CA1112639A - Mono-insulin and method of preparing the same - Google Patents
Mono-insulin and method of preparing the sameInfo
- Publication number
- CA1112639A CA1112639A CA269,730A CA269730A CA1112639A CA 1112639 A CA1112639 A CA 1112639A CA 269730 A CA269730 A CA 269730A CA 1112639 A CA1112639 A CA 1112639A
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- Prior art keywords
- insulin
- extract
- process according
- reverse osmosis
- substances
- Prior art date
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
- C07K14/625—Extraction from natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Abstract of the Disclosure: The present invention relates to monocomponent insulin purified to such a degree that it shows only a single component when analyzed by amide gel electrophoresis using a gel containing 15% poly-acrylamide. It, furthermore, relates to a method of preparing such insulin from an insulin containing raw extract prepared from pancreas glands, consisting in the working up of the extract in such a manner that the insulin is maintained in dissolved state during the whole processing, without change of phase, until the final recovery of the insulin, the undesired substances being from the beginning of the process until the end removed from the different solvents used. A preferred method comprises removal of fat from the extract by cooling to a temperature below -25°C and separation of the crystallized fat, concentration of the extract and simultaneous removal of impurities present in the extract, both substances having a larger molecule than insulin and substances having a smaller molecule than insulin, by means of reverse osmosis plant for removal of colouring substances and salts, further purification by means of ion exchange and recovery of the insulin from the concentrated purified solution resulting from the ion exchange treatment by precipitation with metal ions under strong cooling. The monocomponent insulin may be used in insulin preparations of any kind for clinical use.
Description
The invention relates to insulin having very little or no antigenicity and a process for its production. It, furthermore, relates to preparations containing such insulin.
It is now generally assumed that the antigenicity in insulin preparations is mostly due to im~urities in the preparations, whereas it was praviously believed that the insulin antibodies were-produced by the insulin as such.
These impurities may be accompanying proteins from pancreas distinct from insulin, proin5ulin which is a precursor of insulir, intermediate insulin, the dimer, arginine insulin, ethylester insulin, desamido insulin desamidised to various extents, and other insulin modifications.
Eforts have therefore been made in order to produce ;
;~insulin preparations consisting of pure insulin, the so~
called monocomponent insulin, free of impurities and accompanying substances of any kind.
~;~For that purpose it has been proposed to subject amorphous or crystalline insulin prepared in a conventional ;
manner to an extensive further purification, e.g. by gel filtration and/or ion exchange treatment~
The resulting highly purified insulins show a strong ~;
decrease in antigenicity, but not a complete removal there- ~ ;
of. This is no doubt due to the fact that in spite of the purification steps the purified preparations still contain substances different from insulin. --The invention is based on the recognition that someof the impurities present in insulin are formed during the recovery itself of insulin, the usually used processes re-sulting in-ter alia in a decomposition o the insulin and formation of aggregates. The insulin as it is accumulated in the pa~creas glands, from which the insulin is re-covered, is the pure monomer (having a molecular weight of about 6000), and it is extracted as such by conventional extraction with an acidic aqueous 60-80 per cent alcohol.
In the further conventional processing, which inter alia comprises a separation of fat by vacuum distillat,on of .:
the extract at 25-30 C, whereby the alcohol is evaporated and an aqueous extract with a strongly reduced alcohol . 10 content is obtained, the monomer is, however, subjected to :;
conditions resulting in decomposition - inter alia due to the detrLmental action of the enzymes which can display their activity in aqueous solution (solutions having an al-cohol content less than 50 per cen~), where.as they are not active in alcoholic solution (over 'iO per cent alcohol) -formation of aggregates, etc.
According to the invention it is therefore proposed to carry out the preparation of insulin in such a manner that the insulin, from the extraction from the pancreas :~
glands until tha obtaining of the final product, is only subjected to conditions of such a nature that they do not ~:
cause the formation of decomposition products, aggregates etc.
~he present invention, then, in one aspect, resides in a process for purif.ying an insulin-containiny extract prepared from pancreas glands, whereln the extract is ::~
worked up in a plurality of steps to pure.insulin, characterized in that the insulin is maintained in a solvent environment during the whole process, the undesired substances being from the beginning of the process until the end removed from the solvent or solvents used, said process cvmprising removal ", ,~
t~t.~ 3 of fat by cooling of the extract to a temperature below -25C
followed by separation of the crystallized fat, reduction of the volume of the extract, by use of reverse osmosis or gel filtration or by ion exchange treatment, and further purification of the extract by means of such processes as reverse osmosis or treatment with ion exchangers or molecular sieves,in which the insulin remains in a solvent environment; and, where decired, recovery of the insulin from the purified extract.
Also provided by the present invention ia mono-insulin ~;
purified to such a degree that it shows only a single ` component when analyzed by polyacrylamide gel electrophoresis using a gel containing 15% polyacrylamide.
By "in a solvent environmentl' we mean that the insulin is in solution or a like state. It may remain at all times in the liquid phase in solution or if for example it is ;~ retained on an ion exchange material will be protected by the solvent and hence still in the desired environment.
., ~
Insulin is very easily soluble in an acidic alcoholic extract of from about 55 to about 80 per cent by volume of alcohol, and in such an e~tract the insulin will, i the extract ls treated expediently, no~ decompose and form aggregates with itself or with impurities in the :
-3a-3~
alcoholic extract In a preferred embodlment of the process accordingto the lnvention the insulin is extracted from the pancreas glands with such an aqueous acidic alcohol, and the insulin is maintained in pure and dissolved state during the further ~ processing, without change of phase, until the isolation ;~ of the end product, e.g. by precipitation and possible -~ crystallization.
; Use can of course be made of other e~tractants than acidic aqueous alcohol, extractants in which insulin is easily soluble and in which it will not form aggregates with itself or with impurities in the extract, e~g. aqueous acetone.
In the process in question it is not a question o~
a purification of the insulin itself, but a purification of the obtained insulin extract takes place to remove dissolv-ed impurities and fat and undesirable proteins and deriva-tives of insulin, until the pure insulin remains alone in the extract (or in another liquid phase).
The process may for instance be carried out in the following manner~
Comminuted frozen pancreas glands are extracted with ethyl alcohol (60-80 per cent by volume) acidified with hydrochloric acid to about pH 3. After separation of the pancreas mass the pH of the extract is adjusted to about 8, whereby certain proteins different from insulin are preci-pitated and separated. The pH is again reduced to about 3.
Instead of using this so-called "pH-8 precipitation" for ~J
'~ ~
separating undesirable proteins one can send the acid extract with a p~-value about 3 through an ultrafiltra- -tion/hyperfiltration plant, see below, using an appropriate membrane retaining the said proteins, i.a. enzymes.
