A PROCESS FOR PREPARING AN INJECTABLE INSULIN PREPARATION

Abstract: A quick-acting, injectable insulin preparation of low antigenicity is obtained by a chromatographic process during which the insulin is transferred to an eluant consisting of the same carrier medium as forms part of the desired insulin preparation, or one or more components of said carrier medium. The insulin-containing eluate is admixed with any residual medium components and is adjusted to the desired pH and insulin concentration to provide the finished insulin preparation.

Description: A process for preparing an injectable insulin prepara¬ tion Diabetes may be treated with various types of insulin preparations which may be characterized according to their action, in time as being quick-acting, slow- acting, or as a mixed preparation of intermediary action. The present invention relates to a process for prepa¬ ring a quick-acting injectable insulin preparation of low antigenicity which is a clear solution at neutral pH, comprising subjecting an insulin solution to a chromatographic process. The process is charac¬ terized in that during the chromatographic process the insulin is transferred to an eluant consisting of the same carrier medium as forms part of the de¬ sired insulin preparation or one or more components of said carrier medium following which the insulin con¬ taining eluate, without isolation of the insulin, is admixed with any residual medium components and is adjusted to the desired pH value and insulin con¬ centration to provide the finished preparation. Preparations of intermediary action can be obtained . by a suitable selection of medium components, e.g. by using protamine or another -base containing amino groups, and preparations of intermediary action can be obtained by mixing a quick-acting preparation produced in accordance with the invention with slow- acting preparations. The preparation produced by the process of the in- vention has a surprisingly low antigenicity compared with corresponding preparations which are produced by precipitation of purified insulin from an eluate, zinc crystallization and redissolution of the insulin in a carrier medium. Even though particular interest has been focussed on the slow-acting preparations in the last decades, be¬ cause the individual patient requires fewer injections per day, there.are still fields in which the quick- acting insulin preparations have a raison d'etre. A case,-in point is the use in connection with chock treatments in " psychiatry, in acute emergencies in diabetes therapy, and of course not least for mixing with the slow-acting preparations, providing the mixed preparations mentioned in the foregoing. Finally, attention should be called to the use in connection ' with controlled administration of insulin by means of adjustable pumps where new developments will undoubted¬ ly arouse great interest in particularly neutral and quick-acting insulin preparation in clear solution. It is known that commercial zinc crystallized insulin contains antigenic impurities, and that they may be removed by column chromatography in a. manner known per se, e.g. as described in the Danish Patent no. 129686 (cf. the US Patent No. 3 907 676) which re¬ lates to a process for preparing pure insulin, the resulting product being essentially freed of proteins having a molecular weight above that of the insulin (6000) and of various insulin-like proteins having approximately the same molecular weight as insulin so that this product essentially contains only pure insulin evaluated by DISC-PAGE. Zinc crystallized insulin of this purity may be used for producing quick-acting as well ' as slow-acting pre- parations, in a conventional manner. Clinical tests, conducted in different parts of the world and also by the applicants, show that the an¬ tigenicity of the quick-acting preparations can be greatly reduced by using insulin of said purity for conventional production of preparations. On the other hand, the reduction is less pronounced in respect of the slow-acting preparations, as far as porcine insulin preparations are concerned, while - generally speaking - there is none at all in case of preparations prepared from bovine insulin. The Danish Patent No. 140801 (cf. the US Patent No. 4183849) describes a process for preparing slow- acting insulin preparations of greatly reduced anti- genicity. The process of Danish Patent No. 140 801 is characteristic in that purified insulin is reacted with an organic base containing basic amino groups, such as protamine, the reaction being carried out in a buffer which contains a stabilizer that maintains the insulin in a monomeric form, and that the reaction with the basic organic compound fixes the insulin in a stable monomeric form which is a constituent part of the preparation. The present invention is based on the finding that it is possible - from insulin of the purity mentioned in e.g. the Danish Patent