IE42460B1 - Process for isolating albumin from blood - Google Patents

Description

This invention relates to a process for isolating albumin, from a medium comprising blood, blood products, other body fluids and/or tissue extracts.

Blood is a fluid which consists of solid and 5 liquid constituents. The solid constituents include red and white blood corpuscles and blood platelets.

The plasma or liquid part of the blood contains about 90% of water and 10% of solids. The substances dissolved in the plasma include, inter alia, albumin, which is the sole protein constituent of plasma which is stable to temperatures in excess of about 60°C. It is in itself known to separate the blood plasma from the red and white blood corpuscles and from the blood platelets. Furthermore 43460 it is known to remove γ-globulins as well as coagulationpromoting substances, such as, for example, fibrinogen, from the plasma. For therapeutic or diagnostic purposes it is desirable to obtain as pure an albumin solution or albumin paste as possible, which should, as far as possible, not contain any further protein constituents of the blood plasma.

To isolate the albumin from the blood, a process known by the name of Cohn method is used in particular (J. Am.Chem.Soc.68(1946): p 459); this process gives a yield of about 70% and a purity of the end product of only 95-98%. The process starts from a plasma mixed from different samples of blood. This process is, in principle, a fractional precipitation under different conditions. Xn the first stage, the first sediment, principally fibrinogen, is initially separated off at minus 3°C and a pH value of 7.2, 8% of alcohol being added. The supernatant liquid is freed progressively from γ-globulins and aland β-globulins in three further steps, each at subzero temperatures and using increasing alcohol contents of the fluid. Thereafter, a so-called crude albumin paste is obtained, which is either converted to a 5% strength solution or is subjected to a further purification, which results in a 20% strength albumin solution. The entire fractional separation by the known Cohn method requires about 6-8 days, assuming a normal working day of 8 hours.

A further disadvantage found with this method is that numerous working steps must be carried out at accurately set sub-zero temperatures. This requires either that the entire laboratory is kept at this temperature or that the reaction vessels are cooled, at great expense. Both 2 4 6 3 represent a disadvantage and are in particular found to be a great nuisance by the laboratory personnel.

Accordingly, there exists the problem of improving the known method decisively, and in particular of achieving a situation where the working time is shortened, the number of working steps is reduced, the nuisance to personnel is limited and and the purity is increased without lowering' the yield. In addition it is also desirable that, the technical effort required should be substantially reduced.

It has previously been proposed to recover serum albumin from an aqueous solution of mammalian serum albumin containing other protein constituents of mammalian blood, by a process which comprises selectively coagulating the other protein constituents in the solution by heating the solution to a temperature between approximately 45° and 75°C., the solution being at a pH on the alkaline side of the isoelectric point of the serum albumin, but not above pH 5.7 and containing caprylate ions at a concentration of between approximately 0.0075 and 0.02 M.

It is stated that this process can be carried out even if the aqueous solution contains up to 4% concentration of methanol or ethanol. In this case, ethanol may be added to the blood to 4% concentration and the pH adjusted to 7.3, whereupon fibrinogen is removed. The pH is then adjusted to not above 5.7 and the albumin recovered in the manner just indicated, the 4% concentration of ethanol in the serum not interfering with the process.

In contradistinction to this previously proposed process, the present process is conducted in the presence 2 4 6 0 of more than 4% and preferably 7 to 9% of an alcohol. Accordingly, the present invention provides a process of isolating albumin from a medium comprising blood, blood products, other body fluids and/or tissue extracts, including the steps of a) separating plasma from the solid constituents of the said medium, b) precipitating the globulins by heating the separated plasma in the presence of more than a 4% concentra]_0 tion of an alcohol having the formula CH3-(CH2)n-OH, where n is Ο, 1 or 2, and in the presence of one or more albumin stabilisers (as hereinafter defined) to a temperature of from 60 to 75°C., and separating off the fluid from globulins? and c) recovering the albumin contained in the residual fluid.

The medium is preferably blood and the plasma is separated from the solid constituents of the blood, such 20 as the blood cells and blood platelets. If desired, the dissolved non-albumin constituents may be recovered from the plasma at this stage and prior to the precipitation of the globulins.

Blood, blood products, other body fluids or tissue extracts are to be regarded as starting products of the present process. By blood products, there are meant e.g. intermediate products which have bean obtained in accordance with the Cohn method. By tissue extracts, there are meant 2 4 6 0 . for example, extracts from placentae.

An essential step of the present process is the heating of the fluid containing albumin in the presence of more than 4% of the alcohol. In this process the albumin - in contrast to all other plasma proteins is protected against precipitation by adding stabilisers.

Protein- and albumin-stabilisers are known and are compounds which enable a pure albumin-solution to be heated up to 60°C. without any visual alteration of the albumin. The work of Ballou et al. (J. Clinical Invest, i (1944), pp.454; J. Biol.Chem.153 (1944); pp 589) hint, that propionate, butyrate, valerate, caproate, caprylate, phenylacetate, phenylbutyrate, and tryptophane radicals have the desired stabilising properties.