The resulting clear extract is ccoled down in a special fat ` freezing plant to a temperature of about -30 to -45C, ; preferably a~out -35C, whereby the fat is crystallized in ~ compact fat crystals of small surface. These crystals are .
separated, e.g. by centrifuyation, at the same low tempe-rature. At this low temperature of -30 to -45C and with the constant high alcohol content of 60-80 per cent by volume, no harmful effect on the insulin takes place as is the case in the conventional fat removal by vacuum de-stillation at 25-30C with decreasing alcohol content di-the extract.
Further details regarding this fat separation by ~-freezing out are disclosed in Canadian Patent 1~o4l/9~o2 of W.H. Jensen et al., granted November 7, 1978.
After this gentle separation of the fat a comparative-ly large volume of fat~free extract is available, and this large volume has to be reduced, also in a manner which is harmless to the insulin.
Gel filtration and/or ion exhange treatment may be used, but according to the invention the following method is preferred:
Use is made of an ultrafiltration/hyperfiltration plank working according to the membrane method, the 50-called reverse osmosis system (cf. for instance US Patent 6~
" ~::
Patent No. 3,623,610).
By using this method one can, by an appropriate choice of membrane types, obtain, together with a con-centration of the volume of liquid, a separation of insulin from impurities present in the extract, both substances having a larger molecule than the insulin as well as substances having a smaller molecule.
One achieves the removal not only of undesirable . :
substances of pancreatic origin which are dissolved in the extract, but also of any possible o~her molecules of ~ ~
non-pancreatic origin having a size different from that ~ ;
of the insulin, which the slaughterhouses might by ~
error or inadvertence have included in the pancreas supplies,and thereby also are to be found in the extract.
The separation at that momen1: of by far the major part~of these molecules, both larger and smaller than the insulin molecule, by ultra- and hyperfiltration is of ;~
decisive importance to the obtaining of the mono--.:
~ component insulin, since these impurities are here separat~
ed at a moment when they are in solution and when they have had no posslblity of displaying a detrimental effect ~ ;~
on the dissolved insulin, inter alia on account of the low temperature during the preceding process steps.
The fat-free clear extract is pumped through the ultrafiltration apparatus, which is provided with cooling -means for the extract, for instance at a temperature be- -~
tween a~out ~10C and lCC,preferably between 0C and -10C, and at a pressure of 2-20 atmospheres, preferably 6 f, ?b.~J~
~, .
atmospheres, the type of membrane being chosen so that the '` large un~esirable molecules are separated, down to a size as~ close to the size of the insulin molecule as possible. As * * ~.
membran~s e.g. GRX~6 or GRX-7 (polysulphone membranes manufac-S tured by De Danske Sukkerfabriker A/S) can be used. The large ,~molecules remain in the concentrate, whereas the insulin and the , . ,~-, ~.
impurities of small,er ,molecule size than that of the insulin form part of the permeate. This permeate is thereafter passed through the ultrafiltration apparatus wherein the type of membrane is O chosen so that the major part oi the undesirable molecules smaller than the insulin molecule are separated, up to a size as close ', to the slze of the insulin molecule as possible. As membranes e.g. GR8~P or 930 (~ellulose acetate) both manufactured by ~: .
De Danske Suk~erfabriker A/S can be used. The insulin remains in the concentrate. The extract can be concentrated as strongly ''~
as .lesired, e.g. to 1~5 to 1/40 of the original volume. The , ~' concentration ra~io alcohol : water remains the same as in the starting extract.
The membranes used in the ultra- and hyperfiltration plant must be able to resist cQncentrated alcoh~lic solutions of for instance 62 per cent alcohol. Use can ~or instance - besides of membranes made of polysulphones and of cellulose acetate as mentioned above - be made of membranes of other polymers, such ~, as certain polyamides.
'~ , , Wi~h regard to the construction of the ultra- and hyper filtration plant, reference can, for instance be made to the following patent specifications: US patent No. 3,872,015, British patent No. 1,390,671 and Swiss patent No.- 542,639, * Trade ~arks The resulting concentrate, which contains practically all the insulin from the raw extract, is transparent but more strongly coloured than the raw extract on account of ; the concentration. The colouring substances are washed off ; with, for instance 62 per cent alcohol, over membranes which retain ~he insulin but allow the colouring substances to pass through~ The whole volume of washing liquid passes together wit~ the ;-`
said substances in the permeate, while the volume of the concen-trate remains unaltered.
Simultaneously with the washing off of the colouring sllbstances, the salts present in the extract and originating from the pancreas extr~action are washed of.
The rather few dissolved impu.rities,which still re~
main in the insulin-containing extract, can suitably be ~ :
separated by means of ion exchangers, cation exchangers as well as anion exchangers. The separation can also be ~- -effected by means of molecular sieve, e.g.'lSephadex G-50"
manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden, and other suitable separation methods known per se, but the use of ion exchangers is preferred. -~
Ion exchanging processes for the cleaning of insulin by the column chromatography method as well as by the batch method are known per se, quite particularly the column chromatography method.
It is however hitherto unknown to apply these methods to a concentrated alcoholic raw insulin extract free from fat The conventional alcoholic raw insulin extracts having all the fat dissolved therein will, especially by the column ; chromatography method, contaminate the ion exchangers with * Tracle Mark the impurities - the colouring substances and the fat -to such an extent that the ion exchangers after adsorp- -tiOII and elution once or twice will be so contaminated that it will be extremely difficult to purify and regenerate s the exchangers for re-use in the separation of insulin.
When using the ion exchanging or molecular sieve `~
processes it is a condition that the extract being treated has been freedîrom fat and partially also from colouring substances, undesirable proteins as well as other impurities to such an extent that the ion exchangers and the molecular sieve substances can be regenerated and purified so that they are fully applicable for a substantial period of time, as their use otherwise will be economically prohibitive.
Such a sufficient and necessary purification is 15- just obtained by the method described above for prepurifica-tion of the alcoholic raw insulin extract comprising crystalli~
zation of the fat at a temperature as low as about -40C and removal by ultrafiltration through a reverse osmosis system of part of the colouring substances as well as an substantlal ~ ;
part of the molecules which are larger than or smaller than the insulin molecule, including molecules quite close to the size of the insulin molecule.
