Method to screen high affinity antibody

Abstract

The current invention reports a method for producing an antibody comprising the steps of a) providing a plurality of hybridoma cells each expressing an antibody, b) determining the time dependent amount of said antibody bound to the respective antigen by surface plasmon resonance at different temperatures and different antibody concentrations, c) calculating with the time dependent amount determined in b) based on equations (II) to (XIII) at least the thermodynamic parameters (i) standard association binding entropy (ΔS°‡ass), (ii) standard dissociation binding entropy (ΔS°‡diss), (iii) standard binding entropy (ΔS°), (iv) free standard binding enthalpy (ΔG°), (v) standard dissociation free binding enthalpy (ΔG°‡diss), (vi) standard association free binding enthalpy (ΔG°‡ass), (vii) −TΔS°, (viii) dissociation rate constant k d , (ix) equilibrium binding constant K D , and (x) association rate constant k a , d) selecting a hybridoma cell producing an antibody with at least two of the following: i) a standard association binding entropy of less than 10 J/K*mol, ii) an absolute standard dissociation binding entropy of 100 J/mol*K or more, iii) an absolute standard binding entropy of 100 J/mol*K or more, e) producing an antibody by cultivating said selected cell under conditions suitable for the expression of said antibody and recovering said antibody from the cells or/and the cultivation medium.

1 - 30 . (canceled) 
     
     
         31 . A method for the selection of a temperature-independent antigen binding antibody from a plurality of antibodies, wherein the antibody selected comprises a standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol. 
     
     
         32 . The method according to  claim 31 , wherein the antibody selected comprises at least two of a), b), and c):
 a) a standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol,   b) an absolute standard dissociation binding entropy (ΔS°‡diss) of 100 J/mol*K or more, and   c) an absolute standard binding entropy (ΔS°) of 100 J/mol*K or more.   
     
     
         33 . The method according to  claim 32 , wherein the antibody selected comprises a standard association binding entropy (ΔS°‡ass) of less than 5 J/K*mol. 
     
     
         34 . The method according to  claim 32 , wherein the antibody selected comprises a standard association binding entropy (ΔS°‡ass) of less than 0 J/K*mol. 
     
     
         35 . The method according to  claim 32 , wherein the antibody selected comprises an absolute standard dissociation binding entropy (ΔS°‡diss) of 125 J/mol*K or more. 
     
     
         36 . The method according to  claim 32 , wherein the antibody selected comprises an absolute standard dissociation binding entropy (ΔS°‡diss) of 150 J/mol*K or more. 
     
     
         37 . The method according to  claim 32 , wherein the antibody selected comprises an absolute standard binding entropy (ΔS°) of 125 J/mol*K or more. 
     
     
         38 . The method according to  claim 32 , wherein the antibody selected comprises an absolute standard binding entropy (ΔS°) of 150 J/mol*K or more. 
     
     
         39 . The method according to  claim 31  wherein the antibody selected comprises a free standard binding enthalpy (ΔG°) of −50 kJ/mol or less. 
     
     
         40 . The method according to  claim 39 , wherein the antibody selected comprises a ratio of the standard dissociation free binding enthalpy (ΔG°‡diss) to the standard association free binding enthalpy (ΔG°‡ass) of more than 2.3. 
     
     
         41 . The method according to  claim 40 , wherein the antibody selected comprises a −TΔS° value of
 a) −80 kJ/mol or less, or 
 b) of +40 kJ/mol or more. 
 
     
     
         42 . The method according to any one of  claims 31 - 41 , wherein the method comprises a temperature in the range of from 13° C. to 37° C.