DK149616B - METHOD OF MANUFACTURING INSULINES - Google Patents

Description

in 149616

The present invention relates to a process of the kind set forth in claim 1 for the manufacture of insulins. The insulin derivatives used as starting materials may be prepared by the method of the parent application DK Patent Specification No. 148,714 or by other methods as further explained below.

The invention is explained below in connection with the specific cleavage of derivatives of human insulin, but it is readily seen that the method described can equally well be applied to derivatives of other types of insulin, such as bovine insulin and porcine insulin.

It is known, cf. Morihara et al. (Ref. 1), a synthesis of human insulin from des-alanine (B-30) insulin (DAI) obtained by digestion of porcine insulin with carboxypeptidase A for 8 hours. 10 mM DAI was incubated with a large excess (0.5 M) of threonine-OBu1 ester at 37 ° C for 20 hours in the presence of trypsin and high concentrations of organic 20 co-solvents. The resulting [Thr - 0Bu * "-Β-3θ] insulin was unblocked with trifluoroacetic acid, in order to avoid racemization it is necessary to work in the presence of anisole.

In a similar experiment, Morihara et al. (Ref.

2) used Achromobacter Protease I as the enzymatic catalyst for coupling large excess DAI with Thr -0Bu * · to generate [Thr - OBu ^ - B-30j insulin which was isolated and unblocked as above.

30

A similar experiment with bovine insulin leads to [Thr -OBu ^ - B-30 [] bovine insulin.

The procedures described in Ref. 1 and 2 are also mentioned 55 in EP publication no. 17938 and DK-ans. 2556/80, cf. Petition No. 146,482. In both cases, a chemical unblocking as mentioned above was performed with trifluoroacetic acid in the presence of anisole to avoid racemization.

The object of the present invention is to provide a method for unblocking insulin derivatives which are B-30 carboxyl group protected with amide or ester groups, under gentle conditions, whereby it is possible, in short reaction times, to obtain pure insulin in good yield. and without special precautions to avoid racialization.

This is achieved by the method according to the invention, which is characterized by the characterizing part 15 of claim 1.

Of course, the insulin derivatives useful as starting materials are not only insulin derivatives prepared by the first transpeptidation step of the process 20 according to the parent application, but the method of the invention can be applied to any such derivative irrespective of the origin, in particular derivatives prepared according to EP Publication No. 17938 as shown. below in Example 2.

25

It has recently been shown that the enzyme carboxypeptidase-Y is an effective catalyst for peptide syntheses (Widmer and Johansen (Ref. 3)). Furthermore, it has been shown that, under certain conditions, the enzyme catalyzes the replacement of C-terminal amino acids in a peptide with another amino acid or amino acid derivative in a trans-peptidation reaction (cf. International Application no.

W0 80/02151, continued as Danish patent application no.

5202/80).

The invention according to the parent application rests on the surprising recognition that the aforementioned enzyme carboxy-peptidase-Y can convert porcine insulin to human insulin by replacing B-30 alanine with threonine in a single step optionally without isolating an intermediate and subsequent unblocking processing.

As further illustrated below, the most suitable derivative for this conversion is threoninamide. However, if an intermediate is not isolated, the conversion will result in a mixture of human insulin and a certain amount of unreacted porcine insulin, which is difficult to separate, which is why the human insulin-amide intermediates formed are preferably separated from the reaction mixture. contains unreacted swine insulin, and then deamidates.

15 by the method of the invention, advantageously by the same enzyme carboxypeptidase-Y, as also explained below. .

In DK-ans. 5202/80, the desired enzymatic characteristics of a general peptide synthesis 20 are described in detail, and it is stated that a number of carboxypeptidases exhibit various enzymatic activities which are highly pH dependent, so that, e.g. in alkaline environment at pH 8 - 10.5, predominantly esterase or 25 amidase activity and at pH 9 - 10.5 exhibit no or only negligible carboxypeptidase activity, which, however, becomes more pronounced at pH values falling below 9. These properties may also are advantageously utilized in the method of the invention since they contribute 30 to the implementation of a gentle deblocking process with good yields.

