CS206546B1 - Peptide cleaning method by liquid chromatography - Google Patents

Description

(54) A method for purifying peptides by liquid chromatography

The invention relates to a method for purifying peptides by liquid chromatography.

Synthetic peptides are used as drugs in a variety of indications, both human and veterinary;

medicine. Their effects are very pronounced and often quite specific. Since it acts intensively in very small quantities, it is important to ensure high purity in production and to eliminate as much as possible those substances that could have undesirable or uncontrolled effects on the living organism.

Purification of peptides having a sequence of 10 to 20 amino acids is a complicated problem. During the synthesis, a series of by-products with very similar structural features and often with a molecular weight close to the desired substance is produced.

A countercurrent shaking method is still used to purify the peptides of this type. However, the method is laborious and time consuming and has limited capacity. Emulsions are often formed during separation, which complicates the separation process very much. The purity of the peptides obtained is generally 90%.

Purification of peptides by electrophoresis is slow, and even when using complicated instrumentation, it has insufficient capacity. For the separation of substances with not very different isoelectric point it is necessary to use a very high voltage, sometimes 4 to 5 kV, which increases the working risk and poses a certain energy problem. Purity of peptides purified by electrophoresis is up to 90%.

The most commonly used method of separation of peptides is currently their gel chromatography on polymeric substances. However, only substances whose molecular weight is sufficiently different (e.g., peptide and salt or peptide and higher molecular by-product) can be separated by gel chromatography, so purification only by this technique cannot in many cases guarantee the desired purity of the product.

Therefore, it is necessary to combine several cleaning methods, e.g. gel and ion exchange chromatography, possibly using another separation technique. Thus, separation of peptides becomes a considerable time-consuming and lossy operation. The cleaning conditions cannot be reproduced in many cases.

At present, high pressure liquid chromatography with chemically coupled phases on silica gel is used to purify the peptides. Methanol-water mixtures are used as mobile phases, often having a pH adjusted to the desired value. The advantage is the high purity of the peptides thus obtained. The high cost of chemically bonded phases remains a disadvantage.

These drawbacks are overcome by the liquid chromatography method of the present invention. It consists in subjecting the peptides to liquid chromatography, the stationary phase of which is a silica gel having a particle size of 2 to 200 µm, preferably 5 to 25 µm, and the mobile phase comprises a 95: 5 mixture of alcohol or acetonitrile with water. % to 10:90, preferably 90:10, optionally a mixture of alcohol, acetonitrile and water with a water content of from 5 to 90% by weight, preferably from 10 to 20% by weight.

According to a further aspect of the invention, the alcohol used is methanol and / or ethanol and / or propanol and / or isopropyl alcohol. It is preferred to use a buffer of pH 3 to 8, preferably pH 4.4 to 4.8, in the mobile phase. As buffers alkali metal phosphates and phosphoric acid can be used. Finally, it is possible to add substances based on organic acid salts in an amount of from trace amounts to 5% by weight, forming ion feathers with the peptides.

The process according to the invention yields high purity of the peptides, mostly higher than 95 to 99%, using pure silica gel, which is considerably cheaper and more widely available, in place of expensive ion exchangers and / or chemically bonded phases.

The process according to the invention is described in more detail below according to several specific examples.

He did

1.0 g of crude peptides (l1-acid-3-mercaptopropionic, 8-D-argenine) vasopressin) dissolved in the mobile phase was loaded onto a 40 mm diameter, 270 mm length chromatography column packed with silica gel for particle size 10 chromatography. to 25 µm.

The material was eluted under increased pressure with a mobile phase consisting of 90% methanol, 10% aqueous phosphate buffer pH 4.6. The retention time of the purified peptides was 15 min. 0.72 g of peptides with a purity of 98% were obtained. The purity was verified by high pressure liquid chromatography using two different columns.

Example 2

0.1 g of (8-D-argenine) vasopressin was dissolved in 1 ml of a mixture containing 50% methanol and 50% water and not sprayed onto an 8 mm, 250 mm long column packed with silica gel for chromatography, particle size 7.5 µm. . The flow rate of the mobile phase, containing 90% methanol and 10% aqueous phosphate buffer at pH 4.6, was 100 ml / h. The retention time of the purified peptides was 30 minutes, the total length of the split was 45 minutes. 0.8 g of peptides of 93% purity were obtained.

Example 3

0.1 g of (2-O-methylthyrosine) oxytocin was dissolved in the mobile phase and injected onto a 206546 8 mm, 250 min. Column packed with 10 (ton) silica gel. The mobile phase was a mixture of 80% methanol and 20% The total purification time was 25 minutes, the retention time of the purified peptides was 10 minutes, yielding 0.7 g of peptides of purity greater than 98%.

Example 4

LHRH - releasing factor for luteinizing and follicle stimulating hormone was purified. 0.08 g of this material was injected onto the column as in Case 2 and eluted with a mobile phase containing 80% methanol, 7% acetonitrile and 13% water. 0.055 g of purity hormone higher than 99% was obtained.

Claims (5)

SUBJECT OF THE INVENTION CLAIMS 1. A method for purifying peptides by liquid chromatography, characterized in that the peptides are subjected to liquid chromatography, the stationary phase of which consists of silica gel having a particle size of 2 to 200 [mu] m, preferably 5 to 25 [mu] m. water in a ratio of 95: 5 to 10:90, preferably 90:10, optionally a mixture of alcohol, acetonitrile and water with a water content of from 5 to 90% by weight, preferably 10 to 20% by weight. 2. A process according to claim 1, wherein the alcohol is methanol and / or ethanol and / or propanol and / or isopropyl alcohol. Method according to Claims 1 and 2, characterized in that a buffer of from 3 to 8, preferably pH 4.4 to 4.8, is used in the mobile phase. 4. A process as claimed in any one of claims 1 to 3, wherein the alkali metal phosphate and phosphoric acid buffers are used. Process according to Claims 1 to 4, characterized in that substances which form ionic vapors with the peptides, for example based on salts of inorganic and organic acids, are added in an amount of trace amounts to 5% by weight.