Purification of multi-specific receptors

Abstract

Disclosed is a method for preparing a composition enriched for receptors (typically molecular impringet polymers, MIPs) that bind an agent, where said receptors each specifically bind at least two discrete sites on said agent, by subjecting a sample of receptors to a first step of affinity purification with the agent where one binding site on the agent is non-accessible for binding to the receptors and subsequently subjecting the purified receptors to at least one further step of affinity purification with the agent where a second binding site on the agent is non-accessible. Also disclosed is a method for treatment, amelioration or prophylaxis of a disease selected from the group consisting of phenylketonuria (PKU, Følling's disease), hyperphenylalaninemia (HPA), alcaptonuria (black urine disease), tyrosinemia, hypertyrosinemia, myasthenia gravis, histidinemia, urocanic aciduria, maple syrup urine disease (MSUD), isovaleric acidemia (isovaleryl-CoA dehydrogenase deficiency), homocystinuria, propionic acidemia, methylmalonic acidemia, and glutaric aciduria Type 1 (GA-1), galactosemia, comprising administering to the gastrointestinal tract of a patient in need thereof an effective amount of a composition of molecular imprinted polymers (MIPs), said composition being capable of binding a symptom provoking agent of said disease.

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         3 . A method for treatment, amelioration or prophylaxis of a disease selected from the group consisting of hyperphenylalaninemia (HPA), alcaptonuria (black urine disease), tyrosinemia, hypertyrosinemia, myasthenia gravis, histidinemia, urocanic aciduria, maple syrup urine disease (MSUD), isovaleric acidemia (isovaleryl-CoA dehydrogenase deficiency), homocystinuria, propionic acidemia, methylmalonic acidemia, glutaric aciduria Type 1 (GA-1), and galactosemia, comprising administering to the gastrointestinal tract of a patient in need thereof an effective amount of a composition of molecular imprinted polymers (MIPs), said composition being capable of binding a symptom provoking agent of said disease. 
     
     
         4 . A method for treatment, amelioration or prophylaxis of a disease selected from the group consisting of phenylketonuria, hyperphenylalaninemia (HPA), alcaptonuria (black urine disease), tyrosinemia, hypertyrosinemia, myasthenia gravis, histidinemia, urocanic aciduria, maple syrup urine disease (MSUD), isovaleric acidemia (isovaleryl-CoA dehydrogenase deficiency), homocystinuria, propionic acidemia, methylmalonic acidemia, glutaric aciduria Type 1 (GA-1), and galactosemia, comprising administering to the gastrointestinal tract of a patient in need thereof an effective amount of a composition of molecular imprinted polymers (MIPs), said composition being capable of binding a symptom provoking agent of said disease, wherein the composition of MIPs is prepared by:
 i-a. obtaining a suspension of insoluble molecular imprinted polymers, which bind the symptom provoking agent, and which have been prepared using the symptom provoking agent or a mimic thereof as template molecule,   i-b. subjecting the suspended molecular imprinted polymers to an affinity purification procedure, wherein the template molecule or a fragment thereof or a mimic thereof is used as capture agent,   i-c. recovering the molecular imprinted polymers that bind the capture agent in the affinity purification procedure while substantially excluding the capture agent and molecular imprinted polymers that do not bind the capture agent from the recovered product; or, prepared by a method comprising,   ii-a. providing a sample comprising MIPs that bind said symptom provoking agent,   ii-b. subjecting said sample to a first step of affinity chromatography, where said symptom provoking agent is used as affinity purification agent, and wherein said agent is immobilised to a solid or semi-solid phase via binding to one single of said at least two discrete sites,   ii-c. recovering MIPs binding to the symptom provoking agent,   ii-d. subjecting MIPs recovered in the previous step to at least one further step of affinity chromatography, where said symptom provoking agent is used as affinity purification agent, and wherein said agent is immobilised to a solid or semi-solid phase via binding to another of said at least two discrete sites, and recovering MIPs binding to the agent,   wherein, in each said at least one further step of affinity chromatography, said another of said at least two discrete sites is different from any one of said at least two discrete sites, which has been used previously in steps b and d for immobilization of the agent to a solid of semi-solid phase.   
     
     
         5 . The method according to  claim 3 , wherein the composition of MIPs is essentially free from MIPs which do not bind said symptom provoking agent. 
     
     
         6 . The method according to  claim 4 , wherein the composition of MIPs is essentially free from MIPs which do not bind said symptom provoking agent. 
     
     
         7 . The method of  claim 3 , wherein
 a) said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia; or   b) said symptom provoking agent is L-tyrosine and said disease is tyrosinemia or hypertyrosinemia; or   c) said symptom provoking agent is L-histidine and said disease is myasthenia gravis, histininemia, or urocanic aciduria; or   d) said symptom provoking agent is L-leucine and said disease is maple syrup urine disease or isovaleric acidemia, or   e) said symptom provoking agent is L-methionine and said disease is homocystinuria, or   f) said symptom provoking agent is L-isoleucine or L-valine or L-methionine or L-threonine and said disease is proprionic academia or methylmalonic academia, or   g) said symptom provoking agent is L-tryptophan or L-lysine and said disease is glutaric aciduria   h) said symptom provoking agent is D-galactose or lactose and said disease is galactosemia.   
     
     
         8 . The method of  claim 4 , wherein
 a) said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia, or   b) said symptom provoking agent is L-tyrosine and said disease is tyrosinemia or hypertyrosinemia, or   c) said symptom provoking agent is L-histidine and said disease is myasthenia gravis, histininemia, or urocanic aciduria, or   d) said symptom provoking agent is L-leucine and said disease is maple syrup urine disease or isovaleric acidemia, or   e) said symptom provoking agent is L-methionine and said disease is homocystinuria, or   f) said symptom provoking agent is L-isoleucine or L-valine or L-methionine or L-threonine and said disease is proprionic academia or methylmalonic academia, or   g) said symptom provoking agent is L-tryptophan or L-lysine and said disease is glutaric aciduria Type 1, or   h) said symptom provoking agent is D-galactose or lactose and said disease is galactosemia.   
     
     
         9 . The method of  claim 5 , wherein
 a) said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia, or   b) said symptom provoking agent is L-tyrosine and said disease is tyrosinemia or hypertyrosinemia, or   c) said symptom provoking agent is L-histidine and said disease is myasthenia gravis, histininemia, or urocanic aciduria, or   d) said symptom provoking agent is L-leucine and said disease is maple syrup urine disease or isovaleric acidemia, or   e) said symptom provoking agent is L-methionine and said disease is homocystinuria, or   f) said symptom provoking agent is L-isoleucine or L-valine or L-methionine or L-threonine and said disease is proprionic academia or methylmalonic academia, or   g) said symptom provoking agent is L-tryptophan or L-lysine and said disease is glutaric aciduria Type 1, or   h) said symptom provoking agent is D-galactose or lactose and said disease is galactosemia.   
     
     
         10 . The method of  claim 3 , wherein said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia. 
     
     
         11 . The method of  claim 4 , wherein said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia. 
     
     
         12 . The method of  claim 4 , wherein said symptom provoking agent is L-phenylalanine and said disease is hyperphenylalaninemia, alcaptonuria, tyrosinemia, or hypertyrosinemia.