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WO2006008238A1 - Procede d'acylation selective - Google Patents

Procede d'acylation selective Download PDF

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Publication number
WO2006008238A1
WO2006008238A1 PCT/EP2005/053207 EP2005053207W WO2006008238A1 WO 2006008238 A1 WO2006008238 A1 WO 2006008238A1 EP 2005053207 W EP2005053207 W EP 2005053207W WO 2006008238 A1 WO2006008238 A1 WO 2006008238A1
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Prior art keywords
insulin
water content
reaction mixture
acylation
des
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English (en)
Inventor
Claus Ulrich Jessen
Louis Brammer Hansen
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Novo Nordisk AS
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Novo Nordisk AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins

Definitions

  • the present invention relates to a method for selective acylation of a free ⁇ -amino group in insulin or insulin analogues and precursors thereof.
  • Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.
  • Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.
  • a class of compounds suitable for this task is insulin derivatives in which the ⁇ - amino group in the lysine residue in position 29 of the B-chain is acylated with a hydrophobic moiety are disclosed in EP patents 792,290 and 894,095 and in US patent Nos. 5,693,609, 5,646,242, 5,922, 675, 5,750,497 and 6,011 ,007.
  • Human insulin and closely related insulins have three primary amino groups in the molecule namely the ⁇ -amino groups of Gly A1 and Phe B1 , respectively, and the ⁇ -amino group of Lys B29 .
  • N-Acylation of unprotected insulin may - depending on the conditions - lead to a complex mixture of mono-, di- and even triacylated products.
  • a cer ⁇ tain preference for acylation of a specific position can often be observed the preference is not always sufficiently pronounced to make such direct acylation useful as a method of produc ⁇ ing monoacylated insulins since the formation of the desired product may be accompanied by the formation of considerable amounts of closely related by-products. When by-products are formed, this happens at the expense of the desired product and may lead to problems in the purification of the desired product.
  • a suitable intermediate can be an insulin derivative in which the amino group(s) not to be acylated is (are) blocked with a pro- tection group which can be removed selectively after the acylation has been performed.
  • a protected intermediate can either be an insulin precursor or an insulin derivative in which it has been possible posttranslationally to introduce one or two protection groups in a specific way. For economic reasons, it is however very attractive to avoid the use of specific protec ⁇ tion groups if possible.
  • the present invention is related to an improved process for obtaining high yields of insulin being acylated in an ⁇ -amino group, in particular the ⁇ -amino group in Lys B29 .
  • the present invention is related to a method for selectively acylation of a free ⁇ -amino group of a lysine residue in insulin or insulin analogues or precur ⁇ sors thereof by reacting the insulin in question with an acylation agent under neutral condi ⁇ tions, i.e. without addition of a base, and at a water content in the reaction mixture of below about 5% w/w.
  • the water content in the reaction mixture is from about 0.01 to about 5% w/w.
  • the water content in the reaction mixture is from about 0.01 to about 4% w/w or from about 0.01 to about 2.5 w/w. In a further embodiment the water content in the reaction mixture is from about 0.1 to about 5, from about 0.1 to about 4,from about 0.1 to about 2.5% w/w or from about 0.1 to about 2% w/w .
  • the water content in the reaction mixture is from about 0.2 to about 5, from about 0.2 to about 4, from about 0.2 to about 2.5 or from about 0.2 to about 2% w/w.
  • the acylation agent is an activated ester or an activated amide.
  • the activated ester is an ONSu ester and in a still further aspect the activated ester is an ONSu ester of a fatty acid.
  • the activated amide is an azolide of the acid corresponding to the acyl group to be introduced and in a further aspect the azolide is benzotri- azole or substituted benzotriazole.
  • the acyl group may be selected from the group consisting of unbranched, aliphatic monocarboxylic acids having from 6 to 24 carbon atoms, dicarboxylic acids and lithocholic ac ⁇ ids and derivatives thereof.
  • the acyl group is a fatty acid and in par ⁇ ticular a long chain fatty acid having from 6 to 18 C-atoms, from 10 to 18 C-atoms or from 10 to 14 C-atoms.
  • the fatty acid is selected from the group consisting of capric acid, lauric acid, tetradecanoic acid (myristic acid), pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid.