The carboxypeptidases useful in the process of the invention are L-specific serine or thiol-carboxypeptidases, such enzymes may be produced by yeast fungi or they may be of animal, vegetable yeast fungi or they may be of animal, vegetable or other microbial origin. .

A particularly suitable enzyme is, as mentioned, carboxypeptidase-Y 5 from yeast fungi (CPD-Y). This enzyme is described in DK-ans. No. 5202/80, i.a. citing Johansen et al. (Ref. 4), which developed a particularly convenient purification method by affinity chromatography on an affinity resin comprising a polymeric resin backbone with benzylsuccinyl groups attached. CPD-Y, which is a serine enzyme, is distinguished by having the aforementioned relationships between the various enzymatic activities at pH 9 and by not exhibiting endopeptidase activity. Another advantage of CPD-Y is that it is available in large quantities and exhibits a relatively high stability. Further details are given in Ref. 3 and 6.

in addition to CPD-Y, which is the preferred enzyme at present, the method of the invention can be carried out with 20 other carboxypeptidases, e.g. the following:

Enzyiji Origin

Mushrooms

Penicillocarboxypeptidase S-1 Penicillium janthinellum 25 "S-2" "

Carboxypeptidase (s) from Aspergillus saitoi “Aspergillus oryzae

plants

Carboxypeptidase (s) C Orange Leaves 30 Orange Shells

Carboxypeptidase C ^ Citrus natsudaidai Hayata

Phaseolain Bean Leaves

Carboxypeptidase (s) from germinating barley

Sprouting cotton plants 35 Tomatoes

watermelons

Bromelain (pineapple) powder 5 149616

The close relationship between a number of the above carboxypeptidases is discussed by Kubota et al. (Ref.

7).

It should be noted that ionizable groups present in the individual amino acids contained in the insulin starting material may, if desired, be blocked in a manner known per se depending on the nature of the group. However, this is absolutely not necessary, which is precisely one of the advantages of the present process. If for some reason it should be desirable to protect the functional groups, suitable protecting groups may be selected as stated in the above DK case.

5202/80.

15

The amino acid B contained in the insulin derivative, which is the starting material for the process of the invention, can be any of the known L-amino acids, e.g. Leu, Ile, Ala, Gly, Ser, Val, Thr, Lys, Arg, Asn, Glu, Gln, Met, Phe, Tyr, Trp or His, but are preferably Thr.

For the group, "alkyl" means straight or branched alkyl, preferably having from 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, amyl or hexyl.

As stated in the characterizing part of claim 1, the process according to the invention is carried out at pH 7.0 - 10.5, preferably at pH 9 - 10.5. The preferred pH, which is often within a very narrow range, depends on the pH maxima and pH minima, respectively, of the various enzymatic activities of the enzyme used, since it is understood that the pH value must be chosen so that the activities are mutually coordinated as explained above.

U9616 6

If CPD-Y is used as an enzyme, the pH is preferably 7.5 - 10.5, especially 9.0 - 10.5 due to the more pronounced amidase and esterase activity and the negligible carboxy peptidase activity.

For example, pH control can e.g. is obtained by incorporating a suitable buffer for the desired pH range in the reaction medium, such as a bicarbonate buffer.

The pH may also be maintained by the addition of an acid such as HCl or a base such as NaOH during the reaction. This can be conveniently done using a pH state.

15 Based on the information given above and in Ref. 3 and 6, those skilled in the art will be able to select the most suitable reaction conditions, especially with respect to the pH at which the amidase, esterase or peptidylamino acid amide hydrolase activity can best be utilized depending on the insulin starting material.

However, these conditions can also be affected by variation in enzyme concentration, reaction time, etc.

The reaction is carried out in an aqueous reaction medium which, if desired, may contain up to 50 µm of an organic solvent. Preferred organic solvents are alkanols, e.g. methanol and ethanol, glycols, e.g. ethylene glycol or polyethylene glycols, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dioxane and dimethoxyethane.

