Process for recovering insulin

Insulin can be recovered for processing and re-use from an insulin-protamine complex by bringing the complex into contact with an aqueous medium having a pH of from about 2 to about 5 and containing an insulin dissociating or depolymerizing agent, a cation exchanger, and a salt at a concentration of from about 0.2 M to about 0.6 M.

BACKGROUND OF THE INVENTION 
In the development of insulin formulations suitable for treatment of 
diabetes mellitus, it became desirable to alter the characteristics of 
insulin to prolong its blood-sugar-lowering effect. It was discovered that 
this could be achieved by conversion of the insulin to a complex which was 
only partly soluble at the pH of body fluids but which, over an extended 
period of time, would dissociate with release of insulin. The result is 
much less rapid insulin absorption. 
When insulin is mixed with a properly buffered solution containing 
protamine, a protamine-insulin precipitate results which has poor 
solubility and therefore is only slowly absorbed from body tissue. As a 
result of this finding, several protamine-containing commercial 
proinsulins are available, including protamine zinc insulin (PZI) and NPH 
insulin. Either of these insulins, upon subcutaneous injection makes 
available a depot of supply from which insulin is slowly made available 
and paid out into the body fluids. 
Due to the large quantities of protamine-containing insulin formulations 
that are produced for use by diabetics, it is natural that certain amounts 
are returned for any of several reasons, including, for example, 
outdating, lack of refrigeration, and miscellaneous cosmetic defects. It 
is highly desirable to have a facile and efficient method available for 
recovering high purity insulin from formulations containing the 
protamine-insulin complex. Such insulin then could be re-processed and 
formulated for distribution to diabetics. 
An examination of the literature indicates that, although it contains in 
abundance papers directed to the extraction and purification of insulin 
from pancreas glands, it fails to address methods for recovering insulin 
from protamine-insulin complexes. It is to such a method that this 
invention is directed. 
SUMMARY OF THE INVENTION 
Therefore, this invention defines a process for recovering insulin from an 
insulin-protamine complex, which comprises (1) bringing the 
insulin-protamine complex into contact with an aqueous medium having a pH 
of from about 2 to about 5 and containing an insulin dissociating or 
depolymerizing agent, a cation exchanger, and salt at a concentration of 
from about 0.1 M to about 0.6 M, and (2) recovering insulin having reduced 
protamine content. 
DETAILED DESCRIPTION OF THE INVENTION 
As noted, this invention comprises a process for recovering high purity 
insulin from an insulin-protamine complex. The process permits recovery of 
insulin at levels representing 90-100% of total insulin with substantially 
complete removal of the protamine. 
In general, the process of this invention involves the use of a cation 
exchanger. Typical cation exchangers include, for example, SP 
(sulfopropyl) Sephadex, CM (carboxymethyl) Sephadex, SE (sulfoethyl) 
cellulose, CM Trisacryl and the like. Of the above, SP Sephadex is 
preferred. 
Essentially, insulin is recovered from insulin-protamine by contact under 
suitable conditions with the cation exchanger. 
Insulin molecules tend to associate forming complexes and polymers, 
typically as dimers and hexamers. Suitable conditions under which contact 
with the cation exchanger is effected include, therefore, the use of an 
aqueous medium containing an agent that exhibits an insulin dissociating 
or depolymerizing effect. Examples of suitable dissociating and 
depolymerizing agents are urea; guanidine; lower alkyl alcohols, such as 
methanol and ethanol; amides, including N,N-dialkylamides such as 
N,N-dimethylformamides and N,N-dimethylacetamide, acetamide, N-alkylamides 
such as N-methylacetamide; nitriles, such as acetonitrile, and the like. 
Any of these are effective in producing monomeric insulin or at least in 
promoting insulin dissociation. A requirement of the insulin dissociating 
agent is that it be water soluble or water miscible. Of the foregoing 
non-exhaustive list of insulin dissociating agents, urea and lower alkyl 
alcohols are preferred, and urea is most preferred. When a lower alkyl 
alcohol is used, it preferably is present in an amount, based by volume 
upon the total medium, of about 30% to about 70%, and, preferably, of 
about 40% to about 60%. When urea is used, it generally is present in the 
aqueous medium at a concentration of from about 2 M to about 8 M, 
preferably from about 5 M to about 8 M, and, most preferably, about 7 M. 
