DE2537123A1 - Albumin recovery from blood fractions - by first treating with an anionic ion exchanger, then selective denaturation of other proteins - Google Patents

Abstract

Prepn. of blood albumin (I) comprises first sepg. a (I)-contg. fraction from blood protein, serum or plasma by treatment with a anionic ion-exchange resin having amino, alkylamino, guanidino or quat. ammonium exchanging gps. An aq. soln. of this separated fraction is prepd., an agent (II) capable of stabilising (I) against heat-denaturation is added, then the soln. heated to denature proteins other than (I). The denaturated proteins are removed leaving a soln. of (I). The stabiliser is >=1 of acetyltryptophan; middle chainlength fatty acids or the salts of these cpds. (esp. Na octanoate). Pref. the resin is crosslinked regenerated cellulose or crosslinked dextran contg. diethylaminoethyl gps. I) is used as a plasma-volume extender and to treat hypoproteinaemia. Using the ion exchange treatment gives a more conc. (I) fraction almost completely free of gamma-globulins (which tend to resist the heat-denaturation step), so less unwanted protein has to be removed.

Description

Method for producing albumin The present invention relates to the production of albumin and in particular the production of blood albumin from Blood, blood serum, or blood plasma.

Blood albumin is a water-soluble protein made from the blood, blood serum or blood plasma can be isolated. Human blood, serum or albumin Plasma is used in medicine, for example, as a plasma volume expander and for improvement used by hypoproteinemia. Bovine serum albumin obtained from bovine blood, serum or Plasma extracted has a wide variety of uses in chemistry and Biochemistry. Current methods of making albumin include precipitates or fractionation using inorganic salts and / or organic solvents for blood, blood serum or blood plasma, for example Cohn fractionation. Such working methods require considerable effort to be carried out with associated costs.

The present invention is based on the problem of the disadvantages to bring existing working methods to a minimum.

According to the invention it has now been found that blood albumin can be prepared by taking an albumin-containing fraction of proteins from blood serum or plasma is separated with the aid of anionic ion exchange resins and then the proteins contained therein, which are different from albumin, are selectively denatured, the albumin then being separated from the other denatured proteins can.

Accordingly, the present invention relates to a method of manufacture of blood albumin, which is essentially characterized in that one contains an albumin Fraction of blood proteins from blood serum or plasma with the help of an anionic Ion exchange resin, in which the exchange groups are selected from amino, Alkylamino and guanidino groups and quaternary ammonium ions, separates and a aqueous solution of the albumin-containing fraction of blood proteins forms, 'I too this solution adds an agent that the albumin contained therein against heat denaturation can stabilize and which consists of one or more compounds selected are among acetyltryptophan, fatty acids with medium chain length, salts of acetyltryptophan and salts of fatty acids with medium chain length, the resulting solution of a Subjects to heat denaturation for blood proteins other than albumin and separating the denatured proteins from the product obtained, wherein the blood albumin remains in solution.

Blood serum for use in the method according to the invention can be obtained by letting the whole blood clot and then the liquid Separates the serum from the solids, for example by expressing it.

Blood plasma for use in the method according to the invention can also by centrifuging the whole blood to separate the plasma from the blood cells, preferably obtained by using a conventional high speed centrifuge will.

In the case of animal blood, for example bovine blood, the separation can of the blood plasma conveniently at the freezers or at other locations in a slaughterhouse and the blood is collected directly from the animal. One preferably enters Anticoagulants, such as sodium oxalate, put the blood in a centrifuge and collects the light phase (the plasma) from it. It is of course not essential, the separation at the same time or place as the collection of the Blood carry out, provided that the blood is in a suitable manner treated with an anticoagulant and suitable in the meantime is stored.

When the method according to the invention is carried out, it is all over For the sake of simplicity, it is preferred that the blood serum or plasma be treated first to create a lower ionic strength in the solution. This can be done by any known methods for desalination of the plasma, for example using a Ultrafiltration extraction or electrolytic cell techniques can be achieved. Preferably, however, the ionic strength is reduced by treating the serum or plasma diluted with distilled water. The obtained, increased volume of serum - or Plasma solution for treatment is not disadvantageous for the method according to the invention.