The choice of the composition of the reaction medium depends in particular on the solubility, temperature and pH of the reaction components and the insulin products involved, as well as on the stability of the enzyme.

7 149616

The reaction medium may also contain a component which renders the enzyme insoluble, but retains a substantial part of the enzyme activity, such as an ion exchange resin.

Alternatively, the enzyme may be immobilized in a manner known per se, cf. Methods of Enzymology, Vol. 44, 1976, e.g. by bonding to a matrix, such as a cross-linked dextran or agarose, or to a silica, polyamide or cellulose, or by encapsulation in polyacrylamide, alginates or fibers. Furthermore, the enzyme can be chemically modified to improve its stability or enzymatic properties.

The reaction medium may also contain urea or guanidine hydrochloride at concentrations up to 3 molar. This may also be advantageous at pH values and in media where the insulin starting material has limited solubility.

The enzyme activity may vary, but is preferably 10 ** - 10 3 molar, especially 10 ~ 3 molar.

20

The reaction temperature of the process according to the invention is preferably 20-40 ° C. The most appropriate reaction temperature can be determined by experiment, but depends in particular on the concentration of the enzyme. A suitable temperature 25 is usually approx. 20 to 30 ° C, preferably approx. 25 ° C.

At temperatures below 20 ° C, the reaction time will usually be inappropriately long, while temperatures above 40 ° C often cause problems with the stability of the enzyme and / or the reactants or reaction products.

30

The standard reaction time of the process according to the invention is extremely short, namely as low as 15-20 minutes, as is evident from the examples of deamidation.

Before the process of the invention is further illustrated by examples, the starting materials, measurement methods, etc. will be discussed.

SOURCE MATERIALS

5 Swine insulin was provided by the Nordic Insulin Laboratory, Copenhagen. Carboxypeptidase-Y from bakery yeast, a commercial product of the Carlsberg Breweries, was isolated by a modification of the affinity chromatography method of Johansen et al. (Ref. 4) and obtained as 10 a lyophilized powder (10¾ enzyme in sodium citrate).

(1?)

Prior to use, the enzyme was desalted on a "Sephadex ^ G-25" column (1.5 x 25 cm) equilibrated and eluted with water. The enzyme concentration was determined spectrophoto-1¾ metric at E ^ µg nm = The enzyme preparation 15 used was free of Protease A determined according to Lee and Riordan (Ref. 5). L-threoninamide was purchased from Vega-Fox,

Arizona, USA. L-threonine methyl ester from Fluka, Switzerland and L-threonine, Dansyl chloride, carboxypeptidase A and trypsin were obtained from Sigma, USA. Chromatographic materials were from Pharmacia, Sweden. All other reagents and solvents were of analytical purity from Merck, BRD.

AMINOSYREÅNALVSER

25

Samples for amino acid analysis were hydrolyzed in 6M HCl at 110 ° C in vacuum for 24 hours and analyzed on a Durrum D-500 amino acid analyzer. The amino acid composition was based on the known content of aspartic acid and glycine. Thr, Lys and Ala are the only amino acids affected by the reactions. The values of these amino acids for porcine (human) insulin are: Thr = 1.93 (2.87), Lys = 0.97 (0.98) and Ala = 2.00 (1.05). The amount of unconverted swine insulin in the reaction mixtures was determined from alanine analysis of the "insulin pool" after chromatography on "Sephadex G-50". The coupling yield is indicated as the amount of U9616 9 of a given product divided by the amount of total insulin consumption in the reaction.

ENZYMATIC DEVELOPMENT OF INSULIN DERIVATIVES (Example 2) 5

Degradation of various insulin derivatives with carboxy peptidase A and Y was carried out in a 0.05 M Tris buffer, pH

7.5 at room temperature using approx. 0.5 mM insulin and 5 µM carboxypeptidase. The reaction time was 10 3 hours with CPD-A and 1.5 hours with CPD-Y. Under these conditions, the maximum release of C-terminal amino acid was obtained. After the reaction mixture was acidified with HCl, the amounts withdrawn were used directly in the amino acid analyzer.

