CA1079269A - Process of separating precipitated proteins from albumin - containing suspensions - Google Patents
Process of separating precipitated proteins from albumin - containing suspensionsInfo
- Publication number
- CA1079269A CA1079269A CA268,010A CA268010A CA1079269A CA 1079269 A CA1079269 A CA 1079269A CA 268010 A CA268010 A CA 268010A CA 1079269 A CA1079269 A CA 1079269A
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- Prior art keywords
- albumin
- process according
- filtration
- filter
- filtering
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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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Biochemistry (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
ABSTRACT OF THE DISCLOSURE:
Precipitated proteins are separated from albumin-containing suspensions obtained from the recovery of albumin from blood plasma, by an alluvial filtration of the suspensions through a fabric filter element having a mesh size of between 20 and 200 microns. The process allows to obtain clear filtrates which do not require any additional clarification or purification filtration and which, thus, permit optimum performance of the operations in the recovery of albumin to be obtained.
Precipitated proteins are separated from albumin-containing suspensions obtained from the recovery of albumin from blood plasma, by an alluvial filtration of the suspensions through a fabric filter element having a mesh size of between 20 and 200 microns. The process allows to obtain clear filtrates which do not require any additional clarification or purification filtration and which, thus, permit optimum performance of the operations in the recovery of albumin to be obtained.
Description
The present invention relates to an improved process of separating precipitated proteins from albumin-containing suspensions obtained from the recovery of albumin from blood plasma.
In the recovery of albumin from blood, blood products, albumin-containing body fluids and the like, an albumin-contain-ing suspension is obtained which, in addition to the dissolved albumin, contains precipitated proteinaceous materials, particu-larly glo-bulins. In this connection, Canadian patent app`lica-tion No. 223,373 of March 24, 1975 discloses a process of isolating alumin from human blood and the like, said process comprising the following process steps:
a) Separating of the plasma from the solid constituents of the blood (blood cells and blood platelets) and - if desired - recovering of the dissolved non-albumin-constituents from the plasma:
b) Precipitating of the globulines by heating of the separated liquid in the presence of 7 to 12 % by volume of alcohols having the composition CH3-n (CH2)-OH, with n being 0,1 or 2, and albumin stabilisers, as sodium caprylate, to temperatures ; of between about 60 and 75 C, and separating off the fluid from globulines;
c) Enriching of the albumin contained in the residual fluid.
The separation of the precipitated globulines and of other possibly precipitated proteins by means of thermal preci-pitation is effected in the process disclosed in the above mentioned Canadian patent application at a pH of about 4,4 by centrifuging in a continuous flow. The separated proteins are collected in the rotors, whereas albumin remains in the super-natant liquid.
~ 1079Z69 In the conventional process of separation, it is considered to be unsatisfactory that a relatively great amount of albumin is left back in the separated concentrate such that a plurality of washing and centrifuging operations are required to recover the valuable residual albumin. Besides, the centri- ~ -- fuging step is extremely time consuming and accompanied by high expenditure of work. In addition, the noise of the centrifuges ;~- is a great nuisance. In spite of these drawbacks, a different mode of operation has not yet been taken into consideratio`n because it is customary and necessary in the Cohn method which ~;
has been practised for many years, to separate by a centrifuging step the suspension comprising precipitated globulins and dissolved albumin. Tests have shown, that the suspensions obtained by the Cohn method can substantially not be filtered under normal requirements.
In view of the fact that the substances recovered in accordance with Canadian patent application No. 223,373 and the conventional Cohn method involve such substances which have been obtained from the same starting material and which show sub-stantially the same consistency in the form as an intermediate product and as the final product, it has heretofore not been thought possible that the substances obtained by the more recent process could be filtered at all. Various filtration techniques have been tried to remove precipitated proteins in cold ethanol fractionation procedures, Following experience, these precipi-tates cannot be economically removed by filtration techniques, most probably because of their intrinsic consistency. Never-theless different filtration procedures were investigated, as it had been noticed that the consistency of heat-ethanol precipitated globulins is considerably different form those globulins precipitated with cold ethanol.
Contrary to experience and expectation, it has now
In the recovery of albumin from blood, blood products, albumin-containing body fluids and the like, an albumin-contain-ing suspension is obtained which, in addition to the dissolved albumin, contains precipitated proteinaceous materials, particu-larly glo-bulins. In this connection, Canadian patent app`lica-tion No. 223,373 of March 24, 1975 discloses a process of isolating alumin from human blood and the like, said process comprising the following process steps:
a) Separating of the plasma from the solid constituents of the blood (blood cells and blood platelets) and - if desired - recovering of the dissolved non-albumin-constituents from the plasma:
b) Precipitating of the globulines by heating of the separated liquid in the presence of 7 to 12 % by volume of alcohols having the composition CH3-n (CH2)-OH, with n being 0,1 or 2, and albumin stabilisers, as sodium caprylate, to temperatures ; of between about 60 and 75 C, and separating off the fluid from globulines;
c) Enriching of the albumin contained in the residual fluid.