  • the acylated insulin is N ⁇ B29 tetradecanoyl-des(B30) human insu ⁇ lin.
  • the acylated insulin is N ⁇ B29 -lithocholoyl- ⁇ -glutamyl des(B30) human insulin.
  • the acylated insulin is N ⁇ B29 -(N ⁇ -(HOOC(CH 2 ) 14 CO)- ⁇ - GIu) des(B30) human insulin or N ⁇ B29 -(N ⁇ -(HOOC(CH 2 )i 6 CO)- ⁇ -Glu) des(B30) human insulin.
  • the method according to the present invention may comprise the following steps: a) dissolving insulin or an insulin analogue or a precursor thereof in a polar organic solvent; b) adding an activated acylation agent and conducting the acylation at neutral pH at a tem ⁇ perature from about -2O 0 C to about 5O 0 C and at a water content in the reaction mixture below about 5% w/w at neutral pH; and c) isolating the product being acylated in the desired position.
  • the temperature may vary within a broad range. Suitable temperature ranges for step b) are ranges from about -20 0 C to about 5O 0 C, from about -20 0 C to about 40 0 C, from about -20° t C to about 30 0 C, from about 0 0 C to about 50 0 C, from about 0 0 C to about 40 0 C, from about 0 0 C to about 35 0 C, from about 2O 0 C to about 50 0 C, from about 2O 0 C to about 4O 0 C and from about 20 0 C to about 35 0 C.
  • the starting insulin product to be acylated may typically be dried in a well known manner before it is acylated in the method according to the invention.
  • the insulin or insulin analogue or a precursor thereof is dried to a water content of less that about 10% w/w before being dissolved in the reaction medium (step a) and in another embodiment the insulin or insulin analogue or a precursor thereof is dried to a water content of about 4 to about 5% w/w before being dissolved in the reaction mixture (step a).
  • a suitable drying agent may be added to the reaction mixture before adding the acylation agent.
  • a chemical desiccant is added before the acylation step (step b) to reduce the water content to below about 5 or about 2.5% w/w.
  • the solution from step a) is passed through a physical desiccant before step b) to reduce the water content to below about 5 to 2.5% w/w.
  • the physical desiccant can be a molecular sieve or a silica gel.
  • the polar organic solvent may be any suitable solvent such as DMSO, DMF, NMP and sulpholane.
  • insulin is intended to include human insulin, porcine insulin, insulin ana ⁇ logues and insulin derivatives derived from insulin and having the molecular structure similar to that of human insulin including the disulfide bridges between Cys A7 and Cys B7 and be ⁇ tween Cys ⁇ 0 and Cys B19 and an internal disulfide bridge between Cys A6 and Cys A11 to pre- serve the insulin activity.
  • An insulin analogue is an insulin molecule having one or more mutations, substitu ⁇ tions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule.
  • the insulin analogues are preferably such wherein one or more of the natu ⁇ rally occurring amino acid residues, preferably one, two, or three of them, have been substi- tuted by another codable amino acid residue.
  • position 28 of the B chain may be modi ⁇ fied from the natural Pro residue to one of Asp, Lys, or He.
  • Lys at po ⁇ sition B29 is modified to Pro.
  • Asn at position A21 may be modified to Ala, GIn, GIu, GIy, His, He, Leu, Met, Ser, Thr, Trp, Tyr or VaI, in particular to GIy, Ala, Ser, or Thr and pref ⁇ erably to GIy.
  • Asn at position B3 may be modified to Lys.
  • Further examples of insulin analogues are insulin analogues wherein PheB1 or ThrB30 have been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension.
  • one or two Arg may be added to position B1.
  • insulin precursor is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into insulin.
  • An example of a precursor is an insulin precursor with an amino acid sequence B(1 -29)-Ala-Ala-Lys-A(1 - 21) wherein A(1-21) is the A chain of human insulin and B(1-29) is the B chain of human in- sulin in which Thr(B30) is missing.
  • This insulin precursor may be converted in human insulin by enzymatic cleaving off the Ala-Ala-Lys bridge which connects the amino acid residue in position B29 with the amino acid in position A21 , and enzymatic coupling of a Thr amino acid to the B29 amino acid residue.