15

The amino acid sequence of the C-terminal portion of the various insulin derivatives was determined following trypsin breakdown and reaction of the degraded material with Dansyl chloride, followed by identification of Oansyl peptides. Degradation of insulin derivatives with trypsin was performed in 0.1M

NaHCO₂ at pH 8.2 using 1 mM insulin, 40 µM DPCC trypsin and a 1 hour incubation time. Prior experiments on swine insulin had shown that these conditions were sufficient to completely release the C-terminal alanyl residue from the B chain. The released amino acids or dipeptides were dansylated as follows:

To a 100 µl sample of the trypsin degradation mixture was added 100 µl of 0.5 M HCl, thereby stopping the degradation.

The sample was evaporated to dryness and redissolved in 100 µl of 30 0.1 M NaHCO 3, pH 8.2. 100 µl Dansyl chloride (5 mg / ml) in acetone was added and the reaction mixture was incubated for 2 hours at 37 ° C. The reaction mixture was analyzed by HPLC using a Waters liquid chromatography system consisting of a Model U6K injector, 2 Model 6000 A 35 pumps, a Model 660 Solvent Programmer, a Model 450 UV detector, a Waters Data Module and a Waters Radial Com - 149616 ίο pression Module (RCM 100) chamber equipped with a Waters Radial Pak A (C-18 reverse phase) column.

The following standard compounds were synthesized: 5

Dns-Ala-OH, Dns-Thr-OH, Dns-Ala-Thr-OH, Dns-Thr-Thr-OH, Dns-Thr-NH 2, Dns-Thr-Thr-NH 2, and Dns-Ala-Thr-NH ^. Using the above-described procedure for dancing the insulin degradation product, three of these 10 derivatives could be synthesized from H-Ala-OH, H-Thr-OH

and H-Thr-NI 2. The dansylated dipeptides were synthesized via Dns-Ala-OMe and Dns-Thr-OMe: 1 mmol H-Ala-OMe, HCl was dissolved in 0.1 M NaHCO 3, 5 mmol Dansyl chloride was added and the reaction mixture was incubated for 2 hours. Dns-Ala-OMe 15 was extracted from the reaction mixture with ethyl acetate and evaporated to dryness. HPLC demonstrated that the isolated product was pure. The same procedure was used in synthesizing Dns-Thr-OMe. Using the methods of enzymatic peptide synthesis similar to those previously described (Refs. 3 and 6), these two compounds were then coupled to H-Thr-OH which yielded Dns-Ala-Thr-OH and Dns-Thr-Thr-OH. and H-Thr-NH2, yielding Dns-Ala-Thr-NH2 and Dns-Thr-Thr-NH2. The conditions were as follows: 5 mM substrate, 0.5M nucleophile, 0.1M KCl, 2 mM EDTA, 25 µM CPD-Y, pH 9.0, 10¾ ethanol. All seven Dansyl derivatives could be easily separated by HPLC using two different programs.

The invention is further illustrated in the drawing, wherein:

FIG. Figure 1 shows an elution profile from an ion exchange chromatography of the swine insulin-threoninamide reaction product.

EXAMPLE 1 (Background Studies)

Swine insulin was incubated with carboxypeptidase-Y at 25 ° C and pH 5-7, which is a pH range where the enzyme exhibits maximum peptidase activity. Thereby, the following amino acids were released from the C-terminus of the B chain: 1.0 Alanine, 1.0 Lysine, 1.0 Proline, 1.0 Threonine, 1.0 Tyrosine 5 and 2.0 Phenylalanine. At position B-23 (glycine), carboxypeptidase-Y stops and thus a complete release of the first 7 amino acids of the B chain in insulin can be obtained. Surprisingly, C-terminal asparagine (A-21) in the A-chain is not released at all. At pH 9.5, C-terminal 10 alanine is released from the B chain much faster than the subsequent amino acids. As the purified CPD-Y preparation was free of protease A (endopeptidase) unlike many commercially available preparations from other sources, the (B-15-16) Leu-Tyr bond was not hydrolyzed, which is of great importance.