As a result of the globulins being precipitated at about 68°C, while the albumins exist unharmed in the presence of alcohol and stabilisers, it is possible, surprisingly , to dispense with cooling during the separation, In addition, numerous working steps are saved, since fractional precipitation is dispensed with. Instead, all globulins can be removed in one working step.

A temperature of 68°C+ 3° has proved to be the optimum working temperature for precipitating the globulins 25 in the presence of sodium caprylate and ethyl alcohol.

It may be remarked, that non-albumin-plasmaconstituents , not being separated during the first step, may remain in the albumin-containing-fluid, without disturbing the precipitation of the albumin.

For recovering the albumin from the residual albumin· containing fluid, maleic acid and polyethylene glycol have - 6 42460 proved to be of particular usefulness as precipitating agents; however, it is also possible to use for this concentration step aliphatic polyhydric alcohols, organic acids, specific salts, monomeric alcohols, or other methods suitable for protein (albumin) concentration.

In order to convert the precipitated albumin paste into a solution of the desired concentration, the paste is dissolved - following the abovementioned steps 10 in a preferably buffered liquid. The albumin solution can then preferably be heat-sterilised, without adding a stabiliser.

Variants of the process sequence and further features and advantages of the new process are illustrated I,. with the aid of the drawing. In the Figures: Figure la shows the precipitation process according to the so-called Cohn method; Figure lb schematically shows the new process; and 20 Figures 2a-2h show immuno-electrophoresis diagrams (1EP) of different albumin preparations produced in accordance with known processes or in accordance with the new process.

Figure la schematically shows, in stages, the process of the so-called Cohn method. This starts from a mixed plasma to which 8% of ethanol is added and which is precipitated at a pH value of 7.2 and at minus 3°C. This results in fraction I separating out. 19% of ethanol is then added to the supernatant liquid at minus 5°C. and a pH of 3Q value of 5.8. This causes fraction II/III, which in the main consists of γ-globulins, to separate out. The supernatant liquid is again treated at a higher alcohol content, 216 0 at a pH value of 5.8 and at a temperature of minus 7°C. This gives fraction IV, consisting in the main of a-and β-globulins. The supernatant liquid is subjected to a further treatment, at a pH value of 4.8 and a temperature of minus 7°C (ethanol content 40%) Hereupon the so-called crude albumin separates out as a sediment.

The supernatant liquid is discarded. After freeze-drying to remove the alcohol, the crude albumin can be taken up in, or converted into, a 5% strength solution. However, it is also possible again to take up the crude albumin paste and convert it into a purified albumin paste in two further steps (for which the conditions can be seen from the diagram).

In total, about 6-8 days are required for the pre15 paration of the purified paste if a normal 8 hour day is worked. Furthermore, very careful cooling is required, which demands correspondingly high expenditure on technological apparatus.

In contrast, the new process can be carried out substantially more simply (see Figure lb). Here again, a mixed plasma or other fluids containing albumin and globulin are used as the starting material; it is first subjected — in complete contrast to the known process to a heat treatment at a temperature of 68°C in the pres25 ence of an aliphatic alcohol of the formula CH2-^CH2^n_0H where n is 0, 1 or 2 and of a stabiliser as hereinbefore defined. Suitable stabilisers are, as mentioned, certain aliphatic carboxylic acids and their salts, such as say, sodium caprylate, and other constituents, which are noticed - 8 4 3 4 6 0 in the tables I, and III, respectively, in the articles of Ballou et al., mentioned before. In this first process step, which is carried out at a pH value of 6.5 and an ethanol content of 9%, all globulins are denatured and separated out. After cooling, the supernatant liquid is acidified to pH 4.8 and a precipitation medium, such as, say, 22% of polyethylene glycol (PEG), or 18% maleic acid, is added. After an appropriate reaction time, a very pure albumin paste separates out and this is either freeze-dried or taken up to form a 5% strength solution.

As can be seen from the basic descriptions, the present process requires a substantially shorter time and substantially less technological effort. In addition, however, the albumin obtained is also substantially purer than that obtained according to known methods. Figures 2a to 2h show immuno-electrophoresis diagrams (IEP), from which the following can be discerned; Figures 2a and c show IEP diagrams of a natural plasma, in which the sickle-shaped mark of albumin is formed on the left; the thinner sickle-shaped marks which follow on the right originate from globulins and other plasma constituents which must be regarded as impurities in a pure albumin solution. Albumin solutions prepared according to a known process are shown in Figures 2g and h. It can be seen clearly that a part of the impurities has been removed; however, far from all the undesired protein constituents have been removed.

Figures 2b and d show albumin solutions which have been obtained in accordance with the present process. Here, almost 100% purity is achievable. Figure 2f shows the - 9 42363 supernatant liquid which was obtained after the last process step (precipitation of the albumin). It can be seen that this liquid virtually no longer contains any albumin. It follows that the first process step (heating to 68°C in the presence of alcohol and sodium caprylate) achieves practically complete separation of albumins and other proteins of the plasma.