  • insulin precursors may comprise an N-terminal exten ⁇ sion to the B-chain which is then later on cleaved off by suitable enzymatic or chemical treat- ment, see US patent No. 6521738, WO 97/22706, WO 97/00581 and WO 00/04172.
  • connecting peptide or "C-peptide” is meant the connection moiety "C" of the B-
  • C-A polypeptide sequence of a single chain preproinsulin-like molecule Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the
  • desB30 or “B(1-29) is meant a natural insulin B chain lacking the B30 amino acid residue
  • A(1-21) means the natural insulin A chain
  • B(1-27) means the natural B chain lacking the B28, B29, and B30 amino acid residues etc.
  • acylation is meant an acylation which occurs in a de- sired position at a higher degree, preferably at least at two or three times higher degree than in a not desired position.
  • acylation should preferably only take place in the ⁇ -amino group in the Lys B29 and not in the two N-terminal ⁇ - amino groups at the two chain insulin precursor. The ratio between the mono acylated
  • LysB29 and mono acylated A1 and B1 should be higher than about 4 to about 1, preferably higher than about 10 to about 1.
  • activated acylation agent means an acylation agent which has been acti ⁇ vated using general techniques described in Methods of Enzymology 25, 494-499 (1972). "Neutral” pH is meant to cover a pH value of from about 6.5 to about 8.
  • the present method provides a method of securing a selective acylation in a desired position of an insulin molecule.
  • a selective acylation can be ob ⁇ tained at neutral pH.
  • Neutral pH is advantageous because the less harsh conditions have turned out to give an impurity profile of the acylation reaction mixture which enables a much easier purification and isolation of the desired selectively acylated product from non reacted acylating agent, insulin mono acylated in B1 or A1 and di- and triacylated insulin by-products.
  • An adequately low water content in the reaction mixture con be obtained by either using reactants with a low water content or to remove the excessive water from one or more of the reactants before the acylation reaction.
  • the insulin starting product to be acylated can be dried to a suitable water content. Drying of the insulin starting product can be conducted in any convenient way but is typically conducted in vacuum at room tem ⁇ perature. Alternatively the insulin starting product can be dried under conventional freeze drying conditions.
  • the insulin starting product is typically dried to a water content of less than about 10 % w/w, in particular to a water content below about 6-8% w/w and preferably to a water con ⁇ tent as low as about 4-5% w/w before it is acylated by the claimed method that is before it is dissolved in the polar solvent in step a).
  • the low water content in the reaction mixture can also be obtained by use of a suit ⁇ able chemical desiccant, such as silyl compounds like trimethylchlorsilan, or by use of physi ⁇ cal desiccants such as molecular sieves or silica gels.
  • the polar organic solvent may be any suitable polar solvent such as DMSO, DMF, NMP and sulpholane and mixtures thereof.
  • anhydrous solvent as the polar organic solvents used in the present method still may contain a low amount of water and because it may be difficult to conduct the acylation reaction under conditions where only minimal water is absorbed from the surrounding it may also be advisable to add a desiccant during the acylation step.
  • the acyl group to be introduced into the ⁇ -amino group of a Lys residue is the acyl group of a monocarboxylic acid of the general formula (I):
  • this type are the acyl group of an unbranched, aliphatic monocarboxy ⁇ c acid having from 6 to 24 carbon atoms, in particular an acyl group selected from the group comprising CH 3 (CH 2 ) S CO-, CH 3 (CH 2 ) 9 CO-, CH 3 (CHa) 10 CO-, CH 3 (CH 2 )iiCO-, CH 3 (CH 2 ) 12 CO-, CH 3 (CH 2 ) 13 CO-, CH 3 (CH 2 ) I4 CO-, C H 3 (CH 2 ) 15 C0-, CH 3 (CH 2 ) 16 CO-, CH 3 (CH 2 )I 7 CO-, CH 3 (CH 2 ) 18 CO-, CH 3 (CH 2 ) 19 CO-, CH 3 (CH 2 ) 20 CO-, CH 3 (CH 2 )
  • the acyl group to be introduced into the ⁇ -amino group of a Lys may also be an acyl group of a dicarboxylic acid of the general formula (II):
  • D is a long chain hydrocarbon group which may optionally be interrupted by one or more groups each independently selected from the group consisting of an oxygen atom and a sulphur atom.