Example 2 (Conversion of porcine insulin to human insulin using threoninamide as an amine component and isolating the insulin amide intermediates and subsequent deamidation of the invention)

For a solution of zinc-free porcine insulin (2 mM) in 2 mM EDTA, 0.1M KCl and 1.5M guanidine hydrochloride and containing 0.5M threoninamide (L-Thr-NH 2) at pH 7.5 and 25 ° C CPD-Y (15 µM) was added. The pH was kept constant during the reaction by adding 0.5M NaOH using a pH-stat. To follow the course of the reaction, equal samples were taken at different times and reaction 30 was stopped after 2 hours by adjusting the pH to 1.5 - 2.0 with 1M HCl. The insulin fraction was separated from the enzyme and low molecular weight by chromatography on "Sephadex ® G-50 fine" (1 x 30 cm) equilibrated with 1M acetic acid and lyophilized. Amino acid analysis on the lyophilized "insulin pool" as described above showed that 78 ° ό of the porcine insulin had been used during the reaction.

To further analyze the reactants in the "insulin pool", ion exchange chromatography on "DEAE-5 Sephadex ® A-25" was performed essentially as described by

Morihara et al. (Ref. 1). The lyophilized insulin sample (75 mg) was dissolved in 0.01M Tris, 2.5M urea, 0.05M NaCl, pH 7.5 and applied to a "DEAE Sephadex® A-25" column (2.5 x 25 cm) equilibrated with the same buffer. Insulin 10 was eluted with a NaCl gradient from 0.05 to 0.30M in the same buffer and 8 ml fractions were collected on a "Sephadex ® G-25" column and lyophilized.

The elution profile is shown in FIG. 1, where three peaks can be observed. In addition, the amino acid compositions of the individual peaks and the peak compositions as a result of degradation experiments were performed with CPD-Y and CPD-A as described above. Since only the C-terminus of the B chain in insulin is involved in these reactions, -Pro-20 Lys-Ala-OH is used as an abbreviation for swine insulin. Other insulin derivatives are also abbreviated similarly: -Pro-Lys-Thr-OH = human insulin, -Pro-Lys-Thr-NH 4 = human insulin amide, etc. It is seen that at the pH 7.5 used in the reaction and where the amidase activity of CPD-Y is generally lower than the peptidase activity, the peptidamide formed is sufficiently stable since the peak I comprised ca. 208! of the total "insulin pool". Ca.

75¾ of the porcine insulin starting material was converted in the reaction. However, since the threonine content of the top 30 I (3.65) is greater than that of 3.0 in human insulin, it is evident that the original transpeptidation product (-Pro-Lys-Thr-NH 2) is not sufficiently stable to avoid an oligomerization to give (-Pro-Lys-Thr-Thr-NH 2) ·

This formation of a mixture of amide intermediates could immediately indicate that homogeneous human insulin would not be formed by deamidation by CPD-Y, as it was expected that the deamidation would result in a mixture of -Pro -Lys-Thr-OH and -Pro-Lys-5 Thr-Thr-OH. However, when the top I mixture was subjected to a deamidation treatment of the invention with 10 µM CPD-Y at pH 10.0 in 0.1M KCl, 2mM EDTA for 20 minutes, surprisingly nearly pure human insulin was obtained. The experiment proceeded as follows: 10

After the reacted insulin was separated from the enzyme and (low molecular weight material by chromatography on "Sephadex G-50", the material is chromatographed on "DEAE Sephadex® A-25" using procedures identical to those of FIG. 1. The reaction product was eluted as peak II as expected, while unreacted material (<10%) eluted as peak I. There was no peak III, i.e. no insulin derivatives were formed without lysine, as shown in the results in Table I below obtained with CPD. A, 20 CPD-Y and trypsin breakdown, only the threonine content was significantly affected by these reactions, which is consistent with the expected lack of peptidase activity for CPD-Y at this pH.

