Immunoassay-based determination of in-solution binding kinetics

Abstract

Herein is reported a method for the determination of the binding affinity of a binder and its ligand comprising the step of determining based on the result of an immunoassay the fraction of free binder in a sample comprising binder, ligand and binder-ligand-complexes for at least two different binder:ligand ratios in the sample, and if the determined fraction of free binder is not comparable for all used binder:ligand ratios then the binder:ligand ratio in the sample is lowered and the sample is re-analyzed by the same immunoassay, and calculating based on the fraction of free binder in the previous step the binding affinity for the binder to its ligand.

1 . A method for the determination of the binding affinity of a drug to its target comprising the following step:
 determining the fraction of free drug or free target in a sample comprising drug, target and non-covalent drug-target-complexes in the free drug plateau, and   calculating based on the fraction of free drug or calculating based on the fraction of free target determined in the previous step the binding affinity for the drug to its target.   
     
     
         2 . The method according to  claim 1 , characterized in comprising the following steps:
 determining based on the result of an immunoassay the fraction of free drug in a sample comprising drug, target and drug-target-complexes for at least two different drug:target ratios in the sample, and if the determined fraction of free drug is not comparable for all used drug:target ratios then the drug:target ratio in the sample is lowered and the sample is re-analyzed by the same immunoassay,   or   determining based on the result of an immunoassay the fraction of free target in a sample comprising drug, target and drug-target-complexes for at least two different drug:target ratios in the sample, and if the determined fraction of free target is not comparable for all used drug:target ratios then the drug:target ratio in the sample is increased and the sample is re-analyzed by the same immunoassay,   calculating based on the fraction of free drug or calculating based on the fraction of free target determined in the previous step the binding affinity of the drug to its target.   
     
     
         3 . The method according to  claim 2 , characterized in that one or two or three different drug:target ratios are used. 
     
     
         4 . A method for the determination of the binding affinity of a drug to its target comprising the following steps:
 determining based on the result of an immunoassay the fraction of free drug in a sample comprising drug, target and drug-target-complexes for a first drug:target ratio in the sample,   whereby the drug:target ratio is equal or less than a drug:target ratio for which the determined fraction of free drug is comparable for at least two different drug:target ratios,   determining based on the result of an immunoassay the fraction of free drug in a sample comprising drug, target and drug-target-complexes for at least a second drug:target ratio in the sample,   whereby the second drug:target ratio is different from the first drug:target ratio by increasing or reducing the amount of target in the sample and by maintaining the amount of drug in the sample,   whereby the determined fractions of free drug is comparable for the drug:target ratio with increased or reduced amount of target in the sample for an increased or reduced amount of drug in the sample and the starting drug:target ratio,   calculating based on the fraction of free drug determined in the previous step the binding affinity of the drug to its target.   
     
     
         5 . A method for the determination of the binding affinity of a drug and its target comprising the following steps:
 determining based on the result of an immunoassay the fraction of free target in a sample comprising drug, target and drug-target-complexes for a first drug:target ratio in the sample,   whereby the drug:target ratio is equal or higher than a drug:target ratio for which the determined fraction of free target is comparable for at least two different drug:target ratios,   determining based on the result of an immunoassay the fraction of free target in a sample comprising drug, target and drug-target-complexes for at least a second drug:target ratio in the sample,   whereby the second drug:target ratio is different from the first drug:target ratio by increasing or reducing the amount of drug in the sample and by maintaining the amount of target in the sample,   whereby the fraction of free target is comparable for the drug:target ratio with increased or reduced amount of drug in the sample for an increased or reduced amount of target in the sample and the starting drug:target ratio,   calculating based on the fraction of free target determined in the previous step the binding affinity of the drug to its target.   
     
     
         6 . A method for the determination of the binding affinity of a drug to its target comprising the following steps:
 determining the free drug fraction in at least three samples with different drug:target ratios,
 whereby in the at least three samples the amount of drug is kept constant and the amount of target is different in each sample, with the proviso that the sample comprises excess target compared to drug, 
   or   determining the free target fraction in at least three samples with different drug:target ratios,
 whereby in the at least three samples the amount of target is kept constant and the amount of drug is different in each sample, with the proviso that the sample comprises excess drug compared to target, 
   calculating based on the fraction of free drug or calculating based on the fraction of free target determined in the previous step the binding affinity of the drug to its target for each sample,   whereby the binding affinity has been determined if the at least three K D  values are comparable,   whereby the method is repeated if the at least three K D  values are not comparable using samples in which either   i) the amount of target is reduced at constant amount of the drug, or   ii) the amount if drug is reduced at constant amount of the target, or compared to the samples used in the previous determination.   
     
     
         7 . The method according to any one of  claims 1 ,  4 ,  5 ,  6  characterized in that the sample comprises serum or plasma. 
     
     
         8 . The method according to any one of  claims 1 ,  4 ,  5 ,  6 , characterized in that the drug is an antibody and the target is the antigen that is specifically bound by the antibody. 
     
     
         9 . The method according to any one of  claims 1 ,  4 ,  5 ,  6 , characterized in that the target is immobilized on a solid phase. 
     
     
         10 . The method according to any one of  claims 1 ,  4 ,  5 ,  6 , characterized in that for the calculation of the K D  value the following equation is used:
     K   D =(free drug fraction)*(target concentration [nM])/(1−free drug fraction).