Method of production of graft co-polymer excipient with a superior peptide and protein binding property

1. A method of making a semi-random graft co-polymer, wherein the semi-random graft co-polymer is a protective chain and long fatty acid-grafted polyamine, comprising:

step (a): dissolving a linear polyamine backbone containing W moles of free primary amino groups in an aqueous solvent to provide a solution A having a volume Y;

B1 or B2, wherein:

B1 comprises:

step (b): activating a protective chain selected from polyethylene glycol, polypropylene glycol, polyethylene-polypropylene glycol copolymer, methoxylated polyethylene glycol, methoxylated polypropylene glycol, methoxylated polyethylene-polypropylene glycol copolymer, ethoxylated polyethylene glycol, ethoxylated polypropylene glycol, and ethoxylated polyethylene-polypropylene glycol copolymer, wherein the protective chain contains 0.5-1.2×W moles of carboxyl groups with 1.7-7.0×W moles of N-hydroxysuccinimidesulfate and 1.5-3.6×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in an aqueous buffer, at pH 4-5.5 having a final volume Z, such that W/(Y+Z) has ratio of 30-55 mM, for a period of time of up to 30 min to provide a solution B;

step (c): adding solution B to solution A with continuous mixing to provide a solution C;

step (d): adjusting the pH of solution C to above 6.5; and

step (e): adding 0.5-1.5×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after the pH adjustment in step (d); and waiting until the primary amino groups are 55-40% of W moles;

B2 comprises:

steps (b-d): wherein steps (b-d) comprise adding a protective chain selected from polyethylene glycol, polypropylene glycol, polyethylene-polypropylene glycol copolymer, methoxylated polyethylene glycol, methoxylated polypropylene glycol, methoxylated polyethylene-polypropylene glycol copolymer, ethoxylated polyethylene glycol, ethoxylated polypropylene glycol, and ethoxylated polyethylene-polypropylene glycol copolymer, wherein the protective chain contains 0.5-1.2×W moles of carboxyl groups; 1.7-7.0×W moles of N-hydroxysuccinimidesulfate; and 1.5-3.6×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution A to provide solution C, adjusting the pH of solution C to above 6.5; and

step (e): adding 0.5-1.5×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after steps (b-d); and waiting until the primary amino groups are 55-40% of W moles; and

C1, C2, C3, or C4, wherein:

C1 comprises:

step (f): freezing and lyophilizing solution C to provide a lyophilized material when the primary amino groups are 55-40% of W moles and reconstituting the lyophilized material in one or more organic solvent(s) to provide a solution D;

step (g): adding 0.5-6×W moles of N,N-diisopropylethylamine or other tertiary amine to solution D; and

step (h): adding at least 0.75×W moles of a long chain fatty acid N-hydroxysuccinimide ester to provide a solution E and stirring solution E at room temperature for at least 2 hours or until the primary amino groups are less than 5% of W moles to obtain a crude product, followed by purification to provide a final product;

C2 comprises:

step (f): adding at least W moles of a strong nucleophile to solution C and purifying a resulting immediate reaction product and dissolving the immediate reaction product in acetonitrile to provide a solution D, wherein the strong nucleophile comprises anyone of hydroxyl amine, NaOR, LiR, NaOH, KOH, NaCCR, NaNH 2 , NaNHR, NaNR 2 , NaI, LiBr, KI, and NaN 3 , wherein R is C 1 -C 6 alkyl or C 2 -C 6 alkenyl;

step (g): adding 0.5-6×W moles of N,N-diisopropylethylamine or other tertiary amine to solution D; and

step (h): adding at least 0.75×W moles of a long chain fatty acid-N -hydroxysuccinimide ester to provide a solution E and stirring solution E at room temperature for at least 2 hours until the primary amino groups are less than 5% of W moles to obtain a crude product, followed by purification to obtain a final product;

C 3 comprises:

step (f): increasing the total volume of solution C when the primary amino groups are 55-40% of W moles by adding 1.0-2.5 volume equivalents of acetonitrile to obtain a solution D and heating solution D to 40-70° C. for at least 10 minutes;

step (g): adding 0.5-6×W moles of N,N-diisopropylethylamine or other tertiary amine to solution D; and

step (h): adding at least 0.75×W moles of long chain fatty acid N-hydroxysuccinimide ester in acetonitrile to provide a solution E and stirring solution E at room temperature for at least 2 hours or until the primary amino groups are less than 5% of W moles to obtain a crude product, followed by purification to obtain a final product; and

C4 comprises:

step (f): increasing the total volume of solution C when the remaining primary amino groups is 55-40% of W moles by adding 1.0-2.5 volume equivalents of acetonitrile to obtain a solution D and heating solution D to 40-70° C.;

step (g): adding 0.5-6×W moles of N,N-diisopropylethylamine or other tertiary amine to solution D; and

step (h): adding at least 0.75×W moles of long chain fatty acid-N-hydroxysuccinimide ester in acetonitrile to provide a solution E and stirring solution E at room temperature for at least 2 hours or until the primary amino groups are less than 5% of W moles to obtain a crude product, followed by purification to obtain a final product.