Most of the available types of ion exchangers can be used, including ion exchangers on a resin and a cellulose ~ ;
basis as well as the Sephadex ion exchangers SP and QAE
known per se, which are cation exchangers and anion exchangers, respectively, and are modified crosslinked dextran chains having a tridimensional network of polysaccharide ~anufactur-*Trad~k ,~ "' !
ed by Pharmacia Fine C~emicals AB, Uppsala, Sweden). The ion exchangers"SP-Sephadex C-25"(cation exchanger) and QAE-Sephadex A--25 (anion exchanger) are preferred.
One can choose between carrying out column chromato-graphy or batch separation on the ion exchangers, orpossibly both methods can be used.
The ~atch method has been found to preser.t enormous advantages when used in larger industrial operation as a medium to fine puri~ication process inserted ~etween the se-paration and concentration by reverse osmosis and achromatographic purification using an ion exchanger column.
Batch separation is a very quick technique and the method causes no technical trouble as a consequence of swelling or shrinking of the ion exchanger. Separation on an ion exchanse~ can be carried out by binding the impurities and by allowing only the insulin to remain in solution. After the ion exchanger has been equilibrated in the suitable bu~fer chosen at the isoelectric point of the insulin, pH 5 2, the concentrated prepurified raw insulin extract ~;
is at pH 5 2 brought into contact with the ion exchanger and the whole is stirred for instance for 2 hours, where- ~;
after the mixture is filtered. All the impurities are bonded to the ion exchanger, whereas the pure insulin is present in a dissolved state in the alcoholic filtrate,
It is now generally assumed that the antigenicity in insulin preparations is mostly due to im~urities in the preparations, whereas it was praviously believed that the insulin antibodies were-produced by the insulin as such.
These impurities may be accompanying proteins from pancreas distinct from insulin, proin5ulin which is a precursor of insulir, intermediate insulin, the dimer, arginine insulin, ethylester insulin, desamido insulin desamidised to various extents, and other insulin modifications.
Eforts have therefore been made in order to produce ;
;~insulin preparations consisting of pure insulin, the so~
called monocomponent insulin, free of impurities and accompanying substances of any kind.
~;~For that purpose it has been proposed to subject amorphous or crystalline insulin prepared in a conventional ;
manner to an extensive further purification, e.g. by gel filtration and/or ion exchange treatment~
The resulting highly purified insulins show a strong ~;
decrease in antigenicity, but not a complete removal there- ~ ;
of. This is no doubt due to the fact that in spite of the purification steps the purified preparations still contain substances different from insulin. --The invention is based on the recognition that someof the impurities present in insulin are formed during the recovery itself of insulin, the usually used processes re-sulting in-ter alia in a decomposition o the insulin and formation of aggregates. The insulin as it is accumulated in the pa~creas glands, from which the insulin is re-covered, is the pure monomer (having a molecular weight of about 6000), and it is extracted as such by conventional extraction with an acidic aqueous 60-80 per cent alcohol.
In the further conventional processing, which inter alia comprises a separation of fat by vacuum distillat,on of .:
the extract at 25-30 C, whereby the alcohol is evaporated and an aqueous extract with a strongly reduced alcohol . 10 content is obtained, the monomer is, however, subjected to :;
conditions resulting in decomposition - inter alia due to the detrLmental action of the enzymes which can display their activity in aqueous solution (solutions having an al-cohol content less than 50 per cen~), where.as they are not active in alcoholic solution (over 'iO per cent alcohol) -formation of aggregates, etc.
According to the invention it is therefore proposed to carry out the preparation of insulin in such a manner that the insulin, from the extraction from the pancreas :~
glands until tha obtaining of the final product, is only subjected to conditions of such a nature that they do not ~:
cause the formation of decomposition products, aggregates etc.
~he present invention, then, in one aspect, resides in a process for purif.ying an insulin-containiny extract prepared from pancreas glands, whereln the extract is ::~
worked up in a plurality of steps to pure.insulin, characterized in that the insulin is maintained in a solvent environment during the whole process, the undesired substances being from the beginning of the process until the end removed from the solvent or solvents used, said process cvmprising removal ", ,~
t~t.~ 3 of fat by cooling of the extract to a temperature below -25C
followed by separation of the crystallized fat, reduction of the volume of the extract, by use of reverse osmosis or gel filtration or by ion exchange treatment, and further purification of the extract by means of such processes as reverse osmosis or treatment with ion exchangers or molecular sieves,in which the insulin remains in a solvent environment; and, where decired, recovery of the insulin from the purified extract.
Also provided by the present invention ia mono-insulin ~;
purified to such a degree that it shows only a single ` component when analyzed by polyacrylamide gel electrophoresis using a gel containing 15% polyacrylamide.
By "in a solvent environmentl' we mean that the insulin is in solution or a like state. It may remain at all times in the liquid phase in solution or if for example it is ;~ retained on an ion exchange material will be protected by the solvent and hence still in the desired environment.
., ~
Insulin is very easily soluble in an acidic alcoholic extract of from about 55 to about 80 per cent by volume of alcohol, and in such an e~tract the insulin will, i the extract ls treated expediently, no~ decompose and form aggregates with itself or with impurities in the :
-3a-3~
alcoholic extract In a preferred embodlment of the process accordingto the lnvention the insulin is extracted from the pancreas glands with such an aqueous acidic alcohol, and the insulin is maintained in pure and dissolved state during the further ~ processing, without change of phase, until the isolation ;~ of the end product, e.g. by precipitation and possible -~ crystallization.
; Use can of course be made of other e~tractants than acidic aqueous alcohol, extractants in which insulin is easily soluble and in which it will not form aggregates with itself or with impurities in the extract, e~g. aqueous acetone.
In the process in question it is not a question o~
a purification of the insulin itself, but a purification of the obtained insulin extract takes place to remove dissolv-ed impurities and fat and undesirable proteins and deriva-tives of insulin, until the pure insulin remains alone in the extract (or in another liquid phase).
The process may for instance be carried out in the following manner~
Comminuted frozen pancreas glands are extracted with ethyl alcohol (60-80 per cent by volume) acidified with hydrochloric acid to about pH 3. After separation of the pancreas mass the pH of the extract is adjusted to about 8, whereby certain proteins different from insulin are preci-pitated and separated. The pH is again reduced to about 3.
Instead of using this so-called "pH-8 precipitation" for ~J
'~ ~
separating undesirable proteins one can send the acid extract with a p~-value about 3 through an ultrafiltra- -tion/hyperfiltration plant, see below, using an appropriate membrane retaining the said proteins, i.a. enzymes.
