Transglutaminase mediated high molecular weight hyaluronan hydrogels

A process for forming a hyaluronan hydrogel, comprising the steps of

• a. providing an aqueous solution of a first hyaluronan peptide conjugate comprising transglutaminase donor peptides and a second hyaluronan peptide conjugate comprising transglutaminase acceptor peptides,
• b. adding a thrombin polypeptide to said aqueous solution and allowing equilibration of the resulting mixture;
• c. subsequently, adding a factor XIII polypeptide.

The process according to any one of the preceding claims,

• wherein the aqueous solution of step a. additionally comprises heparin or heparan sulfate, particularly at a concentration from 0.05% to 0.5% (w/v relative to the gel), and
• wherein the heparin or heparan sulfate comprises covalently attached transglutaminase donor and/or acceptor peptides, particularly wherein 10% to 20% of carboxylic acid groups present in said heparin or heparan sulfate are covalently modified, more particularly covalently modified to contain a modification described by general formula (II) as laid out above, with L, S and Pep having the meaning indicated above.

The process according to any one of the preceding claims, wherein the concentration of the sum of said first hyaluronan peptide conjugate and said second hyaluronan peptide conjugate is 0.25% (w/v) to 5% (w/v), particularly 0.75% to 0.95% or from 0.5% to 3%, 0.5% to 2%, or 0.5% to 1.5%.

A process for modification of a hyaluronan polymer, wherein said hyaluronan polymer is composed of n dimers of D-glucuronic acid moieties and D-N-acetylglucosamine moieties, and wherein said D-glucuronic acid moieties bear reactive carboxylic acid moieties, and said process comprises the steps of:

• a. thiolation of 5% to 20%, particularly 8-12%, more particularly approximately 10% of said reactive carboxylic acid moieties to yield partially thiolated hyaluronan;
• b. reacting said partially thiolated hyaluronan with divinylsulfone to yield vinylsulfone-hyaluronan;
• c. reacting said vinylsulfone-hyaluronan with a peptide comprising a cysteine moiety, wherein said peptide comprises a sequence selected from a transglutaminase donor peptide sequence and a transglutaminase acceptor peptide sequence.

The process according to any one of the preceding claims, wherein said hyaluronan and/or said hyaluronan peptide conjugate and/or said second hyaluronan peptide conjugate are characterized by a mean molecular weight equal or greater than (≥) 250 kg/mol, 500 kg/mol, particularly greater than 1000 kg/mol (1 MDa);

• more particularly between 1000 kg/mol and 4000 kg/mol, even more particularly between 1000 kg/mol and 2000 kg/mol.

A process for modification of a heparin or heparan sulfate polymer, wherein said heparin or heparan sulfate polymer comprises D-glucuronic acid moieties and L-iduronic acid moieties, each of which bearing reactive carboxylic acid moieties, and said process comprises the steps of:

• a. thiolation of 5% to 20%, particularly 8-12%, more particularly approximately 10% of said reactive carboxylic acid moieties to yield partially thiolated heparin or heparan sulfate;
• b. reacting said partially thiolated hyaluronan with divinylsulfone to yield vinylsulfone-heparin or vinylsulfone -heparan sulfate;
• c. reacting said vinylsulfone-heparin or -heparan sulfate with a peptide comprising a cysteine moiety, wherein said peptide comprises a sequence selected from a transglutaminase donor peptide sequence and a transglutaminase acceptor peptide sequence.

The process according to any one of the preceding claims, wherein

• a. the transglutaminase donor peptide sequence is or comprises a sequence selected from SEQ ID NO 01 to SEQ ID 14;
• b. the transglutaminase acceptor peptide sequence is or comprises a sequence selected from SEQ ID NO 15 to SEQ ID 29.

A composition comprising or essentially consisting of a hyaluronan polymer of general formula I,

• wherein 5% to 20%, particularly 8-12%, more particularly approximately 10% of R1 moieties are represented by a general formula II,
• wherein
• L is a linker moiety, particularly a linker consisting of 2, 3, 4, 5 or 6 C, N and/or O atoms in the linking chain, more particularly L is NH-Alk or O-Alk or NH—NH—CO-Alk with Alk being a C1 to C5 alkyl, even more particularly L is —N—NHCO—(CH2)2—, and —Pep is a transglutaminase donor peptide or a transglutaminase acceptor peptide, respectively, and the rest of R1 moieties are represented by COOH.