2. The method of claim 1 , wherein the linear polyamine backbone is polylysine with a degree of polymerization of 35-150, as determined by light scattering or nuclear magnetic resonance analysis;

wherein the protective chain is methoxypolyethyleneglycol chain having a single carboxyl terminus and a number average molecular weight of 4-12 kDa, as determined by gel permeation chromatography,

wherein in solution A, the aqueous solvent is a buffer having a buffering range from pH 7 to pH 8,

wherein purification of the crude product comprises S1 or S2, wherein

S1 comprises:

step (i): extracting organic solvent(s) and excess fatty acids from the crude product in an aqueous solution using ethyl acetate, discarding the ethyl acetate extract, repeating the extraction of the aqueous solution at least once, to provide an aqueous solution containing the product;

step (j): washing the aqueous solution containing the product by ultrafiltration with at least 10 volume changes of ethanol and water to provide the final product; and

step (k): freezing or lyophilizing the final product;

S2 comprises:

step (i): exchanging the organic solvent in solution E to provide the product in water;

step (j): washing the product by ultrafiltration with at least 10 volume changes of ethanol and water to provide the final product; and

step (k): freezing or lyophilizing the final product.

3. The method of claim 2 , wherein the method comprises C3;

wherein a degree of polymerization of polylysine is 35-85, as determined by light scattering or nuclear magnetic resonance analysis;

wherein the protective chain is methoxypolyethyleneglycol having a number average molecular weight of 4-6 kDa, as determined by gel permeation chromatography, and

wherein the long chain fatty acid N-hydroxysuccinimide ester, when present, is stearic acid N-hydroxysuccinimide ester.

4. The method of claim 2 , wherein solution A in step (a) has a concentration of primary amine groups of between 0.1-0.14M;

wherein in step (b), solution B has 0.8-1×W moles of carboxyl group, 1.7-3.4×W moles of N-hydroxysuccinimidesulfate, and 2.5-2.9×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein pH of solution B is 4.5-4.9;

wherein in step (b), activating a protective chain proceeds for 18-22 min;

wherein pH of solution C is adjusted to 7-8;

wherein in step (e), 0.8-1.1×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C; and

wherein in step (g), 1-3×W moles of N,N-diisopropylethylamine or other tertiary amine is added to solution D,

wherein the long chain fatty acid N-hydroxysuccinimide ester is stearic acid N-hydroxysuccinimide ester.

5. The method of claim 4 , wherein in step (b), solution B has 0.9×W moles of carboxyl group, 2.7×W moles of N-hydroxysuccinimidesulfate, and 2.7×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; and

wherein in step (e), 0.9×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C.

6. The method of claim 2 , wherein in step (b), solution B has 0.75-0.85×W moles of carboxyl group, 2.3-2.8×W moles of N-hydroxysuccinimidesulfate, and 2.3-2.6×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein pH of solution B is 4.5-4.9;

wherein in step (b), activating a protective chain proceeds for 18-22 min;

wherein pH of solution C is adjusted to 7-8;

wherein in step (e), 0.75-0.85×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C; and

wherein in step (g), 1-3×W moles of N,N-diisopropylethylamine or other tertiary amine is added to solution D.

7. The method of claim 6 , wherein in step (b), solution B has 0.8×W moles of carboxyl group; 2.4×W moles of N-hydroxysuccinimidesulfate; and 2.4×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein pH of solution B is 4.5-4.9;

wherein in step (b), activating a protective chain proceeds for 18-22 min;

wherein pH of solution C is adjusted to 7-8;

wherein in step (e), 0.75-0.85×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C;

wherein in step (h), at least 0.75×W moles of stearic acid N-hydroxysuccinimide ester is added to solution D, and

wherein the long chain fatty acid N-hydroxysuccinimide ester is stearic acid N-hydroxysuccinimide ester.