The top II amino acid composition of the deamidation reaction is close to the human insulin assay:

Thr = 2.87, Ala = 1.05, Lys = 0.98. The degradation of deamidated product with CPD-Y, CPD-A and trypsin shows that the sample contained 90-95% pure human insulin. This indicates that the insulin derivative -Pro-Lys-Thr-Thr-NH2 reacts almost exclusively via the peptidyl-amino acid-amide hydrolase activity, while -Pro-Lys-Thr-NH2 reacts predominantly via the amidase activity. The total yield of the conversion of swine insulin to human insulin is about 35%. 30% based on the amount of converted insulin.

TABLE I

14 149616

Amino acid composition of peaks I, II and III of FIG.

1 and peak II obtained after deamidation treatment of the peak I fraction

Top II achieved after deamidation _ Top I Top II Top III of Top I_

Aspartic Acid 2.99 2.99 3.01 3.01

Threonine 3.62 2.52 2.50 2.79

Serine 2.93 2.92 2.92 2.93

Glutamic Acid 6.88 7.16 7.18 7.01

Proline 1.00 0.98 0.71 0.87

Glycine 4.00 4.00 4.00 4.00

Alanine 1.16 1.52 1.03 1.09

Selected 2.42 2.82 2.81 2.50

Ileucine 0.94 1.04 1.09 1.19

Leucine 5.80. 6.21 6.18 6.10

Tyrosine 3.8C 3.86 3.67 4.02

Phenylalanine 2.74 2.82 2.65 2.64

Histidine 1.96 1.90 1.93 1.89

Lysine 0.99 0.74 0.06 1.01

Arginine 0.96 1.00 0.98 0.89 15 EXAMPLE 3 (Deamidation of human insulin amide obtained by trypsin-catalyzed condensation of Des-alanine (B30) porcine insulin (DAI) with threonine amide).

5

To demonstrate that the advantageous CPD-Y catalyzed deamidation of insulin amides by the process of the invention is not limited to insulin amides obtained by the transpeptidation process of the stock application 10, human insulin amide was prepared in accordance with the prior art of Morihara et al. (EP 17938 and Refs. 1 and 2). However, it is noted that while the amides are mentioned in EP 17938 as one among other protecting groups for the carboxyl group in threonine, its usefulness has not been demonstrated by examples, the only example being about threonine tert-butyl ester.

The turnover can be illustrated by the following scheme: 7 n

Trypsin / ThrNH? CPD-Y

DAI -4 DAI-Thr-NhL - * DAIThr

pH 6.5 L

This process has the significant advantage over the method exemplified in EP 17938 that the enzymatic deamidation is much milder than acid-catalyzed deesterification according to Morihara et al.

Preparation of DAI-ThrNHp 30

Threoninamide, hydrochloride (400 mg) was suspended in 60 µm dimethylformamide (DMF) (2 ml). The pH was adjusted to 6.5 with pyridine (20 µl) and 6M NaOH (50 µl). Trypsin (100 mg) and DAI (100 mg) were added. After 1/2 hour, the reaction was quenched by the addition of formic acid (1 ml).

o (R)

The reaction mixture was fractionated on "Sephadex w G-50" with 1M acetic acid. The following fractions were collected:

Pressure infraction: 60 mg

Insulin fraction: 102 mg Rest: 371 mg

The insulin fraction (102 mg) was purified by ion exchange chromatography on "DEAE Sephadex ® A-25", equilibrated with 7M urea and eluted with a NaCl gradient in accordance with EP 17938.

Pure human insulin amide (DAI-Thr-NH 4) in an amount of 59.2 mg (determined without amino acid analysis) was obtained.

Preparation of DAI-Thr (human insulin) DAI-Thr-N1 (11 mg) was dissolved in ImM EDTA and 2mM KCl (2ml). The pH was adjusted to 9.0 by means of 0.5 M NaOH (51 µl) and CPD-Y (50 µl, 13.6 mg / ml) was added.