The stage of separation of blood proteins from serum or plasma, preferably after having lowered the ionic strength in solution, it is carried out, by using an anionic ion exchange resin in which the exchange groups under amino Alkylamino and guanidino groups and quaternary ammonium ions are selected. The anionic ion exchange resin can be based on a synthetic resin, a polysaccharide or a cellulose matrix, the large organic molecules, such as proteins, can be built up. The ion exchange resin must be able to bind macromolecules, such as proteins, without distorting them or denature. DEAE resins, i.e. diethylaminoethyl resins such as DEAE cellulose and DEAE-dextran; Ion exchangers have proven to be particularly advantageous and preferred such resins are the crosslinked, regenerated cellulosic resins with diethylaminoethyl exchange groups established in New Zealand by the Tasman Vaccine Laboratory Limited are sold under the name "Protion", and the networked Dextran resins with diethylaminoethyl exchange groups, which are available from the Pharmacia of Sweden under the name DEAE-Sephadex.

The separation step can be carried out by taking the serum or the plasma or a solution thereof in contact with the ion exchange resin any of the usual methods, for example in columns, countercurrent distribution, or a stirred batch container. The resin then becomes one Subjected to washing stage and then with a suitable buffer solution, essentially eluted according to conventional ion exchange separation techniques. However, if you have that preferred ion exchange resin t'Protion "is used, it is possible to simply use the resin to stir in the diluted serum or plasma and then remove the treated resin from the Separate liquid, for example by decanting or filtering.

After washing, the treated resin can then with a suitable Moving or stirring the buffer solution to release the proteins in solution, and the resulting solution then from the regenerated resin, for example by decanting or filtration.

The solution thus obtained consists of an albumin-containing one or Albumin-rich fraction of blood proteins, consisting essentially of albumin and other blood proteins such as <- and ß-globulins. To this solution there an agent that is able to stabilize albumin against heat denaturation. Suitable stabilizers include acetyltryptophan and fatty acids with medium chain length, (for example C7 to C11) or the salts thereof, for example Alkali metal or alkaline earth metal octanoates in low concentrations. It was found that sodium octanoate is a particularly suitable stabilizing agent is. Mixtures of two or more such stabilizers can be used will.

After the addition of the stabilizer and the preferred adjustment to a pH of about 7, for example to pH 7.0 + 0.1, the solution becomes a Subjected to heat denaturation processes by, for example, the solution brings a sufficiently high temperature, but which is so controlled that the present proteins other than albumin are selectively denatured. The resulting solution is then cooled and acidified and the denatured proteins are separated from the solution, leaving the stabilized albumin in solution.

The separation of the denatured blood proteins is preferably carried out by Filtration or centrifugation or a combination of both measures carried out. Filtering can be done using a filter aid with a plate filter press or preferably a previously coated drum filter.

Centrifugation can be done with a high speed continuous centrifuge be performed.

The diluted albumin solution obtained is preferably further from Low molecular weight material cleaned and applied simultaneously concentrated by ultrafiltration techniques.

The albumin concentrate obtained can then either be freeze-dried to supply the albumin in the form of an amorphous powder, or for production a solution of any suitable concentration of albumin can be used. Alternatively The concentrate can be further purified by washing it with distilled water diluted and again ultrafiltered, using an even smaller proportion of materials with low molecular weight in the concentrate obtained and in that obtainable therefrom Powder or solution.

The method according to the invention can be used for the production of any blood albumin from whole blood, blood serum or plasma, for example for the production of albumin from human blood and for the production of bovine serum albumin from bovine blood, be applied.

The use of an anionic ion exchange resin in the early The stage of the process according to the invention has two important aspects. It allows first, the separation of a fraction of blood proteins rich in albumin; this will reduce the amount of denatured proteins that will occur after the heat denaturation stage must be separated.

If one uses the "protion" ion exchange resin, one can use a Product obtained with a purity greater than 99%.

In particular, this fraction continues to exclude gamma globulins, which survive the subsequent heat denaturation stage and contaminate the end product could. The fraction to be discarded rich in gamma globulin could be used to obtain gamma globulins.

The following is an example of the method according to the invention which is used to make bovine serum albumin.

Example 1. Collection of Blood and Separation of Plasma. One collects fresh bovine blood with a minimum of mechanical agitation in 13.65 liters (3 gal.) Stainless steel buckets, each of which is made by wetting the inner surface was pretreated with 50 ml of 30% sodium oxalate at pH 7.4.

The blood is then transferred to a 40 liter upper tank that is 121.9 cm (4th ft.) is located above a Westfalia SAOH 205 continuous centrifuge, the adjusted with water to the following approximate flow rates: Inlet 121.9 cm (4 ft) head 240 l / hr.

heavy phase 1.83 kg / cm2 (26 psi) 200 l / hr.

light phase 0.14 kg / cm2 (2 psi) 40 1 / hr.

After starting blood flow under these conditions, the proportion of light phase (plasma) can be increased to up to 50% of the input will.