No. 129 686 - to prepare quick- acting insulin preparations of unprecedented low an¬ tigenicity, evaluated by immunization tests on rabbits, see figs. 1-4 and the discussion of them below. Com- pared with the known process the present process is very simple and is particularly unique in that the injectable insulin preparation is an eluate produced by a chromatographic process, with adjustment, if ne- cessary, of the eluate to neutral pH and readjust¬ ment, if necessary, to obtain the desired composi¬ tion. It is surprising that this reduction in antigenicity can be achieved without using stabilizers in the last phases of the production of the insulin preparation and after the formation of the finished preparation, and it has not yet been possible to find an adequate explanation of the reduction in antigenicity achieved by the process. A preferred embodiment of the invention is characterized by the fact that an insulin-containing fraction, from which antigenic impurities have been wholly or partly removed by a chromatographic purification, is trans- ferred to the carrier medium by a chromatographic process following which the product is readjusted, if necessary, to provide the desired pH and composition. Another preferred embodiment is characterized by the fact that insulin, which may be less pure than describ- ed above, is subjected to a chromatographic purifica¬ tion during which the insulin is eluted with the car¬ rier medium or one or more components thereof, the insulin-containing fraction is readjusted, if neces¬ sary, to provide the desired pH and composition. Thus, the invention is not restricted to the use of insulin of a specific purity as starting material, but it is of course necessary that the insulin in the finished product is wholly or partly freed of impuri¬ ties which are antigenic per se, particularly compo- nents having a molecular weight above 6000, as it is desired to obtain preparations whose antigenicity is low so as to suit the purpose. The process of the invention is illustrated in more detail in the examples below. Examples A and B show how suitable chromatographic fractions may be produced in a. manner known per se, while examples 1, 2 and 5 show how neutral, quick-acting preparations in clear solution can be obtained by the process of the in¬ vention,, using said chromatographic fractions as star¬ ting material. Examples 3 and 4 illustrate embodiments of the process of the invention wherein the chro ato- graphic purification directly yields eluates which provides suitable injectable preparations only by simple adjustment of the concentration of the com¬ ponents and of pH. Example 6 concerns some modifi¬ cations of pH and the conditions of elution in the production of a final product of the same type as those prepared in the preceding examples. Examples 7 and 8 illustrate two different embodiments of the use of protamine as additive. This provides so-called intermediary insulin preparations since the addition brings about a prolonged action, depending upon the amount of protamine. The amount of protamine added (in the form of protamine insulin in example 7 and pro¬ tamine sulfate in example 8) is kept so low, however, ' that the finished preparation still contains a con- siderable amount of quick-acting insulin. Example A 250 mg of freeze dried insulin, which were gel fil¬ trated on "SEPHADEX® " G 50 ( a cross-linked dextran gel) in 1 M acetic acid in.a conventional manner, were dissolved at 4°C in a 5.2 ml buffer solution consis¬ ting of 7 M deionized urea and 0.02 M tris of a pH of 8.1. The solution was mixed with 5.2 ml of 7 M urea solution, and the pH of the solution was readjusted to 8.1. A column of a diameter of 5 cm was packed with a 2.1 cm layer of DEAΞ cellulose ("Whatman" DE 52) and equilibrated with a buffer solution of said com¬ position. The insulin solution was applied to the co- lumn which was eluted at 4°C at a rate of 75 ml per hour according to following scheme: 2.5 hours with a buffer of said composition, 2 hours with a buffer of said composition, admixed with 0.0045 M sodium chloride pe litre, 12 hours with a buffer of said composition, admixed wit 0.011 M sodium chloride per litre. The eluate was divided into fractions. The highly purified fractions were pooled, and the content of insulin was determined. Example B A column of a diameter of 5 cm was packed at 4°C with an 85 " cm layer of "SEPHADEX ® " G 50 Superfine (a cross- linked dextran gel) which was swollen in a buffer con¬ taining 0.01 M tris(hydroxymethyl)amino methane, 0.1 M sodium chloride and 7 M deionized urea, and which was adjusted to a pH of 8.5 with hydrochloric acid. To 30 ml of said buffer were added 32 mg of ethylene diamine tetraacetic acid disodium salt, and 800 mg of crystalline insulin were dissolved in this mixture. The pH value of the resulting solution was readjusted to 8.5, and the solution was then applied to the co¬ lumn. Elution was effected at 4°C with said buffer at a rate of 60 ml/hour, and the eluate was divided into fractions. The extinction at 277 nm was measured, and the fractions containing the central portion of the main peak were pooled. Example 1 A column of a diameter of 2.6 cm was packed at 4°C with a 45 cm layer of "SEPHADEX^ " G 25 fine (a cross-linked dextran gel) which was ' swollen in a buffer of pH = 8.3, consisting of 1/60 M disodium phosphate and 0.15 M so¬ dium chloride. Into 50 ml of urea-containing insulin solution, produced by the procedure of example A, was introduced a zinc chloride solution in an amount cor- responding to 7 mg of zinc per g of insulin, and the mixture was applied to the column. The column was ... eluted with said buffer at 4°C at a rate of 30 ml per hour. The eluate was divided into fractions. The insulin- containing fractions were pooled, and the insulin con¬ tent was determined. There was added 1/4 volumen of a solution containing 0.012 M hydrochloric acid and 0.1 M -cresol, and the pH of the solution was readjusted to 7.3. By addition of a buffer containing 1/75 M disodium phosphate, 0.12 M sodium chloride and 0.02 M m-cre¬ sol and adjusted to pH = 7.3 with hydrochloric acid, the insulin concentration of the solution was adjusted to ' 40 IU/ml and filled in vials after sterile filtra- tion. Example 2 A column of a diameter of 5 cm was packed at 4°C with 30 cm layer of "BIO-GEL P-6" (100 to 200 mesh) (a po- lyacrylamide gel) which was swollen in a buffer con- sisting of 1/100 M sodium acetate, 0.12 M sodium chlo- ride and 1/150 M methyl-p-hydroxybenzoate and which was adjusted to pH = 7.3 with hydrochloric acid. To 100 ml of urea-containing insulin solution produced by the procedure of example B, was added a zinc chlo- ride solution in an amount corresponding to 7 mg of zinc per g of insulin, ' and then the mixture was ap¬ plied to the column..The column was eluted at 4°C with said buffer at a rate of 100 ml/hour. The eluate was divided into fractions, the insulin- containing fractions were pooled and the content of insulin was determined. The pH value of the solution ' was readjusted to 7.3 with hydrochloric acid, and by adding a buffer which contains 1/100 M sodium acetate, 1/150 M methyl-p-hydroxybenzoate and 0.12 M sodium chloride and which was also adjusted to pH = 7.3 with hydrochloric acid, the insulin content of the solution was adjusted to 40 IU/ml and filled in vials after sterile filtration. Example 5 A column with a diameter of 10 cm was packed with 90 cm layer of "BIO-GEL P-30" (100 to 200 mesh) (a po- lyacrylamide gel) which was swollen in a buffer con¬ taining 1/75 M phosphoric acid and adjusted to pH = 2.5 with a sodium hydroxide solution. 2 g of crystalline insulin were dissolved in 150 ml of the same buffer, and pH was readjusted with hydro¬ chloric acid to 2.5. • Following filtration to provide a particle free solu¬ tion, the insulin solution was applied to the column, -^R OM which was then eluted with said buffer at a rate of 300 ml per hour. The eluate was divided into fractions. The extinction at 277 nm was measured, the fractions containing the central portion of the main peak were then pooled and the content of insulin was determined. The ' solution was admixed with a zinc chloride solution in an amount corresponding to 5 mg of zinc per g of . insulin, and then glycerol was added until a concen- tration of 1/6 M and m-cresol until a concentration of 1/50 M, followed by adjustment of pH to 7.3 with a sodium hydroxide solution. By addition of a buffer containing 1/75 M disodiu phosphate, 1/50 M m-cresol and 1/6 glycerol and ad- justed to pH = 7.3 with hydrochloric acid, the insulin concentration of the solution was adjusted to 40 IU/ml, and then it was sterile filtrated and filled in vials. Example 4 A column of a diameter of 5 cm was packed at 4°C with an 88 cm layer of » SEPHADEX £y » G 50 Superfine which was swollen in a buffer containing 0.2 M glycine and adjusted to pH = 9.1 with a sodium hydroxide solution. In 40 ml of the same buffer were dissolved 20 mg of ethylene dia ine tetraacetic acid and then 500 mg of insulin crystallized once, followed by readjustment of pH to 9.1 with a sodium hydroxide solution. After filtration to provide a particle free solution, the solution was applied to the column, which was eluted, still at 4°C, with said buffer at a rate of 80 ml per OMPI hour. The eluate was divided into fractions. The extinction at 277 nm was measured, and then the fractions con¬ taining the central portion of the main peak were pooled and the content of insulin was determined. The solution was admixed with phosphoric acid until a concentration of 1/75 M and with methyl-p-hydroxy¬ benzoate until a concentration of 1/150 M, and then pH was adjusted to 7.3 with hydrochloric acid. By di- lution with a buffer containing 1/75 M trisodium phosphate, 0.2 M glycine and 1/150 M methyl-p-hydroxy¬ benzoate and adjusted to pH = 7.3 with hydrochloric acid, the insulin content of the solution was adjusted to 40 lU/ml and filled in vials after sterile filtra- tion. Example 5 A column of a diameter of .1.6 cm was packed at 4°C with a 3 cm layer of "QAE-SEPHADEX^ » A-25 which was swollen and equilibrated in an aqueous buffer that contains 0.02 M tris(hydroxymethyl)amino methane and 7 M urea and was adjusted to pH = 8.1 with hydrochloric acid. "QAE-SEPHADEX® " A-25 is a dextran gel which is cross-linked with epichlorohydrin and acts as a strongly basic anion exchanger. 100 ml of urea containing insulin solution obtained by the procedure of example B were applied to the column at a rate of 25 ml per hour. Elution with dis¬ tilled water was effected at the same rate for 3 hours, and elution was continued with a buffer containing 1/75 M phosphoric acid and 0 r 12 M sodium chloride. The elu- OM tion was monitored by measuring the UV-absorption of the eluate at 277 nm, and when said absorption began to increase, indicating that insulin was being eluted from the column, 25 ml of eluate were collected, which were recirculated through the column, and the pH was constantly kept between 2.0 ' and 2.5 by means of hy¬ drochloric acid. When the content of insulin in the recirculating eluate became constant, the recircula- tion was interrupted and the elution was continued for 1 hour with a fresh buf er under continued col¬ lection. The concentration of insulin in the eluate was deter¬ mined, and a zinc chloride solution was added in an amount corresponding to 5 mg of zinc per g of insulin. Then m-cresol was added until a concentration of 0.02 M and pH was adjusted to 7.3 with a sodium hydroxide solution. By addition of a buffer containing 1/75 M sodium phosphate, 0.12 M sodium chloride and 0.02 M m-cresol and adjusted to pH = 7.3 with hydrochloric acid, the insulin concentration of the solution was adjusted to 40 IU/ml and filled in vials after sterile fil¬ tration. Example 6 A column of a diameter of 5 cm was packed with a 2 cm layer of DEAE cellulose ("Whatman" DE-52) which was swollen and equilibrated at 4°C in an aqueous buffer containing 0.01 M tris(hydroxymethyl)amino methane and adjusted to pH = 7.6 with hydrochloric acid.. 200 ml of urea-containing solution of insulin produced by the procedure of example A and containing 460 ml OMPI insulin, were diluted 1:1 with distilled water and applied to the solumn at a rate of 200 ml per hour at 4°C. Elution was effected with 300 ml of 0.01 M sodium chloride solution and then with a solution containing 0.02 M sodium acetate and 0.24 M sodium chloride (pH~7.8) at a rate of 100 ml per hour. The extinction of the eluate at 280 run was measured and when it began to increase, indicating that the insulin was being eluted from the column, the collection was begun. A total of 150 ml was collected. The concentra¬ tion of insulin in the pooled eluate was determined, and a zinc acetate solution was added in an amount . corresponding to 5 Ug of zinc ' per mg of insulin. Then there were added 150 ml of 0.2% methyl-p-hydro- xybenzoate, and pH was adjusted to 7.3 with hydro¬ chloric acid. By addition of a buffer containing 0.01 M sodium acetate, 0.12 M sodium chloride and 0.1% methyl-p- hydroxybenzoate and adjusted to pH = 7.3 with hydro- chloric acid, the concentration of insulin was ad¬ justed to 40 IU/ml, and then the resulting solution was sterile filtrated and filled in vials. Example 7 A preparation of intermediary action was produced by mixing 100 ml of quick-acting 40 IU/ml insulin pre¬ paration, produced by the procedure of example 1, with 100 ml of slow-acting 40 IU/ml of protamine in¬ sulin preparation (e.g. produced by the process of the Danish Patent 140 801) and subsequent aseptic filling of the obtained suspension in vials. The ob¬ tained preparation contained 50% quick-acting and 50% slow-acting insulin. OI.-I Example 8 An insulin preparation of intermediary action was pro¬ duced by admixing 200 ml of neutral quick-acting 50 IU/ml insulin preparation, produced by the procedure of example 5, with 50 ml of a solution adjusted to a pH of 7.3 and containing 1/75 M disodium hydrogen phosphate, 0.12 M sodium chloride, 0.02 M m-cresol and protamine sulfate in an amount corresponding to 70% of the quantity necessary to provide isophany. The suspension formed was aseptically filled in vials to provide a 40 IU/ml insulin preparation containing 30% quick-acting and 70% slow-acting insulin. To illustrate the changed immunogenic properties of the insulin preparation produced in accordance with the invention, comparative tests were conducted on rabbits which are the animals normally used for examina¬ tion of the antigenic properties of insulin and insulin- line components. In all the tests porcine insulin was used for the production of the preparations which is further described below. In the comparative tests the following preparations were used, reference being made to the figures shown in the drawings. Fig. 1 concerns a conventionally produced, neutral, quick-acting preparation. The insulin was purified as stated in example A, and desalted at pH 4 and zinc crystallized. The crystals were redissolved in a con¬ ventional manner in the carrier medium of the prepa¬ ration. Fig. 2 concerns likewise a conventionally produced, neutral, quick-acting preparation. The insulin was treated as in example A and then as in example 1, with the exception that a zinc crystallization was performed after desalting. Then the crystals were redissolved in a conven- tional manner in the carrier medium of the preparation. Fig. 3 concerns an NPH insulin preparation, i.e. prolonged with protamine, produced as described in the Danish Patent No. 140801. NPH represents "Neutral Protamin Hagedorn". Fig. 4 is a preparation produced by the process of the invention, cf. example 1. The rabbits were injected subcutaneously every 10th day with a constant dose of 20 IU of the insulin preparation to be tested. Normally, it is not possible to show any essential antibody formation after the injection of conventional, highly purified insulin preparation without adjuvants, and with a view to comparison it was therefore decided to adopt the im- munization procedure normally used in the preparation of antibodies, that is to inject the insulin prepa¬ rations of the 1st injection emulsified in Freund's Complete Adjuvant (FCA) and in subsequent injections emulsified in Freund's Incomplete Adjuvant (FIA), which differ from FCA in that it does not contain killed bacteria. Both products contain mineral oils and emulsifiers. The insulin antibody formation was examined at inter¬ vals of 10 days using the so-called PEG method which has been described in the Danish Patent No. 140801. Method of antibody determination by means of polyethy- lene glvcol (PEG) 100 Ul of rabbit serum, 100 /ul of 125 I insulin, a- bout 2 uU/ml, 100 ul of insulin solution, 250 uU/ml, OMF ' and 700 /ul of phosphate buffer, 0.04 M pH 7.4 with 0.15 M NaCl and 0.5% of human albumin were incubated for 48 hours at 4°C. 500 /ul of PEG 6000 (polyethylene glycol having an average molecular weight of 6000) 360 g/l were added, were mixed in a rotamixer and the sample was left to stand for 1 hour at 20°C. After centrifuging for 10 minutes at 3000 rpm, the ssuuppeerrnnaattaanntt wwaass ddeeccaanntteedd aanndd discarded, and 125I in the precipitate was counted. In each test was included a sample with an excess of guinea pig antiporcine insulin, maximum bonding, and a sample of rabbit serum from non-immunized rab¬ bits, 0-sample. The results were calculated as fol¬ lows: %B = " o . 100 T - T wherein T = counting number of preparation examined • T Q = " » " 0-sample T M = " " for maximum bonding, and %B = bonded 12 J 5 ~ insulin in % of maximum bonding. A comparison reveals that the preparation produced in accordance with the invention (fig. 4) has a signi¬ ficantly lower antigenicity than the corresponding conventionally produced neutral, quick-acting prepara¬ tions (figs. 1 and 2) and approximately just as low an antigenicity as a prolonged NPH preparation produced by the process of the Danish Patent No. 140801 and which is distinguished by an extremely low antigenicity. O FI

Claims: P a t e n t C l a i m s 1. A process for preparing a quick-acting, injectable insulin preparation of low antigenicity which is a clear solution at neutral pH, comprising subjecting an insulin solution to a chromatographic process, c h a r a c t e - r i z e d in that during said chromatographic process the insulin is trans erred to an eluant consisting of the same carrier medium as forms part of the desired insulin preparation, or one or more components of said carrier medium, following which the insulin-containing eluate, without isolation of the insulin, is admixed with any residual medium components and is adjusted to the desired pH and insulin concentration to provide the finished preparation. 2. A process according to claim 1, c h a r a c t e - r i z e d in that an insulin-containing fraction, from which antigenic impurities have been wholly or partly removed by a chromatographic purification, is transferred to the carrier medium by a chromatographic process, following which the product is readjusted, if necessary, to provide an insulin preparation of the desired pH and composition. 3. A process according to claim 1, c h a r a c t e ¬ r i z e d in that insulin is subjected to a chromatogra¬ phic process during which the insulin is eluted with the carrier medium or one or more components thereof, follow¬ ing which the insulin-containing fraction is readjusted, if necessary, to provide the desired pH and composition. Ol.ϊP

Inventors: ANDRESEN F, BALSCHMIDT P, JENSEN J, KRISTENSEN O
Applicants: ANDRESEN F, BALSCHMIDT P, JENSEN J, KRISTENSEN O, NORDISK INSULINLAB
Application year: 1980
Main classification: A61K37
IPC codes: A61K9, A61K38, C07K14