Process for the separation of mixtures of insulin, insulin derivatives and, where appropriate, impurities

A method for separating insulin or certain insulin derivatives from mixtures containing the same by chromatography of the mixture on a column filled with silica gel having a particle size between 40 and 500 microns with an eluant mixture comprising chloroform, methanol, water and triethylamine.

The conversion in a semi-synthetic manner of natural pig insulin into human 
insulin by chemical or enzymic exchange of the alanine in position B 30 
for threonine has been disclosed. Obermeier, R., Geiger, R. Hoppe-Seyler's 
Z. physiol. Chemie 357, 759-767 (1976), Ruttenberg, M. A. Science 177 
623-626 (1972), Morihara, K., Oka, T., Tsuzuki, H. Nature 280, 412-413 
(1979), Inouye, K. J. Am. Chem. Soc. 101, 751-752 (1979), Schmitt, E., 
Gattner, H. G. Hoppe-Seyler's Z. physiol. Chem. 359, 799-802 (1978) and 
Bromer, W. W., Chance, R. E. Biochem. Biophys. Acta 133, 219-223 (1967). 
The aim of this conversion is to be able for immunological reasons to use 
insulin from the same species in the treatment of human diabetes mellitus. 
The advantages of this treatment can only be achieved when it is possible 
reliably to separate insulin derivatives and impurities from human insulin 
in the crude material from the semi-synthetic process. 
The known methods of separation are not suitable for reliably ensuring the 
separation of human insulin from pig insulin components. It can be deduced 
from the aminoacid anylses published in the literature that contamination 
with pig insulin up to an order of magnitude of 25% is possible in 
semi-synthetic human insulin. 
In order to prevent even trace contamination of human insulin with pig 
insulin or enzymes by the repeated use of packing materials for 
chromatography columns, which can be regenerated, a cheap disposable 
material having an optimal separation efficiency is desirable as a column 
packing. In analytical high pressure liquid chromatography (HPLC), 
high-resolution chromatographic separations are achieved on hydrophobic 
silica gel which has been derivatized with organic moieties (reversed 
phase). Pickart, Thaler, Preparative Biochemistry 5 [1975] 397-412 propose 
a method for the purification of somatomedin-C insulin-like activity and 
.sup.125 I-bovine insulin by high pressure liquid chromatography (HPLC) on 
columns prepacked with silica gel of 10 micron size using an eluant 
mixture consisting of methanol, water and formic acid. It is taught at the 
top of page 403 that silica gel of 30-44 micron size was not useful in 
separating peptides or proteins. However, the high costs of these HPLC 
sorbents prohibit their use as disposable column packaging materials. 
It has now been found that the chromatographic separation of crude mixtures 
from enzymic semi-synthetic processes for insulin on columns which are 
packed with commercially available silica gel, using certain mixtures of 
organic solvents, such as, for example, chloroform or methylene 
chloride:methanol:water:triethylamine:formic acid, is superior to all 
other methods of separation. This superiority results, on the one hand, 
from the differences in the retention times, some of which are large, of 
the components of the mixtures to be separated, and on the other hand from 
the rapidity of running a column. The costs of the silica gel, which is 
discarded after one run, can be ignored compared to those of the processed 
insulin. 
Thus the invention relates to a process for the separation of mixtures of 
insulin, insulin derivatives and possible impurities by chromatography of 
the mixture in a column filled with a silica gel having a particle size 
between 40 and 500 microns, preferably between 40 and 125 microns, using 
an eluant mixture comprising chloroform, methanol, water and 
triethylamine. 
"Insulin" is understood to mean human insulin or animal, in particular 
mammalian, insulin (e.g., porcine insulin). 
"Insulin derivative" is understood to mean degradations products of 
insulin, insulin esters and or other protected insulins (e.g. human 
insulin-B30-(But).sub.2 or B.sub.1 -BOC-human insulin-30-(But).sub.2). 
The process is particularly suitable for the purification of insulin and 
for the separation of insulin or its degradation products from trypsin or 
similar enzymes and insulin esters or other insulin derivatives. 
All commercially available silica gels for column chromatography having a 
particle size between 40 and 500 microns are suitable as a column packing, 
for example the commercial product "Kieselgel 60" from Merck & Co., 
Darmstadt, available in particle sizes between 40 and 500 microns. 
For the chromatography, it is possible to use as the eluant all those 
mixtures of eluants in which insulin esters or derivatives on the one hand 
and insulin or its degradation products on the other hand exhibit 
differing Rf values on commercially available thin layer chromatography 
plates. Particularly good separation effects are achieved when the crude 
product is chromatographed in an eluant mixture composed of 1,500-2,100 
parts by volume of chloroform, 1,000-1,500 parts by volume of methanol, 
350-450 parts by volume of water, 35-55 parts by volume of triethylamine 
and 0-15 parts by volume of formic acid. The eluant mixture may 
furthermore contain 0-1800 parts by volume of methylene chloride and/or 
0-350 parts by volume of a 2-[(C.sub.1 -C.sub.4)-alkoxy]-ethanol, 
preferably 2-methoxyethanol(methylglycol). 
The process is particularly advantageous for the separation of human 
insulin-B 30 -di-tert.-butylthreonine from pig insulin and trypsin, a 
mixture which arises in the enzymic conversion of pig insulin into human 
insulin.

