CS203539B1 - Method of cleaning the neurohypophysar hormones and the analogues thereof - Google Patents

Description

The invention relates to a method for purifying neurohypophysic hormones and analogs thereof. As is well known, these modern drugs are increasingly used in human and veterinary medicine, in the form of preparations prepared from substances obtained by peptide synthesis methods. One of the quality requirements is the standard number 2 tota. Synthesis of neurohypophysic hormones and their analogs is a multistep process, at the end of which the protecting groups are cleaved from the protected precursor and oxidative closure of the disulfide bridge according to the following scheme (final stages of l-desamino-8-D-arginine-vasopressin synthesis):

Mpa (Bzl) -Tyr-Phe-Gln-Asn-Cys (Bzl) -Pro-D-Arg (Tos) -Gly-NH 2. sodium and liquid ammonia reduction

SH

SH oxidation, purification

In principle, the final step is the same or very similar for all substances of this type.

A number of side reactions usually take place in the process and the crude product is contaminated with a large amount of salts. To date, non-carrier electrophoresis, countercurrent shaking, and various gel chromatography methods have been used to purify the products. The disadvantage of electrophoretic procedures is their relatively low capacity, the necessity to work in continuous operation, the need of specially trained workers who are exposed to the risk of working with high voltages (4000 VJ).

The countercurrent shaking technique requires the use of a hard-to-reach and very expensive apparatus. Cleaning takes considerable time, especially when working in large volumes. After the operation, the cleaning process must be evaluated in a complicated way.

When using gel filtration, partial (sometimes irreversible J adsorption of material on the gel column) occurs under commonly used conditions. When gel chromatography is carried out in a two-phase system, the products are usually contaminated with pyridine acetate, which can only be completely removed by further purification. Although efficient, it requires a difficult gel column preparation, and the evaluation of the purification process is laborious and yields are low.

In all cases, with the exception of countercurrent shaking, one of the conditions for success in cleaning is the perfect desalination of the raw material. This is usually done either on a gel column or using an ion exchange resin. When desalting on a gel column, the product dissolved in a large volume of water must first be isolated by evaporation. For some peptides, this method of desalting is preferable to ion exchange chromatography, the use of which is limited by the adsorption capacity of the substances and is highly dependent on the exact adherence to the procedure.

In order to ensure the standard purity of the synthetic substances of the neurohypophyseal hormones and their analogs, it was necessary to find and elaborate a purification process sufficiently gentle and simple and with respect to the production volume with a large and continuously variable capacity, but without the disadvantages of the prior art purification methods.

According to the present invention, there is provided a method of purifying neurohypophysic hormones and their analogs contained in a reaction solution after deprotection and oxidation according to the invention, which comprises removing the reaction solution from salts, preferably by contacting a styrene copolymer carboxylate cation exchanger. with divinylbenzene or cross-linked dextran bearing carboxyl groups, then the desired product is eluted with a concentration gradient of acetic acid from 0.25 to 60% by weight, the fraction containing the product is dehydrated, preferably by lyophilization, the obtained product is redissolved in 5 to 20 % acetic acid to a solution having a concentration of 3 to 5 wt. and after removal of the mechanical impurities, the solution is freed of undesirable fractions by adsorption partition chromatography on a molecular network, preferably of the cross-linked dextran type, with a separating capability for substances having a molecular weight of from 100 to 1500, followed by elution with dilute acetic acid 60% by weight, preferably 20% by weight, and the fractions containing the product are processed, for example by lyophilization.

The process of the invention combines the advantages of ion exchange chromatography and dextran gel type molecular sieves, particularly when using a discontinuous acetic acid concentration gradient after desalination and purification. The densely crosslinked gels contain a greater amount of carboxyl groups (some species are a weakly acid cation exchanger containing methylene carboxyl groups bound to the dextran matrix), which strongly adsorb substances of this type. In fractionation, this property can be utilized and operated under conditions where the adsorption and crosslinking properties of the materials complement each other appropriately, as is evident in some dextran gels in the pH range where their ion-exchange properties (pH range 1.5 to 2, 5j, using 15 to 30% acetic acid to elute the peptide, and is thus adsorption partition chromatography.

The combination of ion exchange chromatography and adsorption chromatography on a molecular network by the method of the invention eliminates most of the disadvantages of the above-mentioned processes. The method according to the invention is gentle, has a considerable, continuously variable capacity, is very simple, its laboriousness is negligible compared to other processes and the risk of material loss is minimized as much as possible. Another advantage is safety, low demands on operator qualification and energy.

Further details of the method according to the invention can be seen from the examples which illustrate the method but do not limit it in any way.

Examples

Example 1

Desalination and purification of 1-desamino-8-D-arginine-vasopressin

a) The post-oxidation reaction solution containing 1-desamino-8-D-arginine-vasopressin, a mixture of by-products and salt in 30 liters of water (pH about 4.5), is pumped through a weakly acid cation exchange column in an H ⁺ -cycle (cation exchange volume 2.0 to 2.5 liters). In this operation, the substance is trapped on the column, the salts flow and are discarded. The column is then washed with a concentration gradient of acetic acid from 0.25 to 60 percent by weight. The absorption at 280 nm is monitored. The product-containing eluate is dehydrated, preferably by lyophilization. About 10 g of desalinated product is obtained,

b) Concentrate the reaction solution as above to a volume of 2.5 to 5.0 liters and lyophilize. The resulting mixture of peptides and salts (150-300 g) was stirred with 400-600 ml of 50% acetic acid, filtered and the filtrate was applied to a molecular sieve column of dextran gel syrup (7 liter gel volume) equilibrated in 50% acetic acid. Elute with 20% acetic acid at a rate of 300 to 500 ml / h and monitor the absorption at 280 nm. The eluate was lyophilized. About 10 g of desalted product is obtained.

c] Dissolve the desalted product to a 3-5% solution in 1 M acetic acid, filter and load onto a molecular sieve column (10 x 100 cm] equilibrated in 1 M acetic acid. Elute with 20% acetic acid at a rate of 300-500 ml / h and absorption at 280 nm was monitored and fractions containing sufficiently pure material were further processed, for example, by lyophilization, to give about 3 g of 100% peptide.

d) The desalted product is dissolved in a 3-5% solution in 20% acetic acid and applied to 2 liters of dextran gel equilibrated in 20% acetic acid. The column is eluted with either a 20% acetic acid or a 20 to 70% acetic acid concentration gradient. The absorption at 280 nm is monitored. Fractions containing sufficiently pure material are further processed, for example, by lyophilization. About 3 g of 100% peptide are obtained.

Determination of content: a) from UV spectrum at least 70% b) according to nitrogen content 13,5 - 18,34% N

Optical rotation:

(α>) _η ^2θ - 62,0 (c 0,1 M acetic acid)

Amino acid analysis:

Phe 1.02, Tyr 0.84, Gly 1.00, Pro 0.95,

Glu 1.04, Asp 0.98, Arg 1.01,

Cys - not determined

Example 2

Desalination and purification of oxytocin

a) The oxidation reaction solution containing a mixture of oxytocin, peptides and salts in 2 liters of water (pH about 4.5) is concentrated to a volume of 200 ml and lyophilized. The resulting mixture of peptides and salts (7 g) is mixed with 20-30 ml of 50% acetic acid, filtered and the filtrate is applied to 500 ml of dextran gel equilibrated in 50% acetic acid. Elute with 20% acetic acid at a rate of 60 ml / h and monitor the absorption at 280 nm. The eluate containing the salt-free product is lyophilized. 1 g of desalted product is obtained. This was dissolved in a 3-5% solution in 1 M acetic acid, filtered and loaded onto a dextran gel column (3 x 100 cm < 3 > equilibrated in 1 M acetic acid. Eluted with 20% acetic acid at a rate of 15 ml / h) Fractions containing sufficiently pure material were lyophilized to give 0.65 g of product.

Content determination:

UV spectra 89% (e 15,76, water pH 3,3, HCl)

b) by nitrogen content 13, 63%

Optical rotation:

(α) ϋ ²⁰ -— 23,4 (c 0,1 1 M acetic acid)

Amino acid analysis:

Gly 0.93, Leu 0.97, Pro 0.97, Asp 1.00,

Glu 1.05, Ile 1.05, Tyr 0.93, 1/2 Cys 1.93 Example 3

Desalination and purification of methyloxytocin

(a) The oxidation reaction solution, which contains a mixture of methyloxytocin, peptides and salts in 20 liters of water (pH about 4,5), is pumped through a weakly acidic cation exchange column in a H ⁺ -cycle (2 to 3 liters volume). The column is then washed with a concentration gradient of acetic acid from 0.25 to 60% by weight, and absorption at 280 nm is monitored, and the product-containing eluate is further processed, for example, by lyophilization. 4 g of salt-free product.

b) The oxidation reaction solution containing methyioxytocin, various by-products and salts in 60 liters of water (pH about 4.5) is concentrated to a volume of 2.5 to 5.0 liters and lyophilized. The resulting mixture (150-300 g) is stirred with 400-800 ml of 50% acetic acid, filtered, and the filtrate is applied to a 7 liter dextran gel column equilibrated in 50% acetic acid. Elute with 20% acetic acid at a rate of 300 to 500 ml / h and monitor the absorption at 280 nm. The eluate containing the desalted product is lyophilized. 20 g of desalted product are obtained. Dissolve all of this to a 3-5% solution in 1 M acetic acid, filter and load onto a dextran gel column (10 x 100 cm 3, equilibrated in 1 M acetic acid. Elute with 20% acetic acid at a rate of 300 to 500 ml The absorption at 280 nm was monitored and fractions containing sufficiently pure material were then treated, for example, by lyophilization, to give about 6 g of product.

Content determination:

UV spectrum 88.6% (e 15.76, water, pH 3.3, HCl)

b) by nitrogen content 15.0%

Optical rotation:

(orJ _D ²⁰ - 18.9 (c 0.15, 1 M acetic acid)

Amino acid analysis:

Asp 0.98, Glu 1.02, Pro 1.04, Gly 0.93,

Ile 0.93, Leu 1.02, Tyr 0.92, 1/2 Cys 1.90 Example 4

Desalination and purification (Gly.Gly.Gly.CyshLys ¹ } vasopressin

a) After the oxidation reaction solution containing the product, a mixture of peptides and salts in 30 liters of water (pH about 4.5) is pumped through a weakly acidic cation exchange column in an H ⁺ -cycle (cation exchange volume 2.0 to 2.5 liters) . In this operation, the desired substance is retained on the column, the salts flow and are discarded. The column is then washed with a concentration gradient of acetic acid from 0.25 to 60% by weight. The absorption at 280 nm is monitored. The product-containing eluate is lyophilized. About 10 g of desalted product is obtained.

b) The reaction solution as above was concentrated to a volume of 2.5 to 5.0 liters and lyophilized. The obtained mixture of peptides and salts (150-300 g) is stirred with 400-600 ml of 50% acetic acid, filtered and the filtrate is applied to a column containing 7 liters of dextran gel equilibrated in 50% acetic acid. Elute with 20% acetic acid at a rate of 300 to 500 ml / h and monitor the absorption at 280 nm. The eluate was lyophilized to give about 10 g of desalted product.

c) The desalted product obtained according to a) or bj is dissolved in 1 M acetic acid to a 3-5% solution, filtered and loaded onto a dextran gel column (10 x 100 cm) equilibrated in 1 M acetic acid. Elute with 20% acetic acid at a rate of 300 to 500 ml / h and monitor the absorption at 280 nm. Fractions containing sufficiently pure material are further processed by lyophilization. About 3 g of 100% peptide are obtained.

d) The desalted product obtained according to a] or b) is dissolved in 20% acetic acid to a 3-5% solution and applied to a column containing 2 liters of dextran gel equilibrated in 20% acetic acid. The column is eluted with either a 20% acetic acid or a 20 to 70% acetic acid concentration gradient. The absorption at 280 nm is monitored. Fractions containing sufficiently pure material are lyophilized. About 3 g of 103% of the peptides are obtained. Content determination:

according to nitrogen content 13,5 - 18,25% N Optical rotation:

(a) _D ²⁰ - 95.0 (c 0.2 1 M acetic acid)

Amino acid analysis:

Lys 0.99, Phe 1.04, Tyr 0.90, Cys: _1.72;

Gly 4.03, Glu 1.01, Pro 1.06, Asp 0.98

Claims (1)

SUBJECT Process for purifying neurohypophysic hormones and analogues thereof contained in a reaction solution after deprotection and oxidation, characterized in that the reaction solution is first freed from salts, preferably by contact with a carboxylate cation exchanger based on a styrene copolymer with divinyl benzene or a crosslinked dextran carrying a carboxylic acid %, then the desired product is eluted with a concentration gradient of acetic acid from 0.25 to 60 weight percent. the product-containing fraction is dehydrated, preferably by lyophilization, obtained The product is redissolved in 5 to 20% acetic acid to a solution with a concentration of 3 to 5% by weight. and after removal of the mechanical impurities, the solution is freed of undesirable fractions by adsorption partition chromatography on a molecular network, preferably of the cross-linked dextran type, with a separating capability for substances having a molecular weight of 100 to 1500, then eluting the fractions with dilute acetic acid %, preferably 20 wt. and fractions containing the product are processed, for example by lyophilization.