US2014273279A1PendingUtilityA1

Method for determining the binding constant of high affinity compounds

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Assignee: HOFFMANN LA ROCHEPriority: Feb 25, 2010Filed: Jun 2, 2014Published: Sep 18, 2014
Est. expiryFeb 25, 2030(~3.6 yrs left)· nominal 20-yr term from priority
Inventors:Frauke AssmusHolger Fischer
B01D 61/243C08H 1/00B01L 2300/0858G01N 33/5302B01L 3/50255B01L 2300/0618B01D 61/28G01N 33/68B01L 2300/0829G01N 33/49G01N 33/566
48
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Claims

Abstract

The invention relates to a method for determining the binding constant of a compound of interest to proteins comprising the following steps: a) adding the high affinity compound to a two-chamber system, wherein the two chambers are separated by a semipermeable membrane, which is permeable for the compound of interest, and determining the amount of the high affinity compound of interest in one of the chambers after the distribution equilibrium has been reached, b) adding a sink compound to one of the chambers whereby the sink compound can not permeate the membrane, and determining the distribution coefficient of the compound of interest to the sink compound after the distribution equilibrium has been reached, c) adding an unspecific protein to the other chamber, whereby the unspecific protein can not permeate the membrane, and determining the distribution coefficient of the compound of interest to the unspecific protein in presence of a sink compound after the distribution equilibrium has been reached, and d) determining the binding constant of the test compound with the distribution coefficient of steps b) and c).

Claims

exact text as granted — not AI-modified
1 . Method for determining the binding constant of a compound of interest to proteins comprising the following steps:
 a) adding the high affinity compound to a two-chamber system, wherein the two chambers are separated by a semipermeable membrane, which is permeable for the compound of interest,
 and 
 determining the amount of the high affinity compound of interest in one of the chambers after the distribution equilibrium has been reached, b) adding a sink compound to one of the chambers whereby the sink compound can not permeate the membrane, and 
 determining the distribution coefficient of the compound of interest to the sink compound after the distribution equilibrium has been reached, 
   c) adding an unspecific protein to the other chamber, whereby the unspecific protein can not permeate the membrane, and
 determining the distribution coefficient of the compound of interest to the unspecific protein in presence of a sink compound after the distribution equilibrium has been reached, 
 and 
   d) determining the binding constant of the test compound with the distribution coefficient of steps b) and c).   
     
     
         2 . The method according to  claim 1  wherein the filter is a size selective membrane. 
     
     
         3 . The method according to  claim 1 , wherein the sink compound is PVP or cyclodextrine. 
     
     
         4 . The method according to any one of  claim 2  wherein the sink compound is PVP25. 
     
     
         5 . The method according to any one of  claims 1  wherein the protein is a plasma protein. 
     
     
         6 . The method according to  claim 5  wherein the plasma protein is serum albumin. 
     
     
         7 . The method according to  claim 1  wherein the steps a), b) and c) are performed in parallel. 
     
     
         8 . The method according to  claim 2  wherein the sink compound is PVP or cyclodextrine. 
     
     
         9 . The method according to  claim 3  wherein the sink compound is PVP25. 
     
     
         10 . The method according to  claim 8  wherein the sink compound is PVP25. 
     
     
         11 . The method according to  claim 2  wherein the protein is a plasma protein. 
     
     
         12 . The method according to  claim 2  wherein the plasma protein is serum albumin. 
     
     
         13 . The method according to  claim 11  wherein the plasma protein is serum albumin. 
     
     
         14 . The method according to  claim 2  wherein the steps a), b) and c) are performed in parallel. 
     
     
         15 . The method according to  claim 3  wherein the steps a), b) and c) are performed in parallel. 
     
     
         16 . The method according to  claim 4  wherein the steps a), b) and c) are performed in parallel. 
     
     
         17 . The method according to  claim 11  wherein the steps a), b) and c) are performed in parallel. 
     
     
         18 . The method according to  claim 17  wherein the plasma protein is serum albumin.

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