The separation of the precipitated globulines and of other possibly precipitated proteins by means of thermal preci-pitation is effected in the process disclosed in the above mentioned Canadian patent application at a pH of about 4,4 by centrifuging in a continuous flow. The separated proteins are collected in the rotors, whereas albumin remains in the super-natant liquid.
~ 1079Z69 In the conventional process of separation, it is considered to be unsatisfactory that a relatively great amount of albumin is left back in the separated concentrate such that a plurality of washing and centrifuging operations are required to recover the valuable residual albumin. Besides, the centri- ~ -- fuging step is extremely time consuming and accompanied by high expenditure of work. In addition, the noise of the centrifuges ;~- is a great nuisance. In spite of these drawbacks, a different mode of operation has not yet been taken into consideratio`n because it is customary and necessary in the Cohn method which ~;
has been practised for many years, to separate by a centrifuging step the suspension comprising precipitated globulins and dissolved albumin. Tests have shown, that the suspensions obtained by the Cohn method can substantially not be filtered under normal requirements.
In view of the fact that the substances recovered in accordance with Canadian patent application No. 223,373 and the conventional Cohn method involve such substances which have been obtained from the same starting material and which show sub-stantially the same consistency in the form as an intermediate product and as the final product, it has heretofore not been thought possible that the substances obtained by the more recent process could be filtered at all. Various filtration techniques have been tried to remove precipitated proteins in cold ethanol fractionation procedures, Following experience, these precipi-tates cannot be economically removed by filtration techniques, most probably because of their intrinsic consistency. Never-theless different filtration procedures were investigated, as it had been noticed that the consistency of heat-ethanol precipitated globulins is considerably different form those globulins precipitated with cold ethanol.
Contrary to experience and expectation, it has now
- 2 -.
~ 1079Z69 surprisingly been found that the thermally precipitated proteins can be separated from the suspension with substantially low expenditure, by performing such separation by alluvial filtration of the suspension through a fabric filter element having a mesh size of between 20 and 200 microns.
Alluvial filtration is known per se. Normally, alluvial filtration is performed in centrifugal purification filters comprising essentially a closed pressure vessel wherein circular filter elements are disposed on a centrally rotating hollow shaft, which filter elements are positioned either horizontally or in vertical position in parallel with each other. Customarily, the filter elements are fitted with a metallic twist (wire) or braid fabric.
The substance to be filtered is admixed with filtering aids, normally kieselguhrs or diatomites, which are added to the substance in metered fashion in accordance with the type or turbidity and with the character of the residues.
The filtrate collected on the filter elements is floated away by rotation of the filter elements and counter-current flushing. The residues are discharged as a so-called slurry.
In the present instance, the alluvial filtration offers surprising advantages, since other types of filters and other filtration methods require filtering periods of such a length -that no advantages are obtained at all over the time consuming centrifuging method. Hereby, the following filtering methods have been tested: Purifying filtration by means of carbon and asbestos plate filters; filter layers on the basis of cellulose;
fiberglass filters; sintered glass. The filtering methods ~-employed resulted either in excessive filtering periodsl in clogging of the filter inserts (elements), or in turbid filtered ;
liquids. It is only the use of an alluvial filter that allows to obtain clear filtrates which do not require any additional ~
,:~,.'.
~ 1079Z69 surprisingly been found that the thermally precipitated proteins can be separated from the suspension with substantially low expenditure, by performing such separation by alluvial filtration of the suspension through a fabric filter element having a mesh size of between 20 and 200 microns.
Alluvial filtration is known per se. Normally, alluvial filtration is performed in centrifugal purification filters comprising essentially a closed pressure vessel wherein circular filter elements are disposed on a centrally rotating hollow shaft, which filter elements are positioned either horizontally or in vertical position in parallel with each other. Customarily, the filter elements are fitted with a metallic twist (wire) or braid fabric.
The substance to be filtered is admixed with filtering aids, normally kieselguhrs or diatomites, which are added to the substance in metered fashion in accordance with the type or turbidity and with the character of the residues.
The filtrate collected on the filter elements is floated away by rotation of the filter elements and counter-current flushing. The residues are discharged as a so-called slurry.
In the present instance, the alluvial filtration offers surprising advantages, since other types of filters and other filtration methods require filtering periods of such a length -that no advantages are obtained at all over the time consuming centrifuging method. Hereby, the following filtering methods have been tested: Purifying filtration by means of carbon and asbestos plate filters; filter layers on the basis of cellulose;
fiberglass filters; sintered glass. The filtering methods ~-employed resulted either in excessive filtering periodsl in clogging of the filter inserts (elements), or in turbid filtered ;
liquids. It is only the use of an alluvial filter that allows to obtain clear filtrates which do not require any additional ~
,:~,.'.
- 3 - ~
- ~: . -, , ,. . . , , :
clarification or purification filtration and which, thus, permit optimum performance of the operations in the recovery of albumin to be obtained.
In a particularly preferred embodiment of the invention, the filtration is performed within a centrifugal purification filter comprising a closed (sealed) pressure vessel.