Adjusting this to maintain optimal conditions requires Continuous monitoring, however, should be a considerable refinement operation with permanent equipment, i.e. non-monitored plasma extraction, to be possible.

The light phase is collected in a transport container, and transported from the freezer to the laboratory for further treatment.

2. Preliminary cleaning of the albumin The preliminary separation of the albumin from the plasma is then carried out by removing the albumin as well as other contaminants binds to a "Protion A2" ion exchange resin, and the fraction rich in albumin washes and elutes as detailed below.

After regeneration from a previous cycle, this becomes protion with buffer A (1380 ml of glacial acetic acid, 792 ml of ethylenediamine, diluted to 36 1 and up pH 6.5 adjusted) equilibrated to pH 6.5 and then washed with water until the eluate has a specific conductivity of 4 5.1 m mho / cm at 250C.

The resin is now ready for the plasma.

The plasma is used as it is received from the freezer 1 M acetic acid adjusted to pH 6.5, to a specific conductivity of c 5.1 m mlio / cm set at 25 ° C., stored overnight at 2 ° C., filtered and the equivalent of 11 plasma / kg resin is applied to the column at approximately 2.3 l / hr / kg given up. The resin bed used has a diameter / length ratio of about 2/1, but there are many other equally suitable arrangements. It should it may also be possible to use a cheaper buffer system than the one mentioned. 1 After all of the diluted plasma has entered the resin, it becomes with an equal volume of buffer B (buffer A diluted to 1 part to 20 parts with water) or as much buffer B as to obtain a clear, almost colorless effluent to accomplish. Then the albumin-rich fraction is mixed with solution C (1700 g NaCl + 1 1 buffer A, diluted to 20 1) eluted to 1 l1h / kg resin, 2.0 1 per kg of resin used. While this solution is being processed, one regenerates the column with solution D (1200 g NaCl + 400 g Na0, diluted to 20 1).

3. Removal of other proteins The albumin-rich fraction is added to Dilute 1% albumin and add 15 ml / l sodium octanoate solution (40 g NaOH '80 ml of octanoic acid diluted to 500 ml). After thorough mixing, the pH will rise to 7.0 set. This solution will now be brought to 700C (approx 10 to 15 minutes or preferably even faster) heated, 25 to 30 minutes kept at this temperature and quickly cooled to 300C when the pH is 10 % HCl is adjusted to 4.5.

The exact heating scheme should depend on the device used be adjusted so that the solution during the for complete denaturation of all other proteins is kept at 700C for a minimum time. In some Cases, 20 minutes is adequate, but this seems like the bare minimum.

After mixing for about 1 minute, the solution becomes quick brought down to near freezing point.

The temperature at which the pH drop occurs changes that Type of precipitation, and recent experiments seem to indicate 0 that it is better could be to do this at 2C.

Two alternative ways of working are available to clarify this solution: a) The solution is stored overnight near the freezing point and the clear, Supernatant fluid is withdrawn in the morning. While this put aside the remaining solution is diluted with an equal volume of water Hyflo-Super-Cel filter aid (25 g / l) and filter the slurry on a Frame and plate filter press using previously 2 mm Hyflo Super-Cel coated calico cloths, when using this press, 250 g Filter aid slurried in 20 l of water for a 6400 cm2 plate used.

b) The solution is as above directly, starting with the addition of the Filter aid, filtered.

If the solution comes from method a), the supernatant liquid and the filtrate combined. If it comes from b), the solution becomes as it is further treated. The filtration.

conditions are heavily dependent on the equipment used.

The above solution is then filtered clear by passing it through a 2 mm bed of filter aid passes through it. Finally, the pH is approximated with in NaOH ( 26.4 ml / l) and in acetic acid, if necessary, adjusted to 7.00.

4 Ultrafiltration and desalting The purified albumin solution is at this level tens or less and requires focusing to about 15% before final drying. To concentrate, the bulk solution is nearby from freezing point and with a peristaltic pump at about 200 ml / min.

through a 1 / u line filter and two Dow Miniplants Ultrafilter C / HFU-1o which are connected in series, circulates. The mini plants are operated under a vacuum of 400 to 600 mm Hg, providing an ultrafiltrate flow from7 1 / h for a 1% albumin solution delivers and to: 1.4 1 / hour. at 12% Albumin falls off. After the initial concentration, the albumin content must be and NaCl are measured and based on these values, the concentrate is measured with Water diluted and re-concentrated so that when a powdered product is made the NaCl content is between 0.8% and 1.2%. Because the ultrafiltration device used a nominal "cut-off" of 30,000 mol-wt.

any material with a molecular weight fewer removed than this if it is not bound to the albumin.

