Vitro prediction of in vivo half-life

Patent metadata

Field	Value
Publication number	US-2021190795-A1
Application number	US-202117158431-A
Country	US
Kind code	A1
Filing date	Jan 26, 2021
Priority date	Mar 21, 2014
Publication date	Jun 24, 2021
Grant date	—

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.

First claim

1 - 18 . (canceled)

19 . A method for selecting an antibody, comprising the steps of:
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 according to 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 according to 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 according to 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 relative 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 relative 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 the retention time and a second retention time that are substantially the same, and thereby selecting an antibody with relative 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 relative increased in vivo half-life.

23 . The method according to claim 19 , wherein the positive linear pH gradient is from about pH 5.5 to about pH 8.8.

24 . The method according to claim 19 , wherein the salt is sodium chloride.

25 . The method according to claim 19 , wherein the first salt concentration is about 140 mM.

26 . The method according to claim 19 , wherein the second salt concentration is about 400 mM.

27 . The method according to claim 19 , wherein the linear salt gradient is from 0 mM salt to 250 mM salt.

28 . The method according to claim 19 , wherein the first pH value is about 5.5.

29 . The method according to claim 19 , wherein the second pH value is about 7.4.

30 . The method according to claim 19 , wherein the high salt concentration is about 400 mM.

31 . The method according to claim 19 , wherein the high pH value is about pH 7.4.

32 . The method according to claim 19 , wherein substantially different retention times differ by at least 5%.

33 . The method according to claim 19 , wherein substantially same retention times differ by 3.5% or less.

34 . The method according to claim 19 , wherein if the retention times are substantially different, and the retention times are proportional to one above the square root of the sal

Assignees

• Hoffmann La Roche

Inventors

• Emrich Thomas
• Kettenberger Hubert
• Schlothauer Tilman
• Schoch Angela

Classifications

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• B01D15/3809 Primary

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• B01D15/168 Primary

  pH gradient or chromatofocusing, i.e. separation according to the isoelectric point pI · CPC title

• C07K2317/94

  Stability, e.g. half-life, pH, temperature or enzyme-resistance · CPC title

• C07K2317/52

  Constant or Fc region; Isotype · CPC title