Preferably, the liquids in question are processed at a mesh size of from 70 to 90 microns. This mesh size provides an optimum between the throughflow that can be obtained `
; 10 and complete filtering out, in which case a single filtering `
step only is required to be carried out in each operation.
In order to adequately prepare the filter surface for the subsequent filtration, it is expedient in some instances initially to perform a primary precoating with a neutral liquid wherein the filter element is precoated with a layer of the filtering aid having a thickness of about 0.5 centimeters. As filtering aids, the customary, commercially available kiessel-; guhrs sold under the tradenames Hyflo-Super-Cel, Celite 545 or perlite are suitable. Additionally, cellulose filtering aids ~ 20 of conventional type are also useful, although with lesser ii filtering efficiency. These filtering aids likewise allow to obtain a product being satisfactory for many applications. It ~ is recommendable that the filtering aid is subjected to - pre-swelling or pre-soaking.
Favorable filtering effects are obtained with a ` horizontally disposed filter element of metallic twist (wire) or braid fabric having a mesh size of 80 microns.
A favorable composition compromises a suspension of from about 4 to 6% of plasmatic protein, to which suspension there are added from about 20 to 70 grams of kieselguhr filtering aid per liter. Thereafter, this suspension is filtered. Lower concentrations of protein may require lesser quantities of _ 4 _ .. . .
filtering aids under certain circumstances.
In order to recover also the albumin present within the slurry or turbid volume, it is proposed that this albumin is likewise isolated by means of a residual volume filtering device comprising a small portion of the total filter area, after a cleaning by rotation and countercurrent flushing or a clearing of the vol~me of the filtering apparatus place in a recirculation system has been performed.
In order to increase the yield, the process may`be carried out at a pressure of 4.0 + 2.5 bar (atmospheres) without sacrifice to the filtering capacity.
Below, the present invention is explained in greater detail by means of the following Exampl~s. The two Examples start with two different suspensions. Example 1 starts with a suspension prepared in accordance with the process of the laid-open German patent application 2,415,079.
Example 1 m e solid constituents (blood cells and blood platelets) are separated from human blood, and the coagulation factors are removed. The starting solution contains from about 5 to 6 %
of plasmatic protein. In accordance with the process disclosed in the above publication, the clotting factor VIII and the fibrinogen are removed by cryo ethanol sedimentation. The prothrombin complex is removed by absorption. The original plasma i~ ~lepatitis-activity negative, it shows normal trans- -aminase values, and it does not contain any visible haemoglobin.
Sodium caprylate is added to the original plasma until a concen-tration of 0.004 moles is obtained. The mixture, containing about 9 % of ethanol, is heated at a pH of 6.5. The pH value is adjusted by means of 0.5 n of HCI. The temperature is -~
increased to 68c within a period of about 3 hours, with uniform heat supply. Then, the liquid is cooled to 10C. The .
---- 1079~;9 suspension is further cooled and processed at a temperature of 10C. The solution subjected to further processing contains from about 2 to 2.5 % of albumin.
The enclosed drawina illustrates in schematical form the processing of the staring solution. In the process a modified rotating discharge filter type ZHR-S (Schenk, upright model) to accomodate the sep~ration of a 600 1 plasma batch within a maximum of 5 h filtering time was used. The central device of the complete system is a filter vessel 20, double walled to make both heating and cooling possible, having a capacity of about 220 1 and a filtration surface area of about 3 m3 The circular filter discs 22 are sealed to a central rotating column 21 in such a manner that the filtrate, once having passed through the filter medium coating each disc, flows into the central column and thence to the outlet through line 23 and valve 8. Removal of the residue (filteraid and precipitated globulins) between filtration cycles is accomplished by centri-fugal washing and a backwash of 1 % NaCl, so that the filter is readily prepared for the next cycle. The filter elements are covered with a metallic twisted (wire) or braid fabric.
The unfiltered starting solution is maintained in suspension within a slurry tank 24 with constant agitation (agitator 3). By means of a pump 2, the starting substance is introduced into the filter vessel 20 via the slurry feed valve 10 through a pair of inlet valves 11, 12. The vessel may be pressurized with compressed air by means of compressed air valve 5.
Initially, the filter elements are precoated with filtering aid suspended in distilled water in a suspension of about 1 kilopond of Celite 545 (trademark for a kieselguhr filter-ing aid) in 500 millimeters of water, to form a primary precoat having a thickness of about 0.2 centimeters at atmosperic ~` , ' .
iO792W
pressure. Thereafter, 500 liters of albumin-containing suspen-sion are mixed with 25 kiloponds of Celite 545 and stirred. The mixture is introduced into the filter vessel from the slurry tank, and urged through the filter surfaces of elements 22 at a positive pressure of 2 bar (atmospheres). Prior to the actual dispensing of the liquid to the clear tank, the substance is repeatedly passed through the filter elements in the so-called recirculation process (recirculation valve 6, pump 2), until the filtrate appears to be sufficiently clear at the sight glass 25. Thereafter, the filtrate is discharged to the clear tank through valve 8.