  • the acyl group is one of the acyl groups of a dicarboxylic acid of the general formula (II) wherein D is an unbranched, divalent aliphatic hydrocarbon group having from 6 to 22 carbon atoms, in particular an acyl group selected from the group comprising HOOC(CH 2 ) 6 CO-, HOOC(CH 2 ) 8 CO-, HOOC(CH 2 ) 10 CO-, HOOC(CH 2 ) 12 CO-, HOOC(CH 2 ) I4 CO-, HOOC(CH 2 ) 16 CO-, HOOC(CH 2 ) 18 CO-, HOOC(CH 2 ) 2 oCO- and HOOC(CH 2 ) 22 CO-.
  • the acyl group to be introduced into the ⁇ -amino group of a Lys residue is a group of the general formula (III):
  • x is an integer from 8 to 24 and R 1 is hydrogen or a group which can be exchanged with hydrogen after the acylation has been performed, for example methyl, ethyl or terf-butyl.
  • Preferred values of x are 10, 12 and 14.
  • the ester group of which R 1 is a part can be hydrolysed to give the corresponding free acid and the alcohol R 1 OH by methods known per se.
  • R 1 is methyl or ethyl
  • the hydrolysis can be performed under alkaline conditions and when R 1 is tert- butyl, the hydrolysis can be carried out using trifluoroacetic acid.
  • the acyl group is connected to the lysine residue using an amino acid linker.
  • the acyl group is connected to a lysine residue via a ⁇ - or an ⁇ -glutamyl linker, or via a ⁇ - or an ⁇ -aspartyl linker, or via an ⁇ -amido- ⁇ -glutamyl linker, or via an ⁇ -amido- ⁇ -aspartyl linker.
  • the acyl group is lithocholoyl-Glu(OH)-OR, wherein OH represent the free carboxyl group of the ⁇ -carbon and OR is an ester group protecting the ⁇ -carbon of glutamic acid wherein R is methyl, ethyl, ortert. butyl.
  • the acyl group to be introduced into the ⁇ -amino group of a Lys residue is a group of the general formula (IV):
  • R 2 is hydrogen or a group which can be exchanged with hydrogen after the acylation has been performed, for example methyl, ethyl or fert-butyl.
  • the ester group of which R 2 is a part can be hydrolysed to give the corresponding free acid and the alcohol R 2 OH by methods known perse.
  • R 2 is methyl or ethyl
  • the hydrolysis can be performed under alkaline conditions and when R 2 is ferf-butyl, the hydrolysis can be carried out using trifluoroacetic acid.
  • the acylating agent can be any suitable acylating agent capable of introducing an acyl group in the desired position in the insulin molecule. Suitable acylating agents are acti ⁇ vated esters and activated amides. Further suitable acylating agents are anhydrides and hal- ides.
  • Activated esters are well known and can be prepared using general techniques de- scribed in Methods of Enzymology 25, 494-499 (1972). The most commonly used activated ester type is ONSu esters.
  • the acylation according to the present invention may also be carried out using an acylating agent which is an activated amide, more particularly an azolide of the acid corresponding to the acyl group to be introduced.
  • an acylating agent which is an activated amide, more particularly an azolide of the acid corresponding to the acyl group to be introduced.
  • azolides (1-acylazoles) can be prepared according to known methods, see for example Staab HA Angew. Chem. 74 (1962) 407-423.
  • Examples of azoles which can be used in the preparation of the acylating agents of this invention are pyrazole, imidazole, 1 ,2,3-triazole, 1 ,2,4-triazole, tetrazole, phenyltetrazole and - where possible - the corresponding benzanelated compounds e.g.
  • azoles for the preparation of the acylating agent are 1 ,2,4-triazole, benzotriazole and substituted benzotriazoles.
  • the azoles mentioned can be substituted with one or more substituents selected from the group comprising alkyl (Ci - C 5 , branched or unbranched, in particular methyl, ethyl, propyl and isopropyl), halogen (e.g.
  • acylating agents are 1 -acyl benzotriazoles and a preferred acylating agent is 1 -tetradecanoyl benzotriazole.
