Method of making a water-soluble polymer-factor VIII moiety conjugate
Conjugates of a Factor VIII moiety and one or more water-soluble polymers are provided. Typically, the water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising the conjugates, methods of making the conjugates, and methods of administering compositions comprising the conjugates to a patient.
1. A method of making a conjugate, the method comprising:
(i) contacting, at ambient temperature, a Factor VIII moiety comprised in a buffer solution, wherein the Factor VIII moiety comprises amino groups and is selected from the group consisting of Factor VIII, Factor Villa, Factor VIII:C and B-domain deleted Factor VIII, with a methoxypoly(ethylene glycol) (mPEG) butryaldehyde reagent having a nominal average molecular weight in a range of 6,000 Daltons to about 90,000 Daltons, to thereby form a Schiff base resulting from reaction of amino groups of the Factor VIII moiety with the mPEG butryaldehyde reagent,
(ii) reducing the Schiff base from (i) to thereby form a reaction mixture comprising methoxypoly(ethylene glycol)—Factor VIII moiety conjugates comprising one or more methoxypoly(ethylene) glycol polymer(s) covalently attached to the Factor VIII moiety via a secondary butamine linkage, and
(iii) purifying the reaction mixture from (ii) to isolate mono-mPEGylated Factor VIII moiety conjugates having one methoxypoly(ethylene glycol) polymer covalently attached to the Factor VIII moiety via a secondary butamine linkage.
2. The method of claim 1 , wherein the mPEG butryaldehyde reagent has a nominal average molecular weight in the range of from about 10,000 Daltons to about 85,000 Daltons.
3. The method of claim 1 , wherein the mPEG butyraldehyde polymer reagent is linear.
4. The method of claim 1 , wherein the mPEG butyraldehyde polymer reagent is branched.
5. The method of claim 1 , wherein the Factor VIII moiety is a recombinant Factor VIII moiety.
6. The method of claim 1 , wherein the reducing step comprises addition of a reducing agent that is either sodium borohydride or sodium cyanoborohydride.
7. The method of claim 1 , wherein the contacting step comprises a molar excess of methoxypoly(ethylene glycol) butyraldehyde.
8. The method of claim 1 , wherein in step (ii), the reaction mixture comprises methoxypoly(ethylene glycol)—Factor VIII moiety conjugates comprising from one to three methoxypoly(ethylene glycol) polymers covalently attached to the Factor VIII moiety via a secondary butamine linkage.
9. The method of claim 1 , wherein the purifying step (iii) comprises gel filtration chromatography.
10. The method of claim 1 , wherein the (mPEG) butryaldehyde reagent has a structure,
11. The method of claim 1 , wherein the (mPEG) butryaldehyde reagent has a weight average molecular weight of 30,000 Daltons.
12. The method of claim 1 , wherein the mono-mPEGylated Factor VIII moiety conjugates from step (iii) are bioactive.