As the clarified albumin containing solution leaves the filter, a 1 % ~aCl wash solution is simultaneously pumped through the filter. (Volume of wash solution used is equal to half the volume of the original batch size). m e filtration rate is 150 l/h. The protein concentration of the filtrate at the beginning of the process is about 2,5 %, while the end con- ~
centration of non-albumin constituents is only about 0,05 %. ~ -The resultant solution is clear with a protein concentration of about 1,5 % albumin and an osmolatity of approximately 1.000 mosm.
This clear filtrate is then diafiltered using the Millipore Pellicon Cassette system (filtration surface area 9,0 m3, Millipore GmbH, Neu-Isenburg, Germany). Diafiltration means, that the solution is filtered through a dialysis membrane.
The flow rate at the beginning of the process is about 5 - 6 liters filtrate/min.
Operating on an 8-hour shift per day, the entire fractionation procedure takes 3 days.
day 1: heat-ethanol precipation, subsequent cooling and pH 4,4 adjustment (5 hours), day 2: alluvial filtration (4 hours) and diafiltration (flow rate so regulated that process is finished at beginning . : .............................. : .~
- , . . . . . ..
of 3rd working day). The material is kept at temperatures between + 6C and + 8 C by cooling the double walled vessels in both procedures, day 3: final adjustment of pH (7,o), protein concentration ~5 %, 20 ~) , osmolality (300 mosm) clear filtration:
sterile filtration (membrane 0,2 ,u); bottling: pasteurizing (10 hours, 60C).
While the yield of albumin using the alluvial filtra-tion/diafiltration procedure is 93 % of the albumin present in the original starting material (average from 60 batches), and quality of the final product is the same as that achieved with previous methods, the greatest advantage of this methods lies in the fact that the entire process, with the exception of bottling, may be performed by one person.
Cleaning of the filters is effected by rotating the filter elements by means of the driving motor 1 and by discharg-ing the slurry through discharge valve 9. Through valve 4, the discharged substance may be suspended and filtered again.
After its passage through the filter, the liquid does not show any Tyndall effect any longer, and it is clear as water. m e liquid is free of accompanying substances, such as lipids of foreign proteins. The liquid may be used directly for the albun~in saturation and subje~d to the subsequent filtration process (compare laid-open German patent application 2,415,079).
In principle, the following steps incur in the process:
PLASMA
600 kg: 33,6 kg PROTEI~ :~
18,48 kg ALBUMIN ( = 55 %) 1. + 9 % ETHANOL
+ O,004 M SODIUM CARPRYLATE
+ HCL pH 6.5 2. HEATPRECIPITATION: 30 min. 68C.
3. + HCL pH 4.4
- ~: . -, , ,. . . , , :
clarification or purification filtration and which, thus, permit optimum performance of the operations in the recovery of albumin to be obtained.
In a particularly preferred embodiment of the invention, the filtration is performed within a centrifugal purification filter comprising a closed (sealed) pressure vessel.
Preferably, the liquids in question are processed at a mesh size of from 70 to 90 microns. This mesh size provides an optimum between the throughflow that can be obtained `
; 10 and complete filtering out, in which case a single filtering `
step only is required to be carried out in each operation.
In order to adequately prepare the filter surface for the subsequent filtration, it is expedient in some instances initially to perform a primary precoating with a neutral liquid wherein the filter element is precoated with a layer of the filtering aid having a thickness of about 0.5 centimeters. As filtering aids, the customary, commercially available kiessel-; guhrs sold under the tradenames Hyflo-Super-Cel, Celite 545 or perlite are suitable. Additionally, cellulose filtering aids ~ 20 of conventional type are also useful, although with lesser ii filtering efficiency. These filtering aids likewise allow to obtain a product being satisfactory for many applications. It ~ is recommendable that the filtering aid is subjected to - pre-swelling or pre-soaking.
Favorable filtering effects are obtained with a ` horizontally disposed filter element of metallic twist (wire) or braid fabric having a mesh size of 80 microns.
A favorable composition compromises a suspension of from about 4 to 6% of plasmatic protein, to which suspension there are added from about 20 to 70 grams of kieselguhr filtering aid per liter. Thereafter, this suspension is filtered. Lower concentrations of protein may require lesser quantities of _ 4 _ .. . .
filtering aids under certain circumstances.
In order to recover also the albumin present within the slurry or turbid volume, it is proposed that this albumin is likewise isolated by means of a residual volume filtering device comprising a small portion of the total filter area, after a cleaning by rotation and countercurrent flushing or a clearing of the vol~me of the filtering apparatus place in a recirculation system has been performed.
In order to increase the yield, the process may`be carried out at a pressure of 4.0 + 2.5 bar (atmospheres) without sacrifice to the filtering capacity.
Below, the present invention is explained in greater detail by means of the following Exampl~s. The two Examples start with two different suspensions. Example 1 starts with a suspension prepared in accordance with the process of the laid-open German patent application 2,415,079.
