Granted Patent B2

US 10,166,295 · App. 14/725,883 · Granted Jan 1, 2019

Inventors: Udi Eyal Fima (Dvira, IL); Oren Hershkovitz (M.P. Shikmim, IL)

Assignee: OPKO Biologics Ltd.

A61K47/48215A61K38/22

Abstract

Claims (16)

1. A method for the preparation of an oxyntomodulin (OXM) conjugate represented by the structure of formula II:

wherein R 2 is H or SO 3 H;

said method comprises:

reacting MAL-Fmoc-NHS (R 2 ═H) represented by the following structure:

MAL-FMS-NHS (R 2 ═SO 3 H) represented by the following structure:

with a resin-bound oxyntomodulin of SEQ ID NO:1, wherein the amino side chains of Lys 12 and Lys 30 of said resin-bound oxyntomodulin are each protected by a protecting group, to obtain a resin-bound MAL-Fmoc-protected OXM or a resin-bound MAL-FMS-protected OXM, respectively;

followed by

(b) reaction of the resin-bound MAL-Fmoc-protected OXM or the resin-bound MAL-FMS-protected OXM with sulfhydryl PEG polymer (PEG-SH) and subsequently removing the protecting groups and said resin; or

(c) removal of the protecting group and the resin to provide MAL-Fmoc-OXM and a MAL-FMS-OXM, respectively, and subsequent reaction of the MAL-Fmoc-OXM or the MAL-FMS-OXM with sulfhydryl PEG polymer (PEG-SH);

to yield the structure of formula II, wherein the amino residue of His 1 of said OXM is linked to said Fmoc or FMS.

2. The method of claim 1 , wherein said MAL-FMS-NHS is prepared by mixing MAL-Fmoc-NHS with trifluoroacetic acid and chlorosulfonic acid.

3. The method of claim 2 , wherein MAL-Fmoc-NHS is dissolved in neat trifluoroacetic acid and 6 equivalents of chlorosulfonic acid dissolved in neat trifluoroacetic acid is added to the reaction mixture.

4. The method of claim 1 , wherein said protecting group of said Lys 12 and Lys 30 is 1-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidine)ethyl]), (ivDde).

5. The method of claim 1 wherein said reaction with said sulfhydryl PEG polymer (PEG-SH) is conducted under a buffer condition of pH between 6 and 6.5.

6. The method of claim 1 , wherein said PEG has a molecular weight in the range of 20,000 Da to 40,000 Da.

7. The method of claim 6 , wherein said PEG has a molecular weight with an average molecular weight of 30,000 Da.

Assignments (1)

Continuity (10)