The resulting clear extract is ccoled down in a special fat ` freezing plant to a temperature of about -30 to -45C, ; preferably a~out -35C, whereby the fat is crystallized in ~ compact fat crystals of small surface. These crystals are .
separated, e.g. by centrifuyation, at the same low tempe-rature. At this low temperature of -30 to -45C and with the constant high alcohol content of 60-80 per cent by volume, no harmful effect on the insulin takes place as is the case in the conventional fat removal by vacuum de-stillation at 25-30C with decreasing alcohol content di-the extract.
Further details regarding this fat separation by ~-freezing out are disclosed in Canadian Patent 1~o4l/9~o2 of W.H. Jensen et al., granted November 7, 1978.
After this gentle separation of the fat a comparative-ly large volume of fat~free extract is available, and this large volume has to be reduced, also in a manner which is harmless to the insulin.
Gel filtration and/or ion exhange treatment may be used, but according to the invention the following method is preferred:
Use is made of an ultrafiltration/hyperfiltration plank working according to the membrane method, the 50-called reverse osmosis system (cf. for instance US Patent 6~
" ~::
Patent No. 3,623,610).
By using this method one can, by an appropriate choice of membrane types, obtain, together with a con-centration of the volume of liquid, a separation of insulin from impurities present in the extract, both substances having a larger molecule than the insulin as well as substances having a smaller molecule.
One achieves the removal not only of undesirable . :
substances of pancreatic origin which are dissolved in the extract, but also of any possible o~her molecules of ~ ~
non-pancreatic origin having a size different from that ~ ;
of the insulin, which the slaughterhouses might by ~
error or inadvertence have included in the pancreas supplies,and thereby also are to be found in the extract.
The separation at that momen1: of by far the major part~of these molecules, both larger and smaller than the insulin molecule, by ultra- and hyperfiltration is of ;~
decisive importance to the obtaining of the mono--.:
~ component insulin, since these impurities are here separat~
ed at a moment when they are in solution and when they have had no posslblity of displaying a detrimental effect ~ ;~
on the dissolved insulin, inter alia on account of the low temperature during the preceding process steps.
The fat-free clear extract is pumped through the ultrafiltration apparatus, which is provided with cooling -means for the extract, for instance at a temperature be- -~
tween a~out ~10C and lCC,preferably between 0C and -10C, and at a pressure of 2-20 atmospheres, preferably 6 f, ?b.~J~
~, .
atmospheres, the type of membrane being chosen so that the '` large un~esirable molecules are separated, down to a size as~ close to the size of the insulin molecule as possible. As * * ~.
membran~s e.g. GRX~6 or GRX-7 (polysulphone membranes manufac-S tured by De Danske Sukkerfabriker A/S) can be used. The large ,~molecules remain in the concentrate, whereas the insulin and the , . ,~-, ~.
impurities of small,er ,molecule size than that of the insulin form part of the permeate. This permeate is thereafter passed through the ultrafiltration apparatus wherein the type of membrane is O chosen so that the major part oi the undesirable molecules smaller than the insulin molecule are separated, up to a size as close ', to the slze of the insulin molecule as possible. As membranes e.g. GR8~P or 930 (~ellulose acetate) both manufactured by ~: .
De Danske Suk~erfabriker A/S can be used. The insulin remains in the concentrate. The extract can be concentrated as strongly ''~
as .lesired, e.g. to 1~5 to 1/40 of the original volume. The , ~' concentration ra~io alcohol : water remains the same as in the starting extract.
The membranes used in the ultra- and hyperfiltration plant must be able to resist cQncentrated alcoh~lic solutions of for instance 62 per cent alcohol. Use can ~or instance - besides of membranes made of polysulphones and of cellulose acetate as mentioned above - be made of membranes of other polymers, such ~, as certain polyamides.
'~ , , Wi~h regard to the construction of the ultra- and hyper filtration plant, reference can, for instance be made to the following patent specifications: US patent No. 3,872,015, British patent No. 1,390,671 and Swiss patent No.- 542,639, * Trade ~arks The resulting concentrate, which contains practically all the insulin from the raw extract, is transparent but more strongly coloured than the raw extract on account of ; the concentration. The colouring substances are washed off ; with, for instance 62 per cent alcohol, over membranes which retain ~he insulin but allow the colouring substances to pass through~ The whole volume of washing liquid passes together wit~ the ;-`
said substances in the permeate, while the volume of the concen-trate remains unaltered.
Simultaneously with the washing off of the colouring sllbstances, the salts present in the extract and originating from the pancreas extr~action are washed of.
The rather few dissolved impu.rities,which still re~
main in the insulin-containing extract, can suitably be ~ :
separated by means of ion exchangers, cation exchangers as well as anion exchangers. The separation can also be ~- -effected by means of molecular sieve, e.g.'lSephadex G-50"
manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden, and other suitable separation methods known per se, but the use of ion exchangers is preferred. -~
Ion exchanging processes for the cleaning of insulin by the column chromatography method as well as by the batch method are known per se, quite particularly the column chromatography method.
It is however hitherto unknown to apply these methods to a concentrated alcoholic raw insulin extract free from fat The conventional alcoholic raw insulin extracts having all the fat dissolved therein will, especially by the column ; chromatography method, contaminate the ion exchangers with * Tracle Mark the impurities - the colouring substances and the fat -to such an extent that the ion exchangers after adsorp- -tiOII and elution once or twice will be so contaminated that it will be extremely difficult to purify and regenerate s the exchangers for re-use in the separation of insulin.
When using the ion exchanging or molecular sieve `~
processes it is a condition that the extract being treated has been freedîrom fat and partially also from colouring substances, undesirable proteins as well as other impurities to such an extent that the ion exchangers and the molecular sieve substances can be regenerated and purified so that they are fully applicable for a substantial period of time, as their use otherwise will be economically prohibitive.
Such a sufficient and necessary purification is 15- just obtained by the method described above for prepurifica-tion of the alcoholic raw insulin extract comprising crystalli~
zation of the fat at a temperature as low as about -40C and removal by ultrafiltration through a reverse osmosis system of part of the colouring substances as well as an substantlal ~ ;
part of the molecules which are larger than or smaller than the insulin molecule, including molecules quite close to the size of the insulin molecule.
Most of the available types of ion exchangers can be used, including ion exchangers on a resin and a cellulose ~ ;
basis as well as the Sephadex ion exchangers SP and QAE
known per se, which are cation exchangers and anion exchangers, respectively, and are modified crosslinked dextran chains having a tridimensional network of polysaccharide ~anufactur-*Trad~k ,~ "' !
ed by Pharmacia Fine C~emicals AB, Uppsala, Sweden). The ion exchangers"SP-Sephadex C-25"(cation exchanger) and QAE-Sephadex A--25 (anion exchanger) are preferred.