Example 1 m e solid constituents (blood cells and blood platelets) are separated from human blood, and the coagulation factors are removed. The starting solution contains from about 5 to 6 %
of plasmatic protein. In accordance with the process disclosed in the above publication, the clotting factor VIII and the fibrinogen are removed by cryo ethanol sedimentation. The prothrombin complex is removed by absorption. The original plasma i~ ~lepatitis-activity negative, it shows normal trans- -aminase values, and it does not contain any visible haemoglobin.
Sodium caprylate is added to the original plasma until a concen-tration of 0.004 moles is obtained. The mixture, containing about 9 % of ethanol, is heated at a pH of 6.5. The pH value is adjusted by means of 0.5 n of HCI. The temperature is -~
increased to 68c within a period of about 3 hours, with uniform heat supply. Then, the liquid is cooled to 10C. The .
---- 1079~;9 suspension is further cooled and processed at a temperature of 10C. The solution subjected to further processing contains from about 2 to 2.5 % of albumin.
The enclosed drawina illustrates in schematical form the processing of the staring solution. In the process a modified rotating discharge filter type ZHR-S (Schenk, upright model) to accomodate the sep~ration of a 600 1 plasma batch within a maximum of 5 h filtering time was used. The central device of the complete system is a filter vessel 20, double walled to make both heating and cooling possible, having a capacity of about 220 1 and a filtration surface area of about 3 m3 The circular filter discs 22 are sealed to a central rotating column 21 in such a manner that the filtrate, once having passed through the filter medium coating each disc, flows into the central column and thence to the outlet through line 23 and valve 8. Removal of the residue (filteraid and precipitated globulins) between filtration cycles is accomplished by centri-fugal washing and a backwash of 1 % NaCl, so that the filter is readily prepared for the next cycle. The filter elements are covered with a metallic twisted (wire) or braid fabric.
The unfiltered starting solution is maintained in suspension within a slurry tank 24 with constant agitation (agitator 3). By means of a pump 2, the starting substance is introduced into the filter vessel 20 via the slurry feed valve 10 through a pair of inlet valves 11, 12. The vessel may be pressurized with compressed air by means of compressed air valve 5.
Initially, the filter elements are precoated with filtering aid suspended in distilled water in a suspension of about 1 kilopond of Celite 545 (trademark for a kieselguhr filter-ing aid) in 500 millimeters of water, to form a primary precoat having a thickness of about 0.2 centimeters at atmosperic ~` , ' .
iO792W
pressure. Thereafter, 500 liters of albumin-containing suspen-sion are mixed with 25 kiloponds of Celite 545 and stirred. The mixture is introduced into the filter vessel from the slurry tank, and urged through the filter surfaces of elements 22 at a positive pressure of 2 bar (atmospheres). Prior to the actual dispensing of the liquid to the clear tank, the substance is repeatedly passed through the filter elements in the so-called recirculation process (recirculation valve 6, pump 2), until the filtrate appears to be sufficiently clear at the sight glass 25. Thereafter, the filtrate is discharged to the clear tank through valve 8.
As the clarified albumin containing solution leaves the filter, a 1 % ~aCl wash solution is simultaneously pumped through the filter. (Volume of wash solution used is equal to half the volume of the original batch size). m e filtration rate is 150 l/h. The protein concentration of the filtrate at the beginning of the process is about 2,5 %, while the end con- ~
centration of non-albumin constituents is only about 0,05 %. ~ -The resultant solution is clear with a protein concentration of about 1,5 % albumin and an osmolatity of approximately 1.000 mosm.
This clear filtrate is then diafiltered using the Millipore Pellicon Cassette system (filtration surface area 9,0 m3, Millipore GmbH, Neu-Isenburg, Germany). Diafiltration means, that the solution is filtered through a dialysis membrane.
The flow rate at the beginning of the process is about 5 - 6 liters filtrate/min.
Operating on an 8-hour shift per day, the entire fractionation procedure takes 3 days.
day 1: heat-ethanol precipation, subsequent cooling and pH 4,4 adjustment (5 hours), day 2: alluvial filtration (4 hours) and diafiltration (flow rate so regulated that process is finished at beginning . : .............................. : .~
- , . . . . . ..
of 3rd working day). The material is kept at temperatures between + 6C and + 8 C by cooling the double walled vessels in both procedures, day 3: final adjustment of pH (7,o), protein concentration ~5 %, 20 ~) , osmolality (300 mosm) clear filtration:
sterile filtration (membrane 0,2 ,u); bottling: pasteurizing (10 hours, 60C).
While the yield of albumin using the alluvial filtra-tion/diafiltration procedure is 93 % of the albumin present in the original starting material (average from 60 batches), and quality of the final product is the same as that achieved with previous methods, the greatest advantage of this methods lies in the fact that the entire process, with the exception of bottling, may be performed by one person.
Cleaning of the filters is effected by rotating the filter elements by means of the driving motor 1 and by discharg-ing the slurry through discharge valve 9. Through valve 4, the discharged substance may be suspended and filtered again.
After its passage through the filter, the liquid does not show any Tyndall effect any longer, and it is clear as water. m e liquid is free of accompanying substances, such as lipids of foreign proteins. The liquid may be used directly for the albun~in saturation and subje~d to the subsequent filtration process (compare laid-open German patent application 2,415,079).
