Process for the purification of a crude peptide by preparative medium pressure liquid chromatography

(42) Date of commencement of the patent: 15. 6.1994 (45) Date of issue: 25. 1.1995

<td>(56) Documents:</td><td>(73) Patent owner:</td>

<td>DE-0S3124818 DE-0S3317092 US-PS4623716</td><td>GEBRO BROSCHEK SOCIETY MBH</td>

<td>CHEMICAL ABSTRACTS 99, 64410K (1983)</td><td>A-6391 FIEBERBRUNN, TYROL (AT).</td>

<td>CHEMICAL ABSTRACTS 101, 51009W (1984)</td><td>(72) Inventor: SCHÄFER DORIS DR. ARISOORF (CH).</td>

CQ (54) PROCEDURE FOR PURIFYING A RAW PEPTIDE BY PREPARATIVE MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY (57) To purify a crude peptide by preparative medium-pressure liquid chromatography, the peptide to be purified is applied in as concentrated a form as possible to a chromatographic column containing alkylated highly condensed polysilicic acid with a grain size of 20 to 90 microns and with a pore size of 6 to 30 nanometers as the stationary phase. Then, it is eluted at a pressure of 0 to 40 bar with a mobile phase, with the addition of at least one chelating agent in low concentration and at least one staik polar organic compound in low concentration. The mixture is separated and the resulting eluate is fractionated.

Peptide compounds with a purity of more than 99% can be obtained.

AT 398 767 me-resis

AT 398 767 Β

The present invention relates to a process for purifying a crude peptide by means of preparative medium pressure liquid chromatography, wherein the peptide to be purified is applied to a chromatography column containing reversed-phase silica material ie contains alkylated highly condensed polysilicic acid having a grain size of 20 to 90 micrometers and having a pore size of 6 to 30 nanometers as the stationary phase, at a pressure of 0 to 40 bar with a mobile phase either isocratic or eluted with a gradient, the mixture is separated and the eluate obtained in this way is fractionated, and wherein the peptide to be purified is applied in the most concentrated form possible to the column and the mobile phase of (a) a mixture of at least one organic solvent and water, or (b) a mixture of at least one organic solvent and a buffer solution having a pH of from 2 to 10, or (c) a pure aqueous buffer solution having a pH range of 2 to 10, consists.

The principle of solid phase synthesis of peptides is described by RB Merifield, for example, in Angewandte Chemie, Vol. 97, (1985), pages 801 to 812. In the International Journal of Peptide and Protein Research, Vol. 25, (1985), pages 449 to 474, M.Bodanszky describes the principle of liquid-phase peptide synthesis.

EP-A 37 516 describes a solid-phase synthesis of a vasopressin derivative according to Merrifield by means of purification on molecular sieves (separation according to molecular weight). Similar purification is also described in U.S. Patent 4,148,787 and in Collection Czechoslov. Chem. Comm., Vol. 31, (1966), pages 4581-4591.

In US Pat. No. 4,093,610, purification on ion exchangers or on molecular sieves is described in connection with a liquid-phase synthesis.

Further purification methods are described in DE-OS 27 23 453 and DE-PS 1 643 273, the latter method being feasible only for amounts in the mg range.

The problem of peptide purification has also been described by G.Fouret et al. in Int. J. Peptide Protein Res., 13, (1979), pages 137 to 141.

For the rest, reference is also made to the following references belonging to the prior art:

K.Noda in Int. J. Peptide Protein Res., 19, (1982), pages 413-419;

Collection of Czechoslov. Chem. Comm., Vol. 32, (1967), pages 1250-1257;

EP-A 112 809, which uses non-economic HPLC;

U.S. Patent 4,495,097;

V.du Vigneaud et al., (1960), J. Biol. Chem. Vol.235, No.12, pages 64-66.

More specifically, certain purification methods are described in J. Chromatog., 38, (1968), pages 396-398, and

M. Zaoral et al., Collection Czechoslov. Chem. Comm., Vol. 43, (1978), pages 511-522.

With all these known cleaning methods, generally acceptable purity levels of 92 to 94% can be achieved. This has the consequence that in a medical use of a peptide compound always had to prove that the accompanying by-products have no adverse consequences, effects and disadvantages, which was not easy, since it often did not know what compounds by-products existed. This condition is unsatisfactory, especially as more and more peptide compounds are used in medicine.

Furthermore, it is known from DE-OS 31 24 818 to carry out the purification of a peptide compound by chromatography in a column with reversed phase in a methanol trifluoroacetate buffer mixture. This gives only low yields. Similar disadvantages exist for a method according to DE-OS 33 17 092, in which a peptide compound is dissolved in a mixture of methanol and water and applied to a column of octadecyl-modified silica gel and the elution is carried out with a mixture of methanol and aqueous trifluoroacetic acid becomes.

Finally, it is known (US Pat. No. 4,623,716) to use a high-pressure liquid chromatography process for purifying a peptide compound, in which a gradient of 10-40% of ethanol is eluted in two moles of aqueous acetic acid with an addition of ammonium hydroxide solution. Again, there are low yields.

The invention has for its object to provide an economical purification process for peptide compounds, with which purities of over 99% can be achieved, such a purification process with high yields on an industrial scale, ie with a way to work in the gram range, to be feasible. On the basis of the known literature, it had to be assumed that it is not possible to find such a process which achieves the desired purity in an economical manner.

Completely surprisingly, such a method has now been found.

AT 398 767 Β

The process according to the invention consists hiebei that, starting from the method described above, in which the mobile phase consists of a mixture or a buffer solution according to one of the variants (a), (b) or (c), the mobile phase in all these three cases (a) to (c) additionally

1. as chelating agent at least one aminocarboxylic acid or nitrilocarboxylic acid in an amount of 0.01 to 0.1 vol .-%, in particular 0.05 vol .-%, based on the total volume of the mobile phase, and

Second at least one strongly polar organic compound, namely hexamethylene phosphorus triamide, dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and N-methylpyrrolidone (NMP) in an amount of 0.05 to 1 vol .-%, in particular 0.5 vol .-%, based to the total volume of the mobile phase in combination.

The process according to the invention has the advantage that it is extremely economical. It is furthermore very advantageous that the process according to the invention can be carried out in a low pressure range. Likewise, the column packing material is relatively inexpensive because irregular rather than spherical particles can be used in the process of the present invention. In comparison to corresponding HPLC material, the column filler used according to the invention is currently about 20 times cheaper. During purification, the peptide compounds are neither denatured nor show a loss of activity; the method according to the invention is thus extremely gentle for the peptide compounds. By suitable choice of the flow agent, the peptide compounds can be separated quickly and selectively. The purified peptide compounds separated according to the invention surprisingly have a purity of more than 99%, which is due to the combined addition of the mentioned chelating agent and the mentioned, strongly polar, organic compounds. Furthermore, large amounts of solvents can be saved with the process according to the invention.

It is quite surprising that at high loadings of the columns there are significant improvements in the yield, not only in comparison with a process without chelator and without a highly polar organic compound, but also in comparison with a process in which either only the strong polar organic compound is used as an additive or only the chelating agent alone.

Although the use of EDTA and of DMF in connection with a liquid chromatography method is known from the reference Chemical Abstracts 99, 64410k (1983), these two substances are not added to the mobile phase, but used for the washing of the column, ie for the stationary Phase, and not in combination, but alternatively. Thus, this prior art is a pretreatment of the column with DMF or EDTA before leukotriene is purified by means of a conventional flow agent.

According to a preferred embodiment of the method according to the invention, the mobile phase contains, as a directing agent, ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid (NTA). This results in particularly favorable results:

The detection of the peptide compounds in the fractions is carried out, for example, in UV light at a wavelength of 232 nm or 275 nm.

To control the purity and the structure of the peptide in the fractions, the known methods can be used: for example, HPLC, DC, amino acid analysis, sequence analysis.

If a buffer is used in the flow agent, the corresponding fractions are advantageously desalted before the lyophilization, for example on ion exchangers or by means of molecular sieves.

In order to obtain the fractionated, purified according to the invention peptide as a solid, the corresponding fraction, for example, lyophilized.

With the purification method according to the invention it is possible, for example, to purify 20-50 g of crude peptide within 3 hours.

Peptide compounds which can be used either by Merrifield solid phase synthesis (Angewandte Chemie, Vol. 97, (1985), pages 810 to 812) or in liquid phase synthesis (Int. J. Peptide Protein Res., 25, (1985 ), Pages 449 to 474).

In particular, the following peptides can be purified with the method according to the invention: disulfide compounds, which have been produced according to a new process, which is essentially characterized that at least one oxidizing agent, which is covalently or electrostatically bound to a solid phase and which - is capable of oxidizing SH groups to disulfide bridges, in water while stirring, then slowly adding an aqueous solution of the peptide compound to be oxidized in such a way, that the concentration of the peptide to be oxidized is so low that the probability of an intermolecular reaction of the peptide molecules is negligibly small, and the reaction part 3

AT 398 767 Β participants react with vigorous stirring, and then worked up the crude, a disulfide bridge having peptide compound. However, it is also possible with the method according to the invention to purify peptides such as terlipressin of the formula

61y-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH_?.

CIS-Pressin the formula

Gly-Gly-Gly-Cys-Tyr-Phe-Asn-Cys-Pro-Lys-Gly-HN_?.

Hamburger peptide of the formula Asp-Ser-Asp-Pro-Arg, vasopressin and its derivatives, calcitonin, somatostatin and insulin. It is preferred to purify terlipressin with the method according to the invention; The terlipressin purified by the process of the present invention is extremely clean and has different characteristics than a commercially available, conventional terlipressin, due to the absence of impurities.

Corresponding structural formulas are described in:

Handbook of Biochemistry C-164 to C-188 - Amino Acid Sequences of Proteins, C-265 (Handbook of Biochemistry selected date for Moeecular Biology, 2nd edition (1970), Editor: Herbert A.SOBER, PhD, published by (The Chemical Rubber Co., Cleveland, Ohio, 44, 128), and

Int. J. Peptide Protein Res., 15, (1980), pages 342-354.

The following examples are intended to illustrate the present invention.

Example 1: terlipressin g terlipressin (crude peptide) of the formula

Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH_2&gt; dissolved in 750 ml of distilled water was applied by means of a MPLC system from Labomatic AG on a 45 x 880 mm column under a pressure of 15 bar. Pack: reverse phase material (Cts-60). The peptide was then mixed with a flux mixture of 26% methanol and 74% 0.1 M triethylammonium phosphate (TEAP) pH 2.45, 0.4% DMSO and 0.05% EDTA with a flow of 60 ml / min. eiuiert and fractionated. The detection of the peptide was carried out at 210 nm in the flow. The peptide was then detected by HPLC analysis in the fractions and the corresponding fractions desalted after lyophilization by known methods on an ion exchanger and lyophilized.

The purity of the resulting peptide was determined by HPLC, amino acid and sequence analysis. Yield: 83%, purity 99.8%. The analytical HPLC was carried out as follows:

<td>Säuiengröße:</td><td>4.6 mm x 250 mm</td>

<td>Stationary phase:</td><td>(alkaline highly condensed poly-silicic acid)</td>

<td>Mobile phase:</td><td>74% 0.1 molar triethylammonium phosphate, pH 2.25, 26% methanol</td>

<td>Temperature:</td><td>20 ° C</td>

<td>Print:</td><td>148 bar</td>

<td>River:</td><td>1.5 ml / minute</td>

<td>detection:</td><td>210 nm</td>

FIG. 1 shows the corresponding chromatogram. The ordinate corresponds to the optical density at 210 nm and the abscissa corresponds to the retention time in minutes.

AT 398 767 B

Example 2: CIS-Pressin

The procedure is essentially as in Example 1, but with the following deviations:

<td>Pillar:</td><td>52 x 480 mm</td>

<td>Pack:</td><td>Reverse phase material (Cis 200-100)</td>

<td>Eluent:</td><td>18% acetonitrile / 82% 0.12 Μ TEAP pH 2.9 0.52% DMF, 0.05% NTA</td>

<td>flow:</td><td>18 ml / min.</td>

<td>Print:</td><td>20 bar</td>

<td>detection:</td><td>232 nm</td>

<td>Sample size:</td><td>5 g dissolved in 200 ml dest.Wasser</td>

<td>Yield:</td><td>78%</td>

<td>Purity:</td><td>99.5%</td>

Example 3: Asp-Ser-Asp-Pro-Arg hamburger peptide

The procedure is essentially as in Example 1, but no desalting is necessary, but a direct lyophilization of the collected fractions. Other deviations were:

<td>Pillar:</td><td>37 x 1083 mm</td>

<td>Pack:</td><td>Reverse phase material (Ci s 30-60)</td>

<td>Eluent:</td><td>10% methanol / 0.1% trifluoroacetic acid (TFA) in dist. Water, 0.3% NMP, 0.045% EDTA</td>

<td>Print:</td><td>17 bar</td>

<td>flow:</td><td>25 ml / min.</td>

<td>detection:</td><td>210 nm,</td>

<td>Sample size:</td><td>3 g dissolved in 70 ml of dist. Water</td>

<td>Yield:</td><td>85%</td>

<td>Purity:</td><td>99.7%</td>