One can choose between carrying out column chromato-graphy or batch separation on the ion exchangers, orpossibly both methods can be used.
The ~atch method has been found to preser.t enormous advantages when used in larger industrial operation as a medium to fine puri~ication process inserted ~etween the se-paration and concentration by reverse osmosis and achromatographic purification using an ion exchanger column.
Batch separation is a very quick technique and the method causes no technical trouble as a consequence of swelling or shrinking of the ion exchanger. Separation on an ion exchanse~ can be carried out by binding the impurities and by allowing only the insulin to remain in solution. After the ion exchanger has been equilibrated in the suitable bu~fer chosen at the isoelectric point of the insulin, pH 5 2, the concentrated prepurified raw insulin extract ~;
is at pH 5 2 brought into contact with the ion exchanger and the whole is stirred for instance for 2 hours, where- ~;
after the mixture is filtered. All the impurities are bonded to the ion exchanger, whereas the pure insulin is present in a dissolved state in the alcoholic filtrate,
- 2~ - since the -insulin at its isaelectric point is no~ bonded to the ion exchanger The advantage of this process is that the insulin does not have to be eluted from the ion exchanger and that the 10- . .
.. .. .. .
. ~ :
, , :
insulin extract immediaLely after the filtration can pass to the next process step.
The ion exchangers are regenerated in a conventional manner.
Conversely, the-insulin and the other proteins can alco be bonded to the ion exchanger and subsequently be ;~
eluted in fr~ction~ by resuspending the mixture in a buffer of a higher ion strength or a different p~ value.
By the latter method a smaller loss of insulin can be obtained than by the f~ormer method, for which reason the latter method is preferred.
The method may or instance be carried out substantial-ly in the following manner:
300 g of dry Sp-sephadex C-25 were swelled during 48 hours in 1000 ml of a bufer, 0.1 molar acetic acid ~ ;
(HAc) in 62~ etha~ol, pH 3~0. The buffer was exchanged several- -~
,. ..
times. The weight of the swelled SP-Sephadex was 720 g.
1000 ml of extract, concentrated lO times from the osmosis, having a content of 18000 i. units of insulin, were adjusted to pH 3.0 and sti~red in a rotary container (12 revolutions per minute) for l~ hours together with half the swelled ion exchanger for adsorptlon. After filtratio~
in vacuo thé adsorption of the filtrate was continued on the second half of the ion exchanger (360 g swelled) like-wlse-for. l~ hoNrs. ~he--supernatant showed an insulin conte~t of-288 i. units correspondin~ to a loss of 1.6~ during the adsorption. Such a double adsorption has proved to reduce the Loss to one third of-the loss occ~rr-in~in a single adsorption.
~--r, '' ' ,'' ~: ' : ` ' ' ' A change now was made to buffer: 1000 ml, 0.1 molar HAc in 62% ethanol, pH 3.0, containing 0.075 mol NaC1.
Impuri.ties were eluted by stirring ~or 2 hours, whereas the insulin was not eluted.
After filtration a change was made to buffer: 1000 ml of 0.1 molar tris(hydroxymethyl)aminomethane (Tris) in 62% ethanol, pH 8.0, whereupon the insulin was eluted.
from the ion exchanger SP; insulin conte~t 17000 i. units, the loss during the eluting process hein~ 4%; total loss 5.6%.
The ion exchanger was washed twice for removal of residual insulin attached to the ion exchanger.
In polyac~yl~n~de gel electrophoresis (with 15% polyacrylam~de) 4 bands (some of them weaker than the msulin band) were found abov~
the insulin band, whereas no band WclS to be found below the insulin band. This means that the partially purified insulin has not during the production process been deamldized. It is free from monodesamido insulin, which is an entirely new feature in the production of insulin.
Subsequently to eluting and washmg the ion exhanger was~treated under ~low stirring for 2-3 hours with 62~ ethan~l having added thereto 0.2-Q.3 mol NaCl. -~
Subsequently, the ion exchanger was regenerated with equilibration buffer pH 3, and it was now perfectly clean, chalk white and ready for the next batch. ~ ;~
The batch process can be carried out advantageous-ly in an apparatus of special design, being Y-shaped and having an adjus~able number of revolutions per minute.
~., : . ,: , .
~: '',.' .' :
::.: . : .
Thereby a very efficient and lenient treatment of the ion exchanger during the adsorption an~ eluting is provided whila at the same tLme the loss during the adsorption as well as during the elutmg has been m~mized. me entire process can take place without remo~ing the ion exchanger -~
from the apparat~s, which is provided with a filtering device and a vacuum tank for rapid filtration of the liquid from the ion exchanger.
The process is composed of the following partial processes:
l) Adsorption on the ion exchanger, pH 3, of the insulin and possibly of other proteins from the concentrated raw insulin extract, in 2 steps with j,~
intermediate vacuum iltxation. `~
2) Vacuum filtration and removal. of the protein-free extract.
.. .. .. .
. ~ :
, , :
insulin extract immediaLely after the filtration can pass to the next process step.
The ion exchangers are regenerated in a conventional manner.
Conversely, the-insulin and the other proteins can alco be bonded to the ion exchanger and subsequently be ;~
eluted in fr~ction~ by resuspending the mixture in a buffer of a higher ion strength or a different p~ value.
By the latter method a smaller loss of insulin can be obtained than by the f~ormer method, for which reason the latter method is preferred.
The method may or instance be carried out substantial-ly in the following manner:
300 g of dry Sp-sephadex C-25 were swelled during 48 hours in 1000 ml of a bufer, 0.1 molar acetic acid ~ ;
(HAc) in 62~ etha~ol, pH 3~0. The buffer was exchanged several- -~
,. ..
times. The weight of the swelled SP-Sephadex was 720 g.
1000 ml of extract, concentrated lO times from the osmosis, having a content of 18000 i. units of insulin, were adjusted to pH 3.0 and sti~red in a rotary container (12 revolutions per minute) for l~ hours together with half the swelled ion exchanger for adsorptlon. After filtratio~
in vacuo thé adsorption of the filtrate was continued on the second half of the ion exchanger (360 g swelled) like-wlse-for. l~ hoNrs. ~he--supernatant showed an insulin conte~t of-288 i. units correspondin~ to a loss of 1.6~ during the adsorption. Such a double adsorption has proved to reduce the Loss to one third of-the loss occ~rr-in~in a single adsorption.
~--r, '' ' ,'' ~: ' : ` ' ' ' A change now was made to buffer: 1000 ml, 0.1 molar HAc in 62% ethanol, pH 3.0, containing 0.075 mol NaC1.
Impuri.ties were eluted by stirring ~or 2 hours, whereas the insulin was not eluted.