  • Suitable 1 -acyl benzotriazoles are such which are derived from mono- or disubstituted benzotriazoles, e.g. 5-substituted or 6-substituted or 5,6-disubstituted benzotriazoles such as 5-methylbenzotriazole, 5-chloroben ⁇ otriazole, 6- chloroben ⁇ otria ⁇ ole, 5-nitroben ⁇ otriazole, 5,6-dimethylbenzotriazole and 5,6-dichlorobenzo- triazole.
  • 5-substituted or 6-substituted or 5,6-disubstituted benzotriazoles such as 5-methylbenzotriazole, 5-chloroben ⁇ otriazole, 6- chloroben ⁇ otria ⁇ ole, 5-nitroben ⁇ otriazole, 5,6-dimethylbenzotriazole and 5,6-dichlorobenzo- triazole.
  • the lower limit of the temperature at which the acylation can be carried out is determined by the freezing point of the medium while the upper limit is determined by the temperature at which the starting insulin or the acylated insulin will deteriorate. This again will depend Ia. on the composition of the medium.
  • the reaction may be possible to carry out the reaction at temperatures between about -20 0 C and about 50 0 C it is usually most convenient to carry out the reaction at a temperature between about -5 0 C and about 3O 0 C.
  • the starting product for the acylation, the insulin or insulin analogue or a precursor thereof can be produced by either well-know peptide synthesis or by well known recombinant production in suitable transformed microorganisms.
  • a human insulin precursor B(1 -29)-Ala-Ala-Lys-A(1 -21 ) may e.g. be produced in yeast as disclosed in US patent No. 4,916,212.
  • This insulin precursor can then be converted into desB30 human insulin by ALP cleavage of the Ala-Ala-Lys peptide chain to give desB30 human insulin which can then be acylated according to the present method.
  • Isolation and purification of the acylated products prepared by the method of the present invention can be carried out by methods known per se, including gel filtration, crystallization and chromatography.
  • Des(B30) human insulin (4.0 g, ⁇ 0.60 mmol) with a water content of 4.7% w/w was dissolved in dimethylsulfoxide (170 ml) at 22 0 C.
  • the pH of the solution was checked using water wet indicator paper and found to be between 6 and 7.
  • Tetradecanoic acid 2,5-dioxo- pyrrolidin-1-yl ester (ONSu ester) (0.22g (0.066mmol) dissolved in 12 ml dimethylsulfoxide) was added within 3 minutes.
  • the reaction mixture was stirred at 22 0 C for 30 minutes and then quenched by adding water (170 ml).
  • the reaction mixture was analyzed by RP-HPLC with the following results:
  • Des(B30) human insulin (1.16 g, -0.18 mmol) with a water content of 4,7% w/w was dissolved in dimethylsulfoxide (60 ml) at 22 0 C.
  • the pH of the solution was checked using wa ⁇ ter wet indicator paper and found to be between 6 and 7.
  • N-lithocholyl-glutamo-5-yl methyl ester O-hydroxy succinimide (0.119g (0.019mmol) was added in one portion.
  • the reaction mixture was stirred at 22 0 C for 10 minutes.
  • An in-process sample showed the following re ⁇ sults:
  • Des(B30) human insulin (1.2 g, ⁇ 0.20 mmol) with a water content of about 5% w/w was dissolved in dimethylsu If oxide (21 ml) at about 2O 0 C.
  • the pH of the solution was checked using water wet indicator paper and found to be between 6 and 7.
  • Tetradecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (74 mg ⁇ 0.23mmol) was added.
  • the reaction mixture was stirred at 22 0 C for 1 hour and then quenched by adding water.
  • the reaction mixture was ana ⁇ lyzed by RP-HPLC with the following results:
  • N ⁇ B29 -tetradecanoyl des(B30) human insulin 56% Di and tri acylated des(B30) human insulin 27 %
  • Des(B30) human insulin was dried for 6 days at 22-24 0 C under vacuum (5-10 hP).
  • the water content determined by Karl Fischer was found to be 1.4% w/w.
  • Des(B30) human insulin (1.0 g, ⁇ 0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 21 0 C.
  • the pH of the solution was checked using wa- ter wet indicator paper, and found to be between 7.5 and 8. An aliquot of the solution was diluted with tree times the volume of pH 7.0 demineralised water. The pH of the solution was found to be 7.91 using a calibrated pH-electrode.