After 15 minutes, the reaction was quenched with 6M HCl (25 µl) and the pH decreased to 1.2. The reaction mixture was separated on "Sephadex G-50" and eluted with 1 H acetic acid. 8 mg of human insulin were obtained. The amino acid analysis is shown in Table II below:

TABLE II

17 148616

Amino acid analysis

Human insulin Human _ (formula) _DAI_Amid_insulin

Asp 3 2.97 3.05 3.04

Thr 3 1.91 2.94 2.90

Ser 3 2.86 2.86 2.95

Glu 7 7.27 7.23. 7.04

Pro 1 0.94 0.95 0.85

Gly 4 4,000 4,000 4,000

Ala 1 1.00 1.00 1.04

Cys 6 5.31 5.19 5.02

Choice 4 3.37 3.11 3.24

Ile 2 1.36 1.22 1.40

Leu 6 6.28 6.20 6.13

Tyr 4 3.64 3.97 3.77

Phe 3 2.82 2.92 2.83

His 2 1.96 1.96 1.87

Lys 1 0.94 1.00 0.95 NH3 6 8.12 6.46 6.98

Arg 1 0.98 0.99 0.87

Thr / Ser 1.00 1.003 (x3 / 2) 1.027 0.984 143616 EXAMPLE 4 (Conversion of human insulin methyl ester to human insulin)

Human insulin methyl ester was prepared by coupling 5 of des-Ala (B30) insulin with threonine methyl ester in the presence of trypsin as described by Gattner et al. (Ref. 8).

5 mg Ins-Thr (B30) -0Me was dissolved in 1 ml 0.1M KCl with ImM EDTA by raising the pH from 4.5 to 8.5 with 1M NaOH. This pH was maintained in a pH state with 0.05M NaOH as titrant at room temperature. The reaction was started by adding 1 µg of CPD-Y dissolved in 10 µl of water. The reaction was followed by HPLC; after 1 hour and 10 minutes the hydrolysis was complete. The reaction mixture was gel filtered in 30% HAC 15 over "Sephadex ® G-50". Yield determined by A2qq was 4.8 mg of human insulin.

Amino acid analysis gave the following composition:

TABLE III

19 149616

FORMAL ANALYSIS

Asp 3 3.00

Thr 3 2.83

Looks 3 3.00

Glu 7 7.09

Pro 1.76

Gly · 4 4.00

Ala 1.07

Cys 6 4.67

Mal 4 2.88

Ile 2 1.97

Leu 6 6.18

Tyr 4 3.89

Phe 3 2.89

His 2 1.86

Arg 1 0.97

List 1 0.98 20 149616

REFERENCES

1. Morihara, V.: Semi-synthesis of human insulin by trypsin-catalysed replacement of Ala-B30 by Thr in porcine insulin, Nature, Vol. 280, No. 5721, 1979, 412-13 2. Morihara, K ,, Oka, T., Tsuzuki, H., Tochino, Y.,

Kanaya, T.: Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin, Biochemical and Biophysical Research Comm. Vol. 92, no. 2, 1980, 396-402.

3. Widmer, F., Johansen, J.T .: Enzymatic peptide synthesis carboxypeptidase Y catalyzed formation of peptide bonds, Carlsberg Res. Commun., Vol. 44, April 23, 1979, 37-46.

4. Johansen, J. T., Breddam, K., Ottesen, M.: Isolation of Carboxypeptidase Y by Affinity Chromatography,

Carlsberg Res. Commun., Vol. 41, no. 1, 1976, 1-13.

5. Lee, H.-M., Riordan, J.F .: Does carboxypeptidase Y have intrinsic endopeptidase activity ?, Biochemical and Biophysical Research Comm., Vol. 85, no. 3, 1978, 1135-1142.

6. Breddam, K., Widmer, F., Johansen, J.T .: Carboxypeptidase Y catalyzed transpeptidations and enzymatic peptide synthesis, Carlsberg Res. Comm. Vol. 45, November 4, 1980, pp. 237-247.

7. Kubota et al., Carboxypeptidase C1, J. Biochem.,

Vol. 74, no. 4 (1973), pp. 757-770.

8. Gattner et al., Trypsin catalyzed peptide.de synthesis: Modification of the B-chain C-terminal region of insulin; European Peptide Symposium 1980.