2 ) n -Y, wherein Y is a halogene, such as chloro, and n is 1 or 2. 13. A method according to claim 11, characterized in that the electrophilic side chain to the heteroaromatic group is comprised of a vinyl substituent. 14. Use of an adsorbent according to claim 1 - 11 for fractionation of biopoly ers such as proteins, peptides and nucleic aαids.

Inventors: OSCARSSON SVEN, PORATH JERKER, OSCARSSON, SVEN, PORATH, JERKER
Applicants: OSCARSSON SVEN, PORATH JERKER, OSCARSSON, SVEN, PORATH, JERKER
Application year: 1995
Main classification: B01J20
IPC codes: B01J20, C07K1, G01N30