5. Freeze-drying Drying the 15% solution usually requires 10 hrs / l on an Edwards Centrifugal Freeze Dryer Model 30P2 / 677. The chamber drying temperature is 38 ° C / 0.16 Torr.

When the powder is dry it must be quickly placed in an airtight container be transferred because the water absorption is very fast. If possible, one should Used in a room with low humidity.

It is believed that a "Rotary-Drum Vacuum Filter" (vacuum filter with a rotating drum) with a pre-coat of Hyflo Super-Cel and a squeegee an automatic micrometer inlet for clarifying or for crude filtration of the the solution resulting from heat denaturation are suitable.

This would help make the process semi-automatic, whereby only a minimum of work is required.

The process according to the invention makes it possible to produce beef serum albumin that meets the following B.S.A. requirements; gen | corresponds to: 1. Description: whitish powder 2. Solubility freely soluble in water 3. Color of the solution: Elcm <0.050 (10% aqueous solution c Q, 5 A) at 404 mpi 10 mm Cells 4. pH of a JO% aqueous solution: 7.0 + 0.1 5. Moisture content: 16 hours over P205 <5 mm Hg 110 ° C N.M.T at 3.0% 6.Sulfated ash: N.M.T 2 % 7. Nitrogen content: N.L.T. 15.50% 8. Fatty acids: - 2 moles / mole Albumin 9. Metals - Cu, Zn, Hg, Pb: N.M.T. 20 ppm total 10. Electrophoresis cellulose Acetate: N.L.T. 99.5% acrylamide gel: 2 - 3 bands 11. NaCl content: 0.8 -> 1.2 %

Claims (15)

Claims g method for the production of blood albumin, thereby characterized in that one contains an albumin-containing fraction from blood proteins Blood serum or plasma with the aid of an anionic ion exchange resin, in which the exchange groups are selected from amino, alkylamino and guanidino groups and quaternary ammonium ions, and an aqueous solution of the albumin-containing Fraction of blood proteins produces, to this solution an agent is added that the the albumin contained therein can stabilize against heat denaturation, and that from one or more compounds selected from acetyltryptophan, fatty acids with medium Chain length, salts of acetyltryptophan, salts of fatty acids with medium chain length is, the solution obtained is a heat denaturation of the contained therein, from Albumin subjects various blood proteins and degrades the denatured proteins the product obtained is separated off, the blood albumin remaining in solution. 2. The method according to claim 1, characterized in that the Produces blood albumin from blood serum. 3. The method according to claim 1, characterized in that the Produces blood albumin from blood plasma. 4. The method according to any one of claims 1 to 3, characterized in that that a diethylaminoethyl resin is used as the anionic ion exchange resin. 5. The method according to claim 4, characterized in that as Diethylaminoethyl resin a cross-linked, regenerated cellulose resin with diethylaminoethyl Exchanger groups used. 6. The method according to claim 4, characterized in that as Diethylaminoethyl resin a crosslinked dextran resin with diethylaminoethyl -Exchange groups used. 7. The method according to any one of claims 1 to 6, characterized in that that the stabilizing agent used is an alkali metal or alkaline earth metal octanoate used. 8. The method according to claim 7, characterized in that as Stabilizer sodium octanoate used. 9. The method according to any one of the preceding claims, characterized in that that the ionic strength of the blood serum is measured before the ion exchange separation stage or plasmas lowers. 10. The method according to claim 9, characterized in that one the ionic strength of the blood serum or plasma by diluting it with distilled water lowers. 11. The method according to any one of the preceding claims, characterized in that that the solution of the albumin-containing fraction of blood proteins after addition of the stabilizer to about pH 7, a heat denaturation by bringing the solution to a sufficiently high temperature that however, it is controlled so that the proteins other than albumin are present are selectively denatured and then cooled and acidified. 12. The method according to any one of the preceding claims, characterized in that that the denatured proteins derived from the heat denaturation stage Product, separated by filtration and / or centrifugation and the blood albumin in Solution leaves. 13. The method according to claim 12, characterized in that one the resulting solution of albumin of low molecular weight material continues cleans and at the same time through ultrafiltration techniques concentrated. 14. The method according to claim 13, characterized in that one Freeze-dried the concentrated solution of albumin with distilled water Diluted to a desired concentration or by dilution with distilled water and ultrafiltration further purifies. 15. The method according to any one of the preceding claims, characterized in that that one makes bovine serum albumin.