Pegylated Interleukin-10
Interleukin-10 (IL-10) conjugated via a linker to one or more polyethylene glycol (PEG) molecules at a single amino acid residue of the IL-10, and a method for preparing the same, are provided. The method produces a stable mono-pegylated IL-10, which retains IL-10 activity, where pegylation is selective for the N-terminus on one subunit of IL-10 with little or no formation of monomeric IL-10. The method also provides a substantially homogenous population of mono-PEG-IL-10.
1 . A mono-PEG-IL-10.
2 . The mono-PEG-IL-10 of claim 1 , comprising one or two PEG molecules covalently attached via a linker to one amino acid residue on IL-10, wherein the attachment is at an N-terminal amino acid residue or on a lysine residue.
3 . The mono-PEG-IL-10 of claim 2 :
(a) which comprises a methoxy PEG;
(b) wherein the IL-10 is human IL-10;
(c) wherein the total molecular mass of all PEG covalently attached to the linker is from 3,000 daltons to 60,000 daltons; or
(d) wherein the linker is a linear or branched C 1-11 alkyl.
4 . The mono-PEG-IL-10 of claim 2 , wherein the total molecular mass of all PEG covalently attached to the linker is from 10,000 daltons to 36,000 daltons.
5 . The mono-PEG-IL-10 of claim 2 , wherein the linker is a linear C 3 alkyl.
6 . The mono-PEG-IL-10 of claim 1 , wherein a PEG molecule is covalently attached to the alpha amino group of one N-terminus of IL-10 via a linear C 3 alkyl linker.
7 . A PEG-IL-10 comprising the formula:
[X—O(CH 2 CH 2 O) n ] b -L-NH-IL-10,
where X is H or C 1-4 alkyl, n is 20 to 2300, b is 1 to 9 and L is a C 1-11 alkyl linker moiety which is covalently attached to nitrogen (N) of the alpha amino group at the amino terminus of one IL-10 subunit, provided that when b is greater than 1 the total of n does not exceed 2300.
8 . A PEG-IL-10 of claim 7 , wherein L is —CH 2 CH 2 CH 2 —.
9 . A pharmaceutical composition, comprising a mono-PEG-IL-10 of claim 1 in combination with a pharmaceutically acceptable carrier.
10 . A method of treating inflammation in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition of claim 9 .
11 . A process for preparing a mono-PEG-IL-10, comprising the step of:
reacting IL-10 with an activated PEG-aldehyde linker in the presence of a reducing agent to form the mono-PEG-IL-10,
wherein the linker is covalently attached to one amino acid residue of the IL-10.
12 . The process of claim 11 wherein:
(a) the reducing agent is sodium cyanoborohydride;
(b) the activated PEG-aldehyde linker is PEG-propionaldehyde;
(c) the PEG is a methoxy-PEG;
(d) the linker is multi-armed;
(e) the ratio of IL-10 to the sodium cyanoborohydride is from about 1:0.5 to 1:50;
(f) the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 3,000 daltons to 60,000 daltons; or
(g) the reacting step is performed at a pH of 5.5 to 7.8.
13 . The process of claim 11 , wherein the ratio of IL-10 to the sodium cyanoborohydride is 1:5 to 1:15.
14 . The process of claim 11 , wherein the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 10,000 daltons to 36,000 daltons.
15 . The process of claim 11 , wherein the reacting step is performed at a pH of 6.3 to 7.5.
16 . The process of claim 11 , further comprising a step selected from:
incubating the mono-PEG-IL-10 product in a buffer at pH 5.0 to 9.0; and
treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.
17 . A process for preparing a mono-PEG-IL-10, comprising the step of:
reacting IL-10 with an activated PEG-propionaldehyde linker in the presence of sodium cyanoborohydride, wherein the molar ratio of IL-10 to sodium cyanoborohydride is from about 1:5 to about 1:15, at a pH of about 6.3 to about 7.5 and a temperature of from 18° C. to 25° C. to form the mono-PEG-IL-10,
wherein the linker is covalently attached to one amino acid residue of the IL-10.
18 . The process of claim 17 , wherein the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 10,000 daltons to 36,000 daltons.
19 . The process of claim 17 , further comprising a step selected from:
incubating the mono-PEG-IL-10 product in a TRIS buffer at pH 7.0 to 8.0; and
treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.
20 . A PEG-IL-10 prepared according to a process of claim 11 .