USE EXAMPLES 
1.4 g of a mixture of 70% human insulin-B 30-di-tert.butylthreonine and 30% 
pig insulin are dissolved in 20 ml of a solvent composed of 
chloroform:methanol:water:triethylamine:formic acid=1,800:1,500:375:45:9 
(v/v) and applied using a disposable syringe onto a dry size C Lobar.RTM. 
ready-packed column (particle size of silica gel is between 63 and 125 
microns). The column is developed with the same solvent. After the solvent 
front has appeared, individual fractions (10 ml) are collected. 
Human insulin-B 30-(But).sub.2 appears in fractions 37-53. These are 
combined and concentrated to a volume of 30-50 ml on a rotary evaporator. 
About 500 ml of acetone are added to this solution and the precipitated 
product is isolated by centrifugation. 
The pig insulin is eluted in the fractions 120-137 and is isolated as 
described above. The two products are dried under slight vacuum. 
Yield: 2.59 g of human insulin-B 30-(But).sub.2 ; 1.03 g of insulin (P) 
2. N-.alpha.B.sub.1 
-tert.-Butyloxycarbonyl-B30-di-tert.-butylthreonine-insulin (4 g) are 
dissolved as in Example 1 and applied to a column (2.5.times.50 cm) filled 
with 300 g of silica gel "Kieselgel 60" having a particle size between 40 
and 63 microns, which has been equilibrated with chloroform. The column is 
eluted with the same solvent mixture as in Example 1. The peak appearing 
first contains B.sub.1 -BOC-insulin-B30-(But).sub.2, and pig insulin is 
eluted in the late fractions. The working up is as in Example 1. 
3. A mixture as in Example 1 (4 g) is applied to a Lobar.RTM. ready-packed 
column C equilibrated with 70% ethanol/30% tris buffer (0.05M, pH 8.0), 
and is chromatographed with the same eluant. The appropriate fractions, 
which contain human insulin B30-di-tert.-butylthreonine and pig insulin 
separated from one another, are worked up as in Example 1. 
4. Impure pig insulin (4 g) is dissolved in a mixture of 
chloroform:methanol:water:triethylamine:formic acid=1,200:1,100:370:47:11 
and chromatographed on a silica gel column as in Example 2. The eluate, 
which is fractionated in 10 ml portions, is worked up as in Example 1. 
Fractions which, after checking by HPLC and polyacrylamide gel 
electrophoresis, contain pure pig insulin are combined. 
Yield: 3.61 g. 
5. A mixture as in Example 1 (4 g) is applied to a column (2.5.times.50 cm) 
packed with 500 g Grace.RTM. silica gel 50 (average particle size 50 
microns) in methylene chloride. The eluant is composed of 
chloroform:methylene chloride:methanol:water:triethylamine:formic 
acid=900:900:1500:375:45:9 (vol./vol.). Fractions are collected and 
isolation of the products is performed as described in Example 1. 
Yields: 2.65 g human insulin-B30-(Bu.sup.t).sub.2 ; 1.1 g porcine insulin 
6. 3.8 g of a reaction mixture obtained by splitting off the protective 
groups of semisynthetic human insulin-B30-(Bu.sup.t).sub.2 by means of 
trifluoro-acetic acid is applied to a column (2.5.times.50 cm), filled 
with 300 g "Kieselgel 60" having a particle size between 63 and 200 
microns, which has been equilibrated with chloroform. The column is eluted 
with a mixture composed of chloroform:methanol:water:triethylamine:formic 
acid:2-methoxyethanol=1,500:1,400:450:55:14:190 (vol./vol.). The 
appropriate fraction, which contains human insulin, is worked up as in 
Example 1. Yield: 2.5 g human insulin.