Process for the Preparation of Insulin-Zinc Complexes

A process for preparing a pharmaceutical formulation comprising an insulin derivative, wherein the process comprises:

• (a) dissolving an insulin derivative in water,
• (b) adjusting the pH solution to a pH above 7.2,
• (c) adding a zinc solution during a period longer than seven minutes while stirring continuously,
• (d) adjusting the pH to the target pH of the formulation, and
• wherein the insulin derivative comprises an insulin molecule having a side chain attached to the ε-amino group of a Lys residue present in the B chain of human insulin or an analogue thereof, the side chain being of the general formula:
• —W—X—Y—Z
• wherein W is:
• an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;
• a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain;
• or
• a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;
• X is:
• —CO—;
• —CH(COOH)CO—;
• —CO—N(CH2COOH)CH2CO—;
• —CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;
• —CO—N(CH2CH2COOH)CH2CH2CO—;
• —CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;
• —CO—NHCH(COOH)(CH2)4NHCO—;
• —CO—N(CH2CH2COOH)CH2CO—;
• or
• —CO—N(CH2COOH)CH2CH2CO—;
• that
• a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or
• b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;
• Y is:
• —(CH2)m— where m is an integer in the range of 6 to 32;
• a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32;
• and
• Z is:
• —COOH;
• —CO-Asp;
• —CO-Glu;
• —CO-Gly;
• —CO-Sar;
• —CH(COOH)2;
• —N(CH2COOH)2;
• —SO3H;
• or
• —PO3H.