In addition to the use of an insulin dissociating or depolymerizing agent, 
the process of this invention is carried out in an acidic environment at a 
pH of from about 2 to about 5, preferably from about 3.5 to about 4.5, and 
at a temperature below about 25.degree. C. and above the freezing point of 
the aqueous medium. Preferably, the process is carried out with cooling, 
the temperature being in the range of from about 4.degree. C. to about 
8.degree. C. The process generally is run for a period no longer than 
about 4 hours and usually much less, e.g., for from about 30 minutes to 
about 1-2 hours. 
Any of a wide range of inorganic and organic acids can be used to lower the 
pH of the mixture to the desired range. Preferred acids are hydrochloric 
acid, sulfuric acid, and acetic acid, and, of these, hydrochloric acid or 
a mixture of hydrochloric acid and acetic acid are most preferred. 
The process is carried out in the presence of a salt. The salt is dissolved 
in the aqueous medium at a defined molar concentration. In general, the 
molar concentration ranges from about 0.1 M to about 0.6 M. The preferred 
range is from about 0.2 M to about 0.5 M with specific highly preferred 
ranges being dependent upon the particular cation exchanger that is 
employed. Thus, for example, when SP Sephadex is used the desirable salt 
concentration is from about 0.35 M to about 0.5 M. When a salt 
concentration at the lower end of the range is employed, some insulin loss 
due to retention on the SP Sephadex may be experienced. Conversely, a salt 
concentration at the higher end of the range, although achieving greater 
insulin recovery, may result in the presence of undesirable insulin 
contaminants. For CM Sephadex, the desired range is from about 0.2 M to 
about 0.3 M, and for SE cellulose, from about 0.35 M to about 0.5 M. Any 
of a wide range of inorganic salts can be used; the preferred salt, 
however, is sodium chloride. 
Although the process of this invention can be carried out using a variety 
of techniques and sequences, the following is typical of the general 
procedure. The insulin-protamine complex is placed in an acid aqueous 
medium containing the desired concentration of insulin dissociating or 
depolymerizing agent. The resulting mixture then is brought into contact 
with the selected cation exchanger, the appropriate quantity of salt is 
added, and the resultant mixture is maintained at the desired temperature 
to permit adsorption by the cation exchanger of protamine and insulin from 
the insulin-protamine complex with accompanying selective desorption of 
insulin from the cation exchanger. The protamine-free insulin is readily 
separated by filtering the supernatant from the cation exchanger and, in 
accordance with standard techniques, isolating the insulin from the 
supernate.

EXAMPLE 1 
An accumulated return stock slurry (20 liters) of insulin-protamine 
precipitate was filtered to obtain approximately 5 kg. (wet weight) of 
solids. To the solids were added 8 liters of a 7 M urea, 0.1 M acetic acid 
buffer. The pH of the resulting mixture was adjusted to 3.7 by addition of 
630 ml. of 10% HCl, and the solids were dissolved by agitation. The filter 
papers were washed with 2 liters of the urea-acetic acid buffer, and the 
wash was added to the insulin-protamine solution. 
The resulting mixture was filtered through fiberglass to obtain 16.57 
liters of filtrate, OD.sub.278 =87.0. (Insulin by radioimmunoassay=1503 
U/ml.) 
Dry SP Sephadex (1500 grams) was swelled in 30 liters of 7 M urea, 0.1 M 
acetic acid buffer. Excess buffer was decanted from the swelled SP 
Sephadex, and the insulin-protamine-containing solution was added to the 
SP Sephadex, producing a total volume of about 33 liters. 