The individual process steps are illustrated with the aid of the following Examples.

Example 1 Plasma Separation A plasma from which the coagulation factors have been removed is used for the plasma separation. The coagulation factor VIII and the fibrinogen have been removed by cryoethanol sedimentation. The prothrombin complex is removed by diethylaminoethanol-cellulose adsorption. The original plasma is. Hepatitis-B-Antigen-negative, has normal transaminase values and does not contain any visible haemoglobin. Sodium caprylate is added to the original plasma until the concentration is 0.004 molar. The mixture, which contains about 9% of ethanol, is heated at a pH value of 6.5, which is obtained by adding 0.5 N HCl. The temperature is brought to 68°C in about 3 hours, heat being supplied at an even rate. The liquid is then cooled. The pH value is now lowered to 4.4 with HCl. The acidified plasma is now left to stand overnight (at about 10°C).

Separating off the fraction The plasma which has already been heat-fractionated is pumped into continuous flow centrifuges through silicone tubes. The proteins which have separated out collect in the 42469 rotors and the albumin remains in the supernatant liquid. The proteins which have separated out in the rotors still contain albumin, which can be isolated additionally by re-suspension and renewed centrifuging. Precipitation and concentration of the albumin with simultaneous reduction of the salt content.

The supernatant liquid is filtered to remove remaining lipids and modified proteins. The precipitant is then added - in the present case maleic acid in a concentration of 18%, at room temperature. The albumin precipitates in seconds. The albumin suspension is again centrifuged. This leaves the polyethylene glycol and the salts in the protein-free liquid. The albumin collects as a paste in the rotors. It is taken up in distilled water in a container, and converted into a solution of approx. 8% strength. After a clarifying filtration, the albumin can be converted directly into a 4-5% strength solution for use, the osmolality (osmotically measured molar strength) being adjusted with glucose or other suitable substances.

The product is then sterile-filtered, filled into bottles and pasteurised for at least 10 hours at 60°C.

Example 2 Preparation of a 20% strength albumin solution.

The steps of plasma separation and separation of the fractions are the same as in Example 1. These steps are followed by precipitation of the albumin and preparation of a 20% albumin solution.

The supernatant liquid is filtered in order to remove remaining lipids and modified proteins. Then, polyethylene glycol having a molecular weight of from 43463 about 4000 to 5000 is added as the precipitating agent in a concentration of 22% at room temperature. The albumin precipitates within a period of 30 minutes. The albumin suspension is centrifuged again. Hereby, the polyethylene glycol and the salts are retained in the protein-free liquid. The albumin is collected as a paste in the rotors. The albumin is re-dissolved in a container in distilled water and converted into an about 8% solution. After a clarifying filtration, the 8% albumin is freeze-dried. The dried powder is dissolved in distilled water, whereby the osmolality - as explained in Example 1 - may be adjusted, to 20% as desired. •s.

Examples 3\ and 4 Preliminary remarks: Two currently discarded Cohn plasma fractions are known to contain albumin in amounts which might be economical to salvage if simple technique, like heating, would give an acceptable product. These fractions are fraction IV-1 and fraction IV. Fraction IV-1 is precipitated from Supernatant II-III by the addition of alcohol to 19%', temp.-7°C, pH 5.2. The yield is about 20 to 25 grams of moist paste per liter of starting plasma, containing about 30% protein.

Fraction IV is precipitated from Supernatant II+III in two steps (first: Fraction IV-1 is won as above? two: alcohol is added to 40%, -6°C, pH 6.5, to bring down Fraction IV-4). The precipitates are removed together in a single centrifugation step as Fraction IV, Yield is 30 to 35 grams of paste per liter of plasma. Paste is 30% protein.

Example 3 1.0 kg of Fraction IV-1 paste is suspended with 2.0 L of water containing 0.004 M sodium caprylate stabiliser. The pH is adjusted to 7-7.5 with sodium hydroxide. The mixture is stirred gently at room temperature and the pH is periodically checked and readjusted upwards. Most of the paste will dissolve within one hour. The resulting crude solution will contain about 10% protein and 4-5% alcohol and is adjusted to 9% alcohol.

The mixture is heated to 68°C as described, and centrifuged. The precipitate is washed to remove entrapped albumin as described. The combined supernatants are filtered to clarify and precipitated with a suitable agent to collect the albumin.

Example 4 Likewise, a suspension of 1.0 kg of Fraction IV paste in water containing stabilizers is dissolved similarly, using 3.0 L per kg for a final solution containing about 7.5% protein and about 7% alcohol.

From experience, approximately 20% of the protein in Fraction IV-1 is albumin. If the above procedure is quantitative, 1 to 2 grams additional albumin might be recovered per liter of starting plasma. The yield of albumin frcm Fraction IV is in the same range.

Instead of the concentration of the albumin by precipitation with polyethylene glycol and/or freezedrