Method for thioether conjugation of proteins
The invention relates to methods for obtaining a protein conjugate wherein a cysteine residue of the protein serves as attachment point for the chemical moiety conjugated to the protein.
1. A method for preparing a protein conjugate wherein a protein (P) is covalently bound to a chemical moiety (Z) via a thioether, comprising the steps of:
a) obtaining a composition of a mixed di-sulfide comprising the protein,
b) adding a reducing agent to the composition to obtain a reduction mix, wherein the reducing agent is a phosphine,
c) allowing a reduction reaction to occur,
d) obtaining a solution comprising a reduced protein (P-SH),
e) optionally removing molecules with a molecular weight below 10 kDa from the solution,
f) adding an activated chemical moiety (Z*) to the solution comprising the reduced protein and obtaining a conjugation mix including 1-4 equivalents of the activated chemical moiety (Z*) relative to the reduced protein,
g) allowing a conjugation reaction to occur to generate a conjugated protein (P-S-Z), and
h) obtaining a preparation of the conjugated protein (P-S-Z),
wherein the reduction mix of step b) has a concentration of the mixed di-sulfide of at least Cmin,
wherein Cmin is defined by: Cmin=a*I −a1 exp(−b*T), wherein T is the temperature in degrees Celsius, I is ionic strength (M) of the reduction mix, a=0.137*10 −3 M 1.425 , a 1 =0.425 and b=0.070° C. −1 ,
wherein at least one step is performed in a cross flow filtration system, and
wherein the activated chemical moiety (Z*) is an activated albumin binder.
2. The method according to claim 1 , wherein the mixed di-sulfide is a protein with a capped free cysteine (P-S-S-Cap).
3. The method according to claim 2 , wherein the Cap is derived from cysteine, cysteamine or glutathione.
4. The method according to claim 1 , wherein the conjugation mix of f) has a concentration of the reduced protein of at least Cmin, where Cmin is defined by:
C min= a *exp(− b 1* T−b 2* I )+ d *exp(− d 1* T )
wherein T is the temperature in degrees Celsius, I is the ionic strength (M) of the conjugation mix, a=6.96*10−4 M, b1=0.0396° C.−1, b2=10.9 M−1, d=6.12*10−5 M and d1=0.0289° C.−1.
5. The method according to claim 1 , wherein the reducing agent is a triarylphosphine.
6. The method according to claim 1 , wherein the amount of the reducing agent included in the reduction mix of step b) is at most 10 equivalents of the protein (P-S-S-Cap).
7. The method according to claim 1 , wherein the activated chemical moiety (Z*) is a halogenated albumin binder including Br, I or Cl.
8. The method according to claim 1 , wherein the conjugation mix of step f) includes at most 3 equivalent of the activated chemical moiety (Z*), relative to the mixed di-sulfide.
9. The method according to claim 1 , where step e) is diafiltration performed using a cellulose membrane.
10. The method according to claim 9 , wherein the diafiltration performed in step (e) use a diafiltration buffer which does not include a reducing agent.
11. The method according to claim 1 , wherein the composition of step a), the reduction mix of step b), the solution of step d), the conjugation mix of step f) and/or the preparation of step h) comprise triethanolamine.
12. The method according to claim 1 , wherein the composition of step a), the reduction mix of step b), the solution of step d), the conjugation mix of step f) and/or the preparation of step h) have pH 7.0-8.0.
13. The method according to claim 1 , wherein the protein is a growth hormone polypeptide comprising a L101C point mutation.
14. The method according to claim 5 , wherein the reducing agent is disodium triphenylphosphine-3,3′-disulfonate (TPPDS).
15. The method according to claim 1 , wherein the activated chemical moiety (Z*) is a halogenated albumin binder including Iodine (I).
16. The method according to claim 1 , wherein the activated chemical moiety (Z*) is an iodoacetamide of an albumin binder.
17. The method according to claim 1 , wherein the activated chemical moiety (Z*) is selected from the group consisting of:
18. The method according to claim 1 , wherein the activated chemical moiety (Z*) is selected from the group consisting of:
19. The method of claim 1 , wherein the conductivity of the solution of d) is 10 mS/cm at 22° C.
20. The method of claim 1 , wherein the mixed di-sulfide composition comprises a buffer selected from the group consisting of BES, HEPES, IVIES, Phosphate, Citrate, Bis-Tris and triethanolamine.