  • Acetic acid (81 microliter of a 40.6 milli mo ⁇ lar acetic acid in dimethylsulfoxide, ⁇ 0.15 mmol) was added, and the pH of the solution was found to be 7.20 using a calibrated pH-electrode.
  • Tetradecanoic acid 2,5-dioxo-pyrrolidin-1 -yl ester 64 mg, ⁇ 0.20mmol dissolved in 3 ml dimethylsulfoxide was added within 3 minutes. The reaction mixture was stirred at RT for 45 minutes, and then quenched by adding water (43 ml).
  • reaction mixture was analyzed by RP-HPLC with the following results:
  • N ⁇ BZ9 -tetradecanoyl des(B30) human insulin 60%
  • Des(B30) human insulin (1.0 g, ⁇ 0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 22°C.
  • Acetic acid (240 microliter of a 40.6 milli molar acetic acid in dimethylsulfoxide, -0.45 mmol) was added.
  • An aliquot of the slightly cloudy so ⁇ lution was diluted with three times its volume of water, and the pH was found to be 6.0-6.5 using wet indicator paper, and 6.48 using a calibrated pH-electrode.
  • Tetradecanoic acid 2,5- dioxo-pyrrolidin-1 -yl ester 64 mg, ⁇ 0.20mmol dissolved in 3 ml dimethylsulfoxide was added within 3 minutes.
  • the reaction mixture was stirred at RT for 45 minutes, and then quenched by adding water (43 ml).
  • the reaction mixture was analyzed by RP-HPLC with the following results:
  • N ⁇ B29 -tetradecanoyl des(B30) human insulin 57%
  • Des(B30) human insulin (1.0 g, ⁇ 0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 33-36 0 C.
  • Tetradecanoic acid 2,5-dioxo-pyrrolidin-1 - yl ester 54 mg, ⁇ 0.17mmol dissolved in 3 ml dimethylsulfoxide was added within 3 minutes. The reaction mixture was stirred at 33-36°C for 45 minutes, and then quenched by adding water (43 ml).
  • N A1 -tetradecanoyl des(B30) human insulin 1.4%
  • N ⁇ B29 -tetradecanoyl des(B30) human insulin 75%
  • N ⁇ B29 -tetradecanoyl des(B30) human insulin 61 % Di and tri acylated des(B30) human insulin 28 %
  • the analytical reverse phase HPLC (RP-HPLC) system consisted of a LiChroSpher column run at 50 0 C using an acetonitrile/water gradient at pH2.5 and recorded at 214 nm. The obtained conversions are expressed as area percentage.
  • acylated Des-B30 insulins Purification of the acylated Des-B30 insulins is carried out using anion exchange chromatography (1 x25 cm column) with Source 3OQ (polystyrene based mono disperses 30 ⁇ m particle) from Amersham Bioscience.
  • the acylation mixture is diluted with one volume of water and applied on the anion exchange column.
  • the column is pre-equilibrated in an etha- nol containing Tris buffer at neutral pH. Unbound material is washed out of the column and bound components are subsequently eluted using a linear gradient of ammonium acetate in ethanol and Tris. Fractions are collected and analysed for HPLC-purity. Pure fractions are pooled and analysed for purity and concentration to determine product purity and step yield.
  • the method is based on a RP-HPLC method at pH 2.5 using a mobile phase com- prised of ammonium sulphate. Separation of components is as carried out using an isocratic elutioji with acetonitrile as organic modifier. Detection of components is ,done at 214 nm. Pu ⁇ rity of acylated Des-B30 and the content of impurities are given in percentage by area. De ⁇ termination of the compound concentration is done using an internal standard.

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Abstract

L'invention concerne un procédé simple et efficace d'acylation sélective d'insuline ou d'analogues d'insuline ou de précurseurs de ceux-ci sur un groupe T-amino libre dans un résidu de l'acide aminé lysine dans la molécule d'insuline. La réaction d'acylation est réalisée dans des conditions de réaction douces à pH neutre et avec une faible teneur en eau dans le mélange de la réaction d'acylation.