In principle, the following steps incur in the process:
PLASMA
600 kg: 33,6 kg PROTEI~ :~
18,48 kg ALBUMIN ( = 55 %) 1. + 9 % ETHANOL
+ O,004 M SODIUM CARPRYLATE
+ HCL pH 6.5 2. HEATPRECIPITATION: 30 min. 68C.
3. + HCL pH 4.4
- 4. ALL WIAL FILTRATIO~
FILTRATE
FILTRATE
5. DIAFILTRATION
5.1 CO~CENTRATIO~
5.2 DIALYSIS
5.1 CO~CENTRATIO~
5.2 DIALYSIS
6. + NaOH pH 7.0 .. . . .
7. CLEAR FILTRATION
' ALBUMIN SOLUTION
17.2 kg ALBUMIN ( = 93 % YIELD) Example 2: ~ -From a placenta extract, the haemoglobin contained therein is initially removed with the aid of solvents, such as, for instance, trichloro acetic acid, chloroform. diethylether.
Then, the protein-containing supernatant liquid is heated to a temperature of 60C at a pH of 6.5.Upon cooling and adjusting the pH to a value of 4.4 with the~addition of 0.5 n of HCl, 100 liters of the thus obtained liquid are mixed with 3 kiloponds of filtering aid and stirred (filtering aid: Hyflo-Super-Cel:
trademark for a kieselguhr filtering aid).
At a temperature of 18C and a pressure of 4 bar (atmospheres), the liquid is urged through an alluvial filter of the above-specified type and having a mesh size of 70 microns.
Accordingly, the operation is performed without a primary 3~ precoat. The liquid is passed in recirculation until it appears in the sight glass to be clear as water and free of the Tyndall effect. Thereupon, the albumin contained in the liquid is "
_ g _ .
~ - . : . , , -: . .
-` 1079Z69 adjusted to the desired concentration by conventional methods.
After the entirety of the liquid has passed through the filter, the slurry or turbid volume retained in the filter is re-washed with distilled water or with 0.9 % NaCl solution.
The liquid in this manner is likewise clear as water, and it still contains from about 0.5 to 20 % of albumin. This liquid may likewise be added directly to the albumin concentration, as is explained e.g. in the laid-open German patent application 2,415,079.
Further reference may be made to the fact that the floating filtration may be carried out both with and without a primary precoat. Clearing of the filter elements is effected by rotation and countercurrent flushing the filter elements being rotated and the residues being discharged as slurry.
If a sufficient quantity of washing liquid is employed, (namely distilled water or NaCl solutions), an increase of the yield of up to about 96 % of the originally used quantity of albumin can be obtained. The quantities of impurities of turbidities (slurries) - caused by variation of the density ratio between solvent and solid substance as well as optimum mechanical distribution - as resulting in other separation processes, such as e.g. by centrifuging, particularly in the elution of the initially separated proteins, are avoided by residual volume filtration, either a cleaning of the volume of ~
the filtering apparatus by rotation and countercurrent flushing ~-or after a clearing thereof in the recirculation process has taken place. Filtration prior to the further processing of the diluted albumin solution may be dispensed with in view of the high degree of purity thereof.
The separation of the solids is independent of tempe-rature. The rate of filtration varies only slightly with increas-ing or decreasing temperatures, and such variation may be neglected. Normally, the heat-treated plasma acidified to a pH
of 4.4 is subjected to separation at room temperature within a centrifugal purification filter adapted to be cooled by means of an external jacket. The filtration step may be performed at different temperatures as well. Experience has shown that a temperature range of from 4 to + 40C can be applied as range of operation. Following the primary precoating which is perform-ed almost without pressure, the filtration pressure may be increased to 4.0 1_ 2,5 bar (atmospheres). The resulting filtra-tion capacity amounts to about 150 liters of filtrate per squaremeter per hour. As mentioned above, kieselguhrs prove to be of particular usefulness as filtering aids.
The quantities specified in the Example are variable depending on the capacity of the filter. Tests have been performed successfully, both with small volumes (about 10 liters) and in large vessels (about 500 liters).
-. . , . : .
, . . . . . .
' ALBUMIN SOLUTION
17.2 kg ALBUMIN ( = 93 % YIELD) Example 2: ~ -From a placenta extract, the haemoglobin contained therein is initially removed with the aid of solvents, such as, for instance, trichloro acetic acid, chloroform. diethylether.
Then, the protein-containing supernatant liquid is heated to a temperature of 60C at a pH of 6.5.Upon cooling and adjusting the pH to a value of 4.4 with the~addition of 0.5 n of HCl, 100 liters of the thus obtained liquid are mixed with 3 kiloponds of filtering aid and stirred (filtering aid: Hyflo-Super-Cel:
trademark for a kieselguhr filtering aid).
At a temperature of 18C and a pressure of 4 bar (atmospheres), the liquid is urged through an alluvial filter of the above-specified type and having a mesh size of 70 microns.
