In vitro prediction of in vivo half-life

Abstract

Herein is reported a method for determining the presence of antibody-Fab-FcRn interaction in an antibody-Fc-FcRn complex influencing the in vivo half-life comprising the steps of a) determining the retention time of the antibody on an FcRn affinity chromatography column with a positive linear pH gradient elution in the presence of a first sodium chloride concentration, and b) determining the retention time of the antibody on an FcRn affinity chromatography column with a positive linear pH gradient elution in the presence of a second sodium chloride concentration, whereby the presence of antibody-Fab-FcRn interaction in an antibody-Fc-FcRn complex influencing the in vivo half-life is determined if the retention time determined in step a) and the retention time determined in step b) are substantially different.

1 - 18 . (canceled) 
     
     
         19 . A method for selecting an antibody comprising the following steps:
 i) determining a first retention time of the antibody and a reference antibody on an FcRn affinity chromatography column with a positive linear pH gradient elution in the presence of a first salt concentration, and determining a second retention time of the antibody and the reference antibody on an FcRn affinity chromatography column with the positive linear pH gradient elution in the presence of a second salt concentration, or   ii) determining a first retention time of the antibody and a reference antibody on an FcRn affinity chromatography column with a linear salt gradient elution at a first pH value, and determining a second retention time of the antibody and the reference antibody on an FcRn affinity chromatography column with the linear salt gradient elution at a second pH value, or   iii) determining for the antibody and a reference antibody the K D  value at pH 6 using surface plasmon resonance, and determining the retention time of the antibody and the reference antibody on an FcRn affinity chromatography column with a positive linear pH gradient elution in the presence of a high salt concentration, or   iv) determining for the antibody and a reference antibody the K D  value at pH 6 using surface plasmon resonance, and determining the retention time of the antibody and the reference antibody on an FcRn affinity chromatography column with a linear salt gradient elution, or   v) determining the retention time of the antibody and its Fc-region on an FcRn affinity chromatography column with a positive linear pH gradient elution, or   vi) determining the retention time of the antibody and its Fc-region on an FcRn affinity chromatography column with a linear salt gradient elution at a high pH value, or   vii) determining for the antibody and its Fc-region the K D  value at pH 6 using surface plasmon resonance, and determining the retention time of the antibody and its Fc-region on an FcRn affinity chromatography column with a positive linear pH gradient elution in the presence of a high salt concentration, or   viii) determining for the antibody and its Fc-region the K D  value at pH 6 using surface plasmon resonance, and determining the retention time of the antibody and its Fc-region on an FcRn affinity chromatography column with a linear salt gradient elution at a high pH value,   and by selecting   a) an antibody that has a first retention time that is substantially the same as the second retention time, or   b) an antibody that has a K D  value that differs from the K D  value of the reference antibody by at most a factor of 10 and that has a retention time that is substantially the same as the retention time of the reference antibody, or   c) an antibody that has a retention time that is substantially the same as the retention time of its Fc-region, or   d) an antibody that has a K D  value that differs from the K D  value of its Fc-region by at most a factor of 10 and that has a retention time that is substantially the same as the retention time of its Fc-region.   
     
     
         20 . The method of  claim 19 , wherein the method is for selecting an antibody that is free of antibody-Fab-FcRn interaction influencing the in vivo half-life of the antibody. 
     
     
         21 . The method of  claim 19 , wherein
 the method is for selecting an antibody that has a relative in vivo half-life that is increased compared to an antibody of the IgG1, IgG3, or IgG4 subclass, and   in v), vi), vii) and viii) further the retention time of a reference antibody or reference Fc-region is determined, and   by selecting
 a) an antibody that has a first retention time that is longer than the first retention time of the reference antibody, and a first retention time that is substantially the same as the second retention time, or 
 b) an antibody that has a K D  value that differs from the K D  value of the reference antibody by at most a factor of 10 and that has a retention time that is longer than the retention time of the reference antibody, or 
 c) an antibody that has a retention time that is substantially the same as the retention time of its Fc-region and that is longer than the retention time of the reference antibody, or 
 d) an antibody that has a K D  value that differs from the K D  value of its Fc-region by at most a factor of 10 and that has a retention time that is substantially the same as the retention time of its Fc-region and that is longer than the retention time of the reference antibody. 
   
     
     
         22 . The method of  claim 19 , wherein
 the method is for determining the relative increase or decrease in the in vivo half-life of an antibody to a reference antibody, and   in v), vi), vii) and viii) further the retention time of a reference antibody or reference Fc-region is determined, and   in i) to viii) further the retention time of an IgG Fc-region with the mutation N434A is determined, and   by selecting
 a) an antibody that has a first retention time that is longer than the first retention time of the reference, that has a first retention time and a second retention time that are substantially the same, and that has a first retention time that is shorter than the retention time of the Fc-region with the mutation N434A and thereby selecting an antibody with increased in vivo half-life, or 
 b) an antibody that has a K D  value that differs from the K D  value of the reference antibody by at most a factor of 10, that has a retention time that is longer than the retention time of the reference antibody and that has a first retention time that is shorter than the retention time of the Fc-region with the mutation N434A and thereby selecting an antibody with increased in vivo half-life, or 
 c) an antibody that has a first retention time that is shorter than the first retention time of the reference antibody, and that has a first retention time and a second retention time that are substantially the same, and thereby selecting an antibody with decreased in vivo half-life, or 
 d) an antibody that has a K D  value that differs from the K D  value of the reference antibody by at most a factor of 10, and that has a retention time that is shorter than the retention time of the reference antibody, and thereby selecting an antibody with increased in vivo half-life. 
   
     
     
         23 . The method of  claim 19 , wherein the positive linear pH gradient is from about pH 5.5 to about pH 8.8. 
     
     
         24 . The method of  claim 19 , wherein the salt is sodium chloride. 
     
     
         25 . The method of  claim 19 , wherein the first salt concentration is about 140 mM. 
     
     
         26 . The method of  claim 19 , wherein the second salt concentration is about 400 mM. 
     
     
         27 . The method of  claim 19 , wherein the linear salt gradient is from 0 mM salt to 250 mM salt. 
     
     
         28 . The method of  claim 19 , wherein the first pH value is about 5.5. 
     
     
         29 . The method of  claim 19 , wherein the second pH value is about 7.4. 
     
     
         30 . The method of  claim 19 , wherein the high salt concentration is about 400 mM. 
     
     
         31 . The method of  claim 19 , wherein the high pH value is about pH 7.4. 
     
     
         32 . The method of  claim 19 , wherein the substantially different retention times differ by at least 5%. 
     
     
         33 . The method of  claim 19 , wherein the substantially same retention times differ by 3.5% or less. 
     
     
         34 . The method of  claim 19 , wherein if the retention times are substantially different, the retention times are proportional to one above the square root of the salt concentration (1/SQRT(c(salt))). 
     
     
         35 . The method of  claim 19 , wherein the antibody is a full-length antibody.