PCT/EP2005/053207 2004-07-16 2005-07-05 Procede d'acylation selective Ceased WO2006008238A1 (fr)

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Cited By (27)

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WO2011161125A1 (fr) 2010-06-23 2011-12-29 Novo Nordisk A/S Dérivés d'insuline contenant des liaisons disulfure supplémentaires
US8569231B2 (en) 2009-03-20 2013-10-29 Smartcells, Inc. Soluble non-depot insulin conjugates and uses thereof
US8623345B2 (en) 2009-03-20 2014-01-07 Smartcells Terminally-functionalized conjugates and uses thereof
US8710000B2 (en) 2007-11-08 2014-04-29 Novo Nordisk A/S Insulin derivative
US8722620B2 (en) 2006-02-27 2014-05-13 Novo Nordisk A/S Insulin derivatives
US8796205B2 (en) 2006-05-09 2014-08-05 Novo Nordisk A/S Insulin derivative
US8846103B2 (en) 2009-01-28 2014-09-30 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
US8906850B2 (en) 2009-01-28 2014-12-09 Smartcells, Inc. Crystalline insulin-conjugates
US8933207B2 (en) 2010-07-28 2015-01-13 Smartcells, Inc. Drug-ligand conjugates, synthesis thereof, and intermediates thereto
US8933021B2 (en) 2006-05-09 2015-01-13 Novo Nordisk A/S Insulin derivative
US8940690B2 (en) 2009-01-28 2015-01-27 National Institutes Of Health (Nih) Synthetic conjugates and uses thereof
FR3013049A1 (fr) * 2013-11-14 2015-05-15 You-Ping Chan Analogue de l'insuline glargine
US9034818B2 (en) 2007-06-13 2015-05-19 Novo Nordisk A/S Pharmaceutical formulations comprising an insulin derivative
US9050370B2 (en) 2009-01-28 2015-06-09 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9068013B2 (en) 2010-07-28 2015-06-30 Smart Cells, Inc. Recombinant lectins, binding-site modified lectins and uses thereof
US9074015B2 (en) 2010-07-28 2015-07-07 Smartcells, Inc. Recombinantly expressed insulin polypeptides and uses thereof
US9427475B2 (en) 2013-10-04 2016-08-30 Merck Sharp & Dohme Corp. Glucose-responsive insulin conjugates
USRE46170E1 (en) 2005-02-02 2016-10-04 Novo Nordisk A/S Insulin derivatives
US9603904B2 (en) 2008-10-30 2017-03-28 Novo Nordisk A/S Treating diabetes melitus using insulin injections with less than daily injection frequency
US10040839B2 (en) 2014-02-28 2018-08-07 Novo Nordisk A/S Insulin derivatives and the medical uses hereof
US10137172B2 (en) 2013-04-30 2018-11-27 Novo Nordisk A/S Administration regime
WO2019034726A1 (fr) * 2017-08-17 2019-02-21 Novo Nordisk A/S Nouveaux analogues d'insuline acylés et leurs utilisations
US10335464B1 (en) 2018-06-26 2019-07-02 Novo Nordisk A/S Device for titrating basal insulin
WO2019222072A1 (fr) * 2018-05-15 2019-11-21 Savior Lifetec Corporation Procédé de préparation d'un dérivé du liraglutide
US10596229B2 (en) 2010-10-27 2020-03-24 Novo Nordisk A/S Method of treating diabetes mellitus by administration, at specifically defined intervals, of a derivative of a naturally occurring insulin or insulin analogue, the derivative having a prolonged profile of action
US11167035B2 (en) 2005-12-28 2021-11-09 Novo Nordisk A/S Insulin compositions and method of making a composition
US12343383B2 (en) 2019-07-12 2025-07-01 Novo Nordisk A/S High concentration insulin formulation

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Cited By (35)

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USRE46170E1 (en) 2005-02-02 2016-10-04 Novo Nordisk A/S Insulin derivatives
US11167035B2 (en) 2005-12-28 2021-11-09 Novo Nordisk A/S Insulin compositions and method of making a composition
US8722620B2 (en) 2006-02-27 2014-05-13 Novo Nordisk A/S Insulin derivatives
US8796205B2 (en) 2006-05-09 2014-08-05 Novo Nordisk A/S Insulin derivative
US8933021B2 (en) 2006-05-09 2015-01-13 Novo Nordisk A/S Insulin derivative
US9034818B2 (en) 2007-06-13 2015-05-19 Novo Nordisk A/S Pharmaceutical formulations comprising an insulin derivative
US8710000B2 (en) 2007-11-08 2014-04-29 Novo Nordisk A/S Insulin derivative
US9603904B2 (en) 2008-10-30 2017-03-28 Novo Nordisk A/S Treating diabetes melitus using insulin injections with less than daily injection frequency
US8846103B2 (en) 2009-01-28 2014-09-30 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
US8906850B2 (en) 2009-01-28 2014-12-09 Smartcells, Inc. Crystalline insulin-conjugates
US8940690B2 (en) 2009-01-28 2015-01-27 National Institutes Of Health (Nih) Synthetic conjugates and uses thereof
US10398781B2 (en) 2009-01-28 2019-09-03 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9050370B2 (en) 2009-01-28 2015-06-09 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9579391B2 (en) 2009-01-28 2017-02-28 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9463249B2 (en) 2009-01-28 2016-10-11 Smartcells, Inc. Crystalline insulin-conjugates
US8569231B2 (en) 2009-03-20 2013-10-29 Smartcells, Inc. Soluble non-depot insulin conjugates and uses thereof
US8623345B2 (en) 2009-03-20 2014-01-07 Smartcells Terminally-functionalized conjugates and uses thereof
WO2011161125A1 (fr) 2010-06-23 2011-12-29 Novo Nordisk A/S Dérivés d'insuline contenant des liaisons disulfure supplémentaires
US9074015B2 (en) 2010-07-28 2015-07-07 Smartcells, Inc. Recombinantly expressed insulin polypeptides and uses thereof
US9068013B2 (en) 2010-07-28 2015-06-30 Smart Cells, Inc. Recombinant lectins, binding-site modified lectins and uses thereof
US8933207B2 (en) 2010-07-28 2015-01-13 Smartcells, Inc. Drug-ligand conjugates, synthesis thereof, and intermediates thereto
US10596229B2 (en) 2010-10-27 2020-03-24 Novo Nordisk A/S Method of treating diabetes mellitus by administration, at specifically defined intervals, of a derivative of a naturally occurring insulin or insulin analogue, the derivative having a prolonged profile of action
US10137172B2 (en) 2013-04-30 2018-11-27 Novo Nordisk A/S Administration regime
US9884125B2 (en) 2013-10-04 2018-02-06 Merck Sharp & Dohme Corp. Glucose-responsive insulin conjugates
US9889205B2 (en) 2013-10-04 2018-02-13 Merck Sharp & Dohme Corp. Glucose-responsive insulin conjugates
US9427475B2 (en) 2013-10-04 2016-08-30 Merck Sharp & Dohme Corp. Glucose-responsive insulin conjugates
WO2015071368A1 (fr) * 2013-11-14 2015-05-21 You-Ping Chan Analogue de l'insuline glargine à action prolongée
FR3013049A1 (fr) * 2013-11-14 2015-05-15 You-Ping Chan Analogue de l'insuline glargine
US10040839B2 (en) 2014-02-28 2018-08-07 Novo Nordisk A/S Insulin derivatives and the medical uses hereof
US10919949B2 (en) 2017-08-17 2021-02-16 Novo Nordisk A/S Acylated insulin analogues and uses thereof
WO2019034726A1 (fr) * 2017-08-17 2019-02-21 Novo Nordisk A/S Nouveaux analogues d'insuline acylés et leurs utilisations
WO2019222072A1 (fr) * 2018-05-15 2019-11-21 Savior Lifetec Corporation Procédé de préparation d'un dérivé du liraglutide
US10335464B1 (en) 2018-06-26 2019-07-02 Novo Nordisk A/S Device for titrating basal insulin
US12226458B2 (en) 2018-06-26 2025-02-18 Novo Nordisk A/S System for providing an up-to-date and long-acting or ultra-long-acting insulin dose guidance recommendation to treat diabetes mellitus
US12343383B2 (en) 2019-07-12 2025-07-01 Novo Nordisk A/S High concentration insulin formulation

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