Accordingly, the operation is performed without a primary 3~ precoat. The liquid is passed in recirculation until it appears in the sight glass to be clear as water and free of the Tyndall effect. Thereupon, the albumin contained in the liquid is "
_ g _ .
~ - . : . , , -: . .
-` 1079Z69 adjusted to the desired concentration by conventional methods.
After the entirety of the liquid has passed through the filter, the slurry or turbid volume retained in the filter is re-washed with distilled water or with 0.9 % NaCl solution.
The liquid in this manner is likewise clear as water, and it still contains from about 0.5 to 20 % of albumin. This liquid may likewise be added directly to the albumin concentration, as is explained e.g. in the laid-open German patent application 2,415,079.
Further reference may be made to the fact that the floating filtration may be carried out both with and without a primary precoat. Clearing of the filter elements is effected by rotation and countercurrent flushing the filter elements being rotated and the residues being discharged as slurry.
If a sufficient quantity of washing liquid is employed, (namely distilled water or NaCl solutions), an increase of the yield of up to about 96 % of the originally used quantity of albumin can be obtained. The quantities of impurities of turbidities (slurries) - caused by variation of the density ratio between solvent and solid substance as well as optimum mechanical distribution - as resulting in other separation processes, such as e.g. by centrifuging, particularly in the elution of the initially separated proteins, are avoided by residual volume filtration, either a cleaning of the volume of ~
the filtering apparatus by rotation and countercurrent flushing ~-or after a clearing thereof in the recirculation process has taken place. Filtration prior to the further processing of the diluted albumin solution may be dispensed with in view of the high degree of purity thereof.
The separation of the solids is independent of tempe-rature. The rate of filtration varies only slightly with increas-ing or decreasing temperatures, and such variation may be neglected. Normally, the heat-treated plasma acidified to a pH
of 4.4 is subjected to separation at room temperature within a centrifugal purification filter adapted to be cooled by means of an external jacket. The filtration step may be performed at different temperatures as well. Experience has shown that a temperature range of from 4 to + 40C can be applied as range of operation. Following the primary precoating which is perform-ed almost without pressure, the filtration pressure may be increased to 4.0 1_ 2,5 bar (atmospheres). The resulting filtra-tion capacity amounts to about 150 liters of filtrate per squaremeter per hour. As mentioned above, kieselguhrs prove to be of particular usefulness as filtering aids.
The quantities specified in the Example are variable depending on the capacity of the filter. Tests have been performed successfully, both with small volumes (about 10 liters) and in large vessels (about 500 liters).
-. . , . : .
, . . . . . .
Claims (9)
1. A process of separating precipitated proteins from albumin containing suspensions obtained from the recovery of albumin from blood plasma, characterized by an alluvial filtration of said suspensions through a fabric filter element having a mesh size of between 20 and 200 microns, thereby collecting the non-albumin constituents on the filter elements and gaining a clear albumin solution as a filtered product.
2. The process according to claim 1, characterized by performing said filtration within a centrifugal purification filter comprising a sealed pressure vessel.
3. The process according to claim 2, characterized in that one of said filter elements has a mesh size from 70 to 90 microns.
4. The process according to claim 2, characterized by performing said filtration with horizontally disposed filter elements comprising metallic twisted (wire) or braided fabrics having a mesh size of 80 microns.
5. The process according to claim 1, characterized by mixing a suspension of from about 4 to 6% of plasmatic protein with from about 30 to 70 grams of kieselguhr filtering aid per liter.
6. The process according to claim 1, characterized by employing a cellulose filtering aid in a pre-swollen or pre-soaked condition.
7. The process according to claims 1, 2 or 6, characterized by recovering also the albumin present within the slurry or turbid volume by isolating it by means of a residual volume filtering device comprising a small portion of the total filter area, after a cleaning by rotating and counter-current flushing or a clearing of the volume of the filtering apparatus place in a recirculation system has been performed.
8. The process according to claims 2 or 6, characterized by performing said process at a positive filtration pressure of 4.0 - 2.5 bar (atmospheres).
9. The process according to claim 1, characterized in that the clear albumin-solution is diafiltered (filtered through a dialysis membrane).