After filtration a change was made to buffer: 1000 ml of 0.1 molar tris(hydroxymethyl)aminomethane (Tris) in 62% ethanol, pH 8.0, whereupon the insulin was eluted.
from the ion exchanger SP; insulin conte~t 17000 i. units, the loss during the eluting process hein~ 4%; total loss 5.6%.
The ion exchanger was washed twice for removal of residual insulin attached to the ion exchanger.
In polyac~yl~n~de gel electrophoresis (with 15% polyacrylam~de) 4 bands (some of them weaker than the msulin band) were found abov~
the insulin band, whereas no band WclS to be found below the insulin band. This means that the partially purified insulin has not during the production process been deamldized. It is free from monodesamido insulin, which is an entirely new feature in the production of insulin.
Subsequently to eluting and washmg the ion exhanger was~treated under ~low stirring for 2-3 hours with 62~ ethan~l having added thereto 0.2-Q.3 mol NaCl. -~
Subsequently, the ion exchanger was regenerated with equilibration buffer pH 3, and it was now perfectly clean, chalk white and ready for the next batch. ~ ;~
The batch process can be carried out advantageous-ly in an apparatus of special design, being Y-shaped and having an adjus~able number of revolutions per minute.
~., : . ,: , .
~: '',.' .' :
::.: . : .
Thereby a very efficient and lenient treatment of the ion exchanger during the adsorption an~ eluting is provided whila at the same tLme the loss during the adsorption as well as during the elutmg has been m~mized. me entire process can take place without remo~ing the ion exchanger -~
from the apparat~s, which is provided with a filtering device and a vacuum tank for rapid filtration of the liquid from the ion exchanger.
The process is composed of the following partial processes:
l) Adsorption on the ion exchanger, pH 3, of the insulin and possibly of other proteins from the concentrated raw insulin extract, in 2 steps with j,~
intermediate vacuum iltxation. `~
2) Vacuum filtration and removal. of the protein-free extract.
3) Washing of the ion exchanger twice with 62% ethanol buffer, pH 3, and remo~al b~ filtration of the washing liquids.
4) Elutlng ~rities with buffer pH 3, ion strength O.OZ5 mol NaCl, 2 hours, 12 r.p.m.
5) Filtration of eluting liquid with its content of impurities.
6) Washing twice of the ion exchanger with 62% ethanol buffer, yH 3; filtration.
7) Eluting the insulin with buffer O.l molar Tris ln 62% ethanol, pH 8; the whole mixture, eluting liquid and ion exchanger, is to be adjusted tQ pH 8 ~'ll .
': ,"` . ,:
,,, :: ~ ~
with Tris; the pH o~ ~he ion exchanger was 3.
': ,"` . ,:
,,, :: ~ ~
with Tris; the pH o~ ~he ion exchanger was 3.
8) Filtration of the eluting liquid containing the insulin.
9) Washing twice of the ion exchanger for remcval of residual insulin attached to the ion exchanger.
lO) Purification and regeneration of the ion exchanger.
The total production cycle 1-9 requires 12 hours, and the purification and regeneration of the ion exchanger require 4 hours, making a total of 16 hours.
The elutin~ extract from phase 7, af~er adjustment of the pH and the ion strength, can be applied directly to cation or anion exchangers according to the column ~ ~;
methods known per se. This process is preferably carried out on programmed columns using automatic fractioning, lS wherein the central part of the insulin curve is removed ~;
which part now contains the pure lnsulin showing only one band in polyacrylamide electrophorssis with 15% poly- ;
acrylamide.
From the concentrated insulin-containing eluate from the last column the insulin can be recovered in a con- -ventional manner, i.e. by dilution of the solution and - subsequent precipitation for instance by the addltion of zinc ions.
According to the invention, it has however been , .. . . ... . .
found that it is not necessary to carry out a dilution of the highly concentrated a~ueous alco~.olic extract prior ta precipitation . The extract can be precipitated direct~
~ '~
,. ..
~ .. , , :, : :~
~h.~
ly with for instance zinc ions when the additlon thereof is effected under cooled conditions, for instance at ~30 C
to -45C.
Thus, a zinc acetate solution m~y be added to the filtrate, subsequently leavi~g the solution to stand ~or 24 hours in a cold store at ~35~C. Thereby the insulin is precipitated quantitatively from the for instance 62 alcoholic solution. The precipitated insulin is removed by centrifugation, washed eOg. with acetone, and dried.
; 10 If desired, it may subsequently be cr~stallized.
As mentioned, the insulin produced by the present pxocess is a pure monocomponent insulin showing merely a single component when analyzed by polyacrylamide gel electrophoresis (DISC PAGE~ by the use of a 15~ concentra `~
tion of the polyacrylamide. (Concerning the general prin-clple of this m~thod, reference is made to Ann. N-.-Y. Acad.
Sci., 121, pp. 321 ~49 and 407-427 (1964)).
Insulin of the purity described herein has not to the best oE our knowledge been pro~uced previous~ly.
In the British patent specification Nos. 1,285,023 and 1,285,024 production of a high purlty insulin is de-sc~ibed. It is ~tated that the purified insulin in a poly-r -- -acrylamide gel elactrophoresis shows substantially a single component. This insulin however still contains a smaller amount o desamido ~lsuLinr and- in polyacryla~ide ge~-ielec- -~rophoresis using a gel with 15% polyacrylamide, it will show two bands, i.e. besides the monoinsulin band a des-amido irsulin band. This result was a confirmation of that reported by Yue and Tutle (The Lancet, 26 October, 1974) who used 20~ poly--acrylamide gel.
A smaller content of desamido insulin wlll, on the ~ `~3 other hand, not be ascertainable in polyacrylamide gel electrophoresis using 7~% polyacrylamide. At this content, complete separation of the individual components is not cbtained, but in~er alia an integration of the insulin and the monodesamido insulin into one band is occurring where two bands will appear with 15% polyacrylamide, The insulin produced by the process of the invention -~
ha~s proved to be extremely stable contrary to the hither-to known highly purified insullns, in which for instance during storing additional small amounts of desamido insulin are often produced; why this happens is unknown, - -but the cause may perhaps be that the insulins produced, despit~ extensive purification, still contain traces of substances having a decomposing efect on the insulin.
~; The insulin produced by the process of the invention does not reveal any such decomposition, which thus may be ~due to the more complete removal of all substances of a ~;
molecular size different to that of the insulin.
From the pure insulin described above insulin pre-parations can-be prepared in any manner known per se, for instance by dissoIving and/or sùspension of the insulin, ; -in amorphous and/or crystalline form, in an aqueous medium suitable for injection, infusion or implantation technique.