Sodium chloride (965 grams) was added to produce a 0.5 M solution. The pH 
was adjusted to 3.7 by addition of 130 ml. of 10% HCl. The resulting 
mixture, maintained at about 5.degree. C., was agitated for 2 hours. The 
Sephadex then was washed twice with 5 liters of pH 3 water. The washes 
were added to the filtrate. 
The insulin-containing filtrate (29 liters, OD.sub.278 =42.0), containing 
752 U/ml. insulin as determined by radioimmunoassay, was diluted with 60 
liters of chilled purified water. Ethanol (10 liters) was added, and the 
pH was adjusted to 5.9 with 300 ml. of 28% ammonium hydroxide. To the 
mixture then were added 200 ml. of 20% ZnCl.sub.2 solution. The mixture 
was agitated for 2 hours and allowed to stand overnight at about 5.degree. 
C. Zinc insulin crystals were recovered by filtration. The filtrate (about 
90 liters) had an OD.sub.278 =3.85. Radioimmunoassay showed an insulin 
concentration of 0.1 U/ml. 
EXAMPLE 2 
Wet insulin-protamine return stock precipitate (27.5 kg.) was dissolved in 
47.5 liters of 0.1 N acetic acid-7 M urea buffer. The solution was 
filtered through a Buchner funnel, and 20 liters of the buffer were added 
as a wash. The resulting mixture, 96 liters, had an insulin activity of 
1493 U/ml. 
The above solution and 3.7 kg. of NaCl were added to 20 kg. of swollen SP 
C-25 Sephadex. The pH of the resulting slurry was adjusted to 3.7 with 10% 
Hcl, and the mixture was stirred for 2 hours at 5.degree. C. The slurry 
then was filtered using a 30.mu. mesh Buchner funnel. Water (50 liters) 
adjusted to pH 3 was passed through the filter as a wash and added to the 
filtrate. The resulting filtrate (total volume-158 liters) showed by 
radioimmunoassay an insulin content of 835 U/ml. The product, free of 
protamine, represents an overall insulin recovery of 92.0%. 
The insulin was crystallized according to standard methods using ZnC.sub.2, 
274 liters of water, 48 liters of ethyl alcohol, 6.15 liters of glacial 
acetic acid, and 24.5 liters of 6 N NH.sub.4 OH to provide an adjusted pH 
of 5.9. 
EXAMPLE 3 
Use of Various Cation Exchangers 
Insulin-protamine return stock precipitate (about 150 g.) was dissolved in 
600 ml. of 0.1 N acetic acid-7 M urea. The solution was filtered and the 
pH of the filtrate was adjusted to 3.7. The mixture was divided into 6-100 
ml. portions. A 100 ml. portion, assayed by radioimmunoassay, showed a 
total of 163,200 U insulin (1632 U/ml.), OD.sub.278 =82.7. 
To each 100 ml. portion were added 50 g. of a cation exchanger, the desired 
quantity of NaCl, and 7 M urea to a resulting total volume of 200 ml. The 
mixture was adjusted to pH 3.7. Using this procedure, the following six 
samples were prepared: 
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Cation Exchanger 
NaCl, M 
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A CM Sephadex 0.2 
B CM Sephadex 0.35 
C CM Sephadex 0.5 
D SE Cellulose 0.2 
E SE Cellulose 0.35 
F SE Cellulose 0.5 
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Each of the mixtures was stirred at 5.degree. C. for 1.5 hours, and the 
supernatant then was removed by filtration. Analysis of the resulting 
filtrates gave the results shown in the table following. 
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Total Insulin, 
Insulin Recovery, % by 
Filtrate, 
U Radioimmuno- 
Protamine 
Sample 
ml. Radioimmunoassay 
OD.sub.278 
assay OD.sub.278 
Content 
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A 192 143,808 38.9 
88.1 90.3 
None 
B 182 139,594 40.5 
85.5 89.1 
Trace 
C 185 153,550 42.1 
94.1 94.2 
Minor 
D 173 97,572 20.6 
59.8 43.1 
None 
E 170 117,130 38.8 
71.8 79.8 
None 
F 157 127,327 43.2 
78.0 82.0 
None 
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