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2556733A DE2556733C3 (en) | 1975-12-17 | 1975-12-17 | Method for isolating albumin from blood plasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1079269A true CA1079269A (en) | 1980-06-10 |
Family
ID=5964580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA268,010A Expired CA1079269A (en) | 1975-12-17 | 1976-12-16 | Process of separating precipitated proteins from albumin - containing suspensions |
Country Status (22)
| Country | Link |
|---|---|
| JP (1) | JPS6033089B2 (en) |
| AT (1) | AT346491B (en) |
| AU (1) | AU508955B2 (en) |
| BE (1) | BE849464R (en) |
| CA (1) | CA1079269A (en) |
| CH (1) | CH632277A5 (en) |
| DD (1) | DD128726A6 (en) |
| DE (1) | DE2556733C3 (en) |
| DK (1) | DK149135C (en) |
| EG (1) | EG12610A (en) |
| ES (1) | ES454379A2 (en) |
| FI (1) | FI56844C (en) |
| FR (1) | FR2335521A2 (en) |
| GB (1) | GB1569168A (en) |
| HU (1) | HU181926B (en) |
| IE (1) | IE44048B1 (en) |
| IL (1) | IL51110A (en) |
| MX (1) | MX3915E (en) |
| NL (1) | NL7613906A (en) |
| PL (1) | PL106430B3 (en) |
| SE (1) | SE445517B (en) |
| ZA (1) | ZA767503B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4483807A (en) * | 1981-01-27 | 1984-11-20 | Japan Atomic Energy Research Institute | Process for producing a slow release composite |
| DE3307871C2 (en) * | 1983-03-05 | 1986-10-30 | R & Z Biologicals S.A. (Pty.) Ltd., Cape Town | Process for obtaining high purity albumin |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2705230A (en) * | 1949-09-23 | 1955-03-29 | Allen F Reid | Method of purifying albumin |
| US2765299A (en) * | 1952-06-27 | 1956-10-02 | Armour & Co | Recovery of serum albumin |
| FR2247471A1 (en) * | 1973-10-15 | 1975-05-09 | Ts Institu | Pure serum albumin isolated from biological liquids - by treatment with alcohol and aliphatic carboxylic acid salt |
| DE2415079C3 (en) * | 1974-03-28 | 1980-02-14 | Plasmesco Ag, Zug (Schweiz) | Method for isolating albumin from blood plasma |
-
1975
- 1975-12-17 DE DE2556733A patent/DE2556733C3/en not_active Expired
-
1976
- 1976-12-10 IE IE2712/76A patent/IE44048B1/en unknown
- 1976-12-13 SE SE7613968A patent/SE445517B/en unknown
- 1976-12-14 DK DK559876A patent/DK149135C/en not_active IP Right Cessation
- 1976-12-15 EG EG769/76A patent/EG12610A/en active
- 1976-12-15 DD DD7600196377A patent/DD128726A6/en unknown
- 1976-12-15 GB GB52438/76A patent/GB1569168A/en not_active Expired
- 1976-12-15 IL IL51110A patent/IL51110A/en unknown
- 1976-12-15 FI FI763610A patent/FI56844C/en not_active IP Right Cessation
- 1976-12-15 NL NL7613906A patent/NL7613906A/en not_active Application Discontinuation
- 1976-12-16 PL PL1976194451A patent/PL106430B3/en unknown
- 1976-12-16 CA CA268,010A patent/CA1079269A/en not_active Expired
- 1976-12-16 AU AU20599/76A patent/AU508955B2/en not_active Expired
- 1976-12-16 HU HU76PA1271A patent/HU181926B/en unknown
- 1976-12-16 AT AT932976A patent/AT346491B/en not_active IP Right Cessation
- 1976-12-16 FR FR7637897A patent/FR2335521A2/en active Granted
- 1976-12-16 BE BE173309A patent/BE849464R/en not_active IP Right Cessation
- 1976-12-17 JP JP51150986A patent/JPS6033089B2/en not_active Expired
- 1976-12-17 CH CH1593176A patent/CH632277A5/en not_active IP Right Cessation
- 1976-12-17 MX MX76100452U patent/MX3915E/en unknown
- 1976-12-17 ZA ZA767503A patent/ZA767503B/en unknown
- 1976-12-17 ES ES454379A patent/ES454379A2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| NL7613906A (en) | 1977-06-21 |
| DD128726A6 (en) | 1977-12-07 |
| FI56844B (en) | 1979-12-31 |
| ZA767503B (en) | 1977-11-30 |
| FI763610A (en) | 1977-06-18 |
| AU508955B2 (en) | 1980-04-17 |
| AT346491B (en) | 1978-11-10 |
| DK149135C (en) | 1986-07-14 |
| FR2335521A2 (en) | 1977-07-15 |
| ES454379A2 (en) | 1978-07-16 |
| GB1569168A (en) | 1980-06-11 |
| AU2059976A (en) | 1978-06-22 |
| PL106430B3 (en) | 1979-12-31 |
| SE7613968L (en) | 1977-06-18 |
| SE445517B (en) | 1986-06-30 |
| EG12610A (en) | 1979-06-30 |
| DE2556733A1 (en) | 1977-11-17 |
| DK149135B (en) | 1986-02-10 |
| ATA932976A (en) | 1978-03-15 |
| CH632277A5 (en) | 1982-09-30 |
| JPS6033089B2 (en) | 1985-08-01 |
| DK559876A (en) | 1977-06-18 |
| JPS5283926A (en) | 1977-07-13 |
| IL51110A0 (en) | 1977-02-28 |
| HU181926B (en) | 1983-11-28 |
| FR2335521B2 (en) | 1982-10-29 |
| BE849464R (en) | 1977-04-15 |
| DE2556733C3 (en) | 1986-10-02 |
| IE44048B1 (en) | 1981-07-29 |
| DE2556733B2 (en) | 1981-05-14 |
| FI56844C (en) | 1980-04-10 |
| MX3915E (en) | 1981-09-18 |
| IE44048L (en) | 1977-06-17 |
| IL51110A (en) | 1979-11-30 |
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