The hitherto unknown steps of procedure described ;~
above, viz. the separation of impurities from a fat-free insulin-containing extract by means of reverse osmosis and precipitation of insulin from a highly concentrated ~16-.: ,, . ~ :
. .
alcoholic solution under cooled conditions,may of course be used not only in the described combination of process steps. The principle:separation of impurities from a fat-free insulin-containing extract by reverse osmosis can be used in any combination, and the solvent does not of course need to be a highly concentrated aqueous alcohol, but may be any suitable solvent for insulin. The precipitation under cooled conditions can be applied to any highly con-centrated insulin-containing organic solvent extract, regardless , of the manner in which it has been obtained.
The invention therefore resides not only in the combination of process steps described herein, but it also resides in said two hitherto unknown steps as considered separately~
' . ' ' :, , ~
lO) Purification and regeneration of the ion exchanger.
The total production cycle 1-9 requires 12 hours, and the purification and regeneration of the ion exchanger require 4 hours, making a total of 16 hours.
The elutin~ extract from phase 7, af~er adjustment of the pH and the ion strength, can be applied directly to cation or anion exchangers according to the column ~ ~;
methods known per se. This process is preferably carried out on programmed columns using automatic fractioning, lS wherein the central part of the insulin curve is removed ~;
which part now contains the pure lnsulin showing only one band in polyacrylamide electrophorssis with 15% poly- ;
acrylamide.
From the concentrated insulin-containing eluate from the last column the insulin can be recovered in a con- -ventional manner, i.e. by dilution of the solution and - subsequent precipitation for instance by the addltion of zinc ions.
According to the invention, it has however been , .. . . ... . .
found that it is not necessary to carry out a dilution of the highly concentrated a~ueous alco~.olic extract prior ta precipitation . The extract can be precipitated direct~
~ '~
,. ..
~ .. , , :, : :~
~h.~
ly with for instance zinc ions when the additlon thereof is effected under cooled conditions, for instance at ~30 C
to -45C.
Thus, a zinc acetate solution m~y be added to the filtrate, subsequently leavi~g the solution to stand ~or 24 hours in a cold store at ~35~C. Thereby the insulin is precipitated quantitatively from the for instance 62 alcoholic solution. The precipitated insulin is removed by centrifugation, washed eOg. with acetone, and dried.
; 10 If desired, it may subsequently be cr~stallized.
As mentioned, the insulin produced by the present pxocess is a pure monocomponent insulin showing merely a single component when analyzed by polyacrylamide gel electrophoresis (DISC PAGE~ by the use of a 15~ concentra `~
tion of the polyacrylamide. (Concerning the general prin-clple of this m~thod, reference is made to Ann. N-.-Y. Acad.
Sci., 121, pp. 321 ~49 and 407-427 (1964)).
Insulin of the purity described herein has not to the best oE our knowledge been pro~uced previous~ly.
In the British patent specification Nos. 1,285,023 and 1,285,024 production of a high purlty insulin is de-sc~ibed. It is ~tated that the purified insulin in a poly-r -- -acrylamide gel elactrophoresis shows substantially a single component. This insulin however still contains a smaller amount o desamido ~lsuLinr and- in polyacryla~ide ge~-ielec- -~rophoresis using a gel with 15% polyacrylamide, it will show two bands, i.e. besides the monoinsulin band a des-amido irsulin band. This result was a confirmation of that reported by Yue and Tutle (The Lancet, 26 October, 1974) who used 20~ poly--acrylamide gel.
A smaller content of desamido insulin wlll, on the ~ `~3 other hand, not be ascertainable in polyacrylamide gel electrophoresis using 7~% polyacrylamide. At this content, complete separation of the individual components is not cbtained, but in~er alia an integration of the insulin and the monodesamido insulin into one band is occurring where two bands will appear with 15% polyacrylamide, The insulin produced by the process of the invention -~
ha~s proved to be extremely stable contrary to the hither-to known highly purified insullns, in which for instance during storing additional small amounts of desamido insulin are often produced; why this happens is unknown, - -but the cause may perhaps be that the insulins produced, despit~ extensive purification, still contain traces of substances having a decomposing efect on the insulin.
~; The insulin produced by the process of the invention does not reveal any such decomposition, which thus may be ~due to the more complete removal of all substances of a ~;
molecular size different to that of the insulin.
From the pure insulin described above insulin pre-parations can-be prepared in any manner known per se, for instance by dissoIving and/or sùspension of the insulin, ; -in amorphous and/or crystalline form, in an aqueous medium suitable for injection, infusion or implantation technique.
The hitherto unknown steps of procedure described ;~
above, viz. the separation of impurities from a fat-free insulin-containing extract by means of reverse osmosis and precipitation of insulin from a highly concentrated ~16-.: ,, . ~ :
. .
alcoholic solution under cooled conditions,may of course be used not only in the described combination of process steps. The principle:separation of impurities from a fat-free insulin-containing extract by reverse osmosis can be used in any combination, and the solvent does not of course need to be a highly concentrated aqueous alcohol, but may be any suitable solvent for insulin. The precipitation under cooled conditions can be applied to any highly con-centrated insulin-containing organic solvent extract, regardless , of the manner in which it has been obtained.
The invention therefore resides not only in the combination of process steps described herein, but it also resides in said two hitherto unknown steps as considered separately~
' . ' ' :, , ~
Claims (15)
1. A process for purifying an insulin-containing extract prepared from pancreas glands, wherein the extract is worked up in a plurality of steps to pure insulin, characterized in that the insulin is maintained in a solvent environment during the whole process, the undesired substances being from the beginning of the process until the end removed from the solvent or solvents used, said process comprising removal of fat by cooling of the extract to a temperature below -25°C
followed by separation of the crystallized fat, reduction of the volume of the extract, by use of reverse osmosis or gel filtration or by ion exchange treatment, and further purification of the extract by means of such processes as reverse osmosis or treatment with ion exchangers or molecular sieves,in which the insulin remains in a solvent environment; and, where desired, recovery of the insulin from the purified extract.
followed by separation of the crystallized fat, reduction of the volume of the extract, by use of reverse osmosis or gel filtration or by ion exchange treatment, and further purification of the extract by means of such processes as reverse osmosis or treatment with ion exchangers or molecular sieves,in which the insulin remains in a solvent environment; and, where desired, recovery of the insulin from the purified extract.
2. A process according to Claim 1 wherein the solvent or solvents used is or are such as to suppress detrimental action of enzymes in the impure insulin extract.