8. The method of claim 2 , wherein in step (a), solution A has a concentration of primary amine of 0.10-0.14M;

wherein in step (b), solution B has 0.65-0.75×W moles of carboxyl group, 2.0-2.5×W moles of N-hydroxysuccinimidesulfate, and 2.0-2.3×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein activation proceeds for 15-25 min;

wherein in step (e), 0.65-0.75×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C after 1 -3hrs;

wherein in step (g), 1-3×W moles of N,N-diisopropylethylamine or other tertiary amine is added to solution D; and

wherein in step (h), 0.75-1.25×W moles of stearic acid N-hydroxysuccinimide ester is added to solution D.

9. The method of claim 8 , wherein in step (b), solution B has 0.7×W moles of carboxyl group, 2.1×W moles of N-hydroxysuccinimidesulfate, and 2.1×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein in step (b), activating a protective chain proceeds for 15-25 min;

wherein in step (e), 0.65-0.75×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C after 1 -3hrs; and

wherein in step (h), 0.75-1.25 33 W moles of stearic acid N-hydroxysuccinimide ester is added to solution D.

10. The method of claim 2 , wherein in step (a), solution A has a concentration of primary amine of 0.10-0.14M;

wherein in step (b), solution B has 0.55-0.65×W moles of carboxyl group, 1.7-2.2×W moles of N-hydroxysuccinimidesulfate, and 1.7-2.0×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein in step (b), activating a protective chain proceeds for 15-25 min;

wherein in step (e), 0.55-0.65×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C after 1-3hrs;

wherein in step (f), a total volume of solution C is increased by adding 1.6-1.9 volume equivalents of acetonitrile;

wherein in step (g), 1-3×W moles of N,N-diisopropylethylamine or other tertiary amine is added to solution D; and

wherein in step (h), 0.75-1.25×W moles of stearic acid N-hydroxysuccinimide ester is added to solution D.

11. The method of claim 10 , wherein in step (b), solution B has 0.6×W moles of carboxyl group, 1.8×W moles of N-hydroxysuccinimidesulfate (NHSS), and 1.8×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein in step (e), 0.6×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C after 1-3hrs.

12. The method of claim 2 , wherein in step (a), solution A has a concentration of primary amine of 0.10-0.14M;

wherein in step (b), solution B has 0.65-0.75×W moles of carboxyl group, 2.0-2.5×W moles of N-hydroxysuccinimidesulfate, and 2.0-2.3×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

wherein in step (b), activating a protective chain proceeds for 15-25 min;

wherein in step (e), 0.65-0.75×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride is added to solution C after 1-3hrs;

wherein in step (g) 1-3×W moles of N,N-diisopropylethylamine or other tertiary amine is added to solution D; and

wherein in step (h), 0.75-1.25×W moles of stearic acid N-hydroxysuccinimide ester is added to solution D.

13. The method of claim 1 , wherein in B1, step (b) comprises activating the protective chain containing 0.5-1.1×W moles of carboxyl groups with 1.7-3.7×W moles of N-hydroxysuccinimidesulfate and 1.5-3.3×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in an aqueous buffer; and

step (e) comprises adding 0.5-1.2×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after the pH adjustment in step (d); and

wherein in B2, steps (b-d) comprise adding the protective chain containing 0.5-1.1×W moles of carboxyl groups; 1.7-3.7×W moles of N-hydroxysuccinimidesulfate; and 1.5-3.3×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution A to provide solution C; and

step (e) comprises adding 0.5-1.2×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after steps (b-d).

14. The method of claim 1 , wherein in B1, step (b) comprises activating the protective chain containing 0.5-1.0×W-moles of carboxyl groups with 1.7-3.4×W moles of N-hydroxysuccinimidesulfate and 1.5-3.0×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in an aqueous buffer; and

step (e) comprises adding 0.5-1.1×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after the pH adjustment in step (d); and

wherein in B2, steps (b-d) comprise adding the protective chain containing 0.5-1.0×W moles of carboxyl groups; 1.7-3.4×W moles of N-hydroxysuccinimidesulfate; and 1.5-3.0×W moles of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution A to provide solution C; and

step (e) comprises adding 0.5-1.1×W moles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solution C 2 to 3 hours after steps (b-d).

15. The method of claim 1 , wherein in step “e”, 0.5-1.0×W of additional 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride EDC is added to solution C; and

wherein in step “f”, when present, 1.5-2.0 volume C equivalents of acetonitrile is added when the remaining primary amino groups reaches 55-40% of the original primary amino.