3. A process according to Claim 2 wherein the solvent is an aqueous alcoholic medium whose content of water-miscible alcohol is always >50% v/v.
4. A process according to Claim 3 wherein said alcohol content is always within the range 60-80% v/v.
5. A process according to Claim 3 or Claim 4 wherein the alcohol is ethanol.
6. A process according to Claim 1 wherein the extract after separation of the fat is subjected to reverse osmosis, characterized in that a membrane is used by means of which the extract is simultaneously concentrated and separated from impurities of greater and of lesser molecular size than insulin.
7. A process according to Claim 6, wherein the insulin-containing concentrate from the reverse osmosis is washed in a reverse osmosis plant for removal of colouring substances and salts from the concentrate.
8. A process according to Claim 7, wherein the purified concentrate is subjected to further purification by means of ion exchange, characterized in that a first treatment by ion exchange is carried out as a batch process.
9. A process according to Claim 8, wherein the insulin-containing liquid phase from the batch-method ion-exchange is subjected to one or mare further purifications by means of ion exchange column chromatography.
10. A process according to Claim 9 wherein the insulin remains in the liquid phase in solution.
11. A process according to Claim 1 wherein the insulin is recovered from a concentrated purified solution by precipitation with metal ions at a temperature sufficiently low to ensure a quantitative precipitation of the insulin.
12. A process according to Claim 11 in which the metal ions are Zn++.
13. A process according to Claim 11 or Claim 12 in which the precipitation is effected in the temperature range -30°C to -45°C.
14. A process according to Claim 1 when the reduction of the volume of the extract is effected by means of reverse osmosis.
15. Mono-insulin purified to such a degree that it shows only a single component when analysed by polyacrylamide gel electrophoresis using a gel containing 15% polyacrylamide, when produced by the process of Claim 1, 2 or 11, or by an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB1740/76 | 1976-01-16 | ||
| GB1740/76A GB1581824A (en) | 1976-01-16 | 1976-01-16 | Preparation of insulin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1112639A true CA1112639A (en) | 1981-11-17 |
Family
ID=9727172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA269,730A Expired CA1112639A (en) | 1976-01-16 | 1977-01-14 | Mono-insulin and method of preparing the same |
Country Status (15)
| Country | Link |
|---|---|
| AT (2) | AT364717B (en) |
| AU (1) | AU514888B2 (en) |
| BE (1) | BE850387A (en) |
| CA (1) | CA1112639A (en) |
| DE (1) | DE2701092A1 (en) |
| DK (1) | DK14177A (en) |
| EG (1) | EG13113A (en) |
| ES (1) | ES455065A1 (en) |
| FI (1) | FI64508C (en) |
| FR (1) | FR2338250A1 (en) |
| GB (1) | GB1581824A (en) |
| IE (1) | IE45013B1 (en) |
| NL (1) | NL7700455A (en) |
| NO (2) | NO149875C (en) |
| SE (1) | SE445972B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK311477A (en) * | 1977-07-08 | 1979-01-09 | Leo Lab | PROCEDURE FOR PREPARING PURE INSULIN |
| US4459226A (en) * | 1982-02-26 | 1984-07-10 | Eli Lilly And Company | Process for recovering insulin |
| BG65045B1 (en) * | 2001-12-03 | 2007-01-31 | "Софарма" Ад | Method for the production of highly purified monocomponent insulin |
| KR20140007377A (en) | 2011-02-01 | 2014-01-17 | 노보 노르디스크 에이/에스 | Purification of insulin |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1966573C3 (en) * | 1969-08-07 | 1979-08-30 | Novo Terapeutisk Laboratorium A/S, Kopenhagen | Method for purifying insulin |
| GB1503919A (en) * | 1974-07-19 | 1978-03-15 | Leo Sa Lab | Recovery of insulin |
-
1976
- 1976-01-16 GB GB1740/76A patent/GB1581824A/en not_active Expired
-
1977
- 1977-01-10 AT AT0007977A patent/AT364717B/en not_active IP Right Cessation
- 1977-01-11 IE IE44/77A patent/IE45013B1/en unknown
- 1977-01-12 DE DE19772701092 patent/DE2701092A1/en not_active Withdrawn
- 1977-01-13 AU AU21317/77A patent/AU514888B2/en not_active Expired
- 1977-01-13 SE SE7700322A patent/SE445972B/en unknown
- 1977-01-14 DK DK14177A patent/DK14177A/en not_active Application Discontinuation
- 1977-01-14 FI FI770112A patent/FI64508C/en not_active IP Right Cessation
- 1977-01-14 FR FR7701077A patent/FR2338250A1/en active Granted
- 1977-01-14 CA CA269,730A patent/CA1112639A/en not_active Expired
- 1977-01-14 NO NO770119A patent/NO149875C/en unknown
- 1977-01-14 ES ES455065A patent/ES455065A1/en not_active Expired
- 1977-01-14 BE BE174084A patent/BE850387A/en not_active IP Right Cessation
- 1977-01-15 EG EG30/77A patent/EG13113A/en active
- 1977-01-17 NL NL7700455A patent/NL7700455A/en not_active Application Discontinuation
-
1980
- 1980-11-06 AT AT0546880A patent/AT366578B/en not_active IP Right Cessation
-
1982
- 1982-09-03 NO NO822985A patent/NO822985L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EG13113A (en) | 1983-03-31 |
| BE850387A (en) | 1977-05-02 |
| DE2701092A1 (en) | 1977-07-28 |
| SE7700322L (en) | 1977-07-17 |
| IE45013B1 (en) | 1982-06-02 |
| GB1581824A (en) | 1980-12-31 |
| NL7700455A (en) | 1977-07-19 |
| AT364717B (en) | 1981-11-10 |
| FI770112A (en) | 1977-07-17 |
| SE445972B (en) | 1986-08-04 |
| AU2131777A (en) | 1978-07-20 |
| DK14177A (en) | 1977-07-17 |
| NO770119L (en) | 1977-07-19 |
| NO822985L (en) | 1977-07-19 |
| AT366578B (en) | 1982-04-26 |
| FI64508C (en) | 1983-12-12 |
| IE45013L (en) | 1977-07-16 |
| FR2338250A1 (en) | 1977-08-12 |
| FR2338250B1 (en) | 1980-04-04 |
| ES455065A1 (en) | 1978-05-01 |
| FI64508B (en) | 1983-08-31 |
| NO149875B (en) | 1984-04-02 |
| ATA7977A (en) | 1981-04-15 |
| ATA546880A (en) | 1981-09-15 |
| AU514888B2 (en) | 1981-03-05 |
| NO149875C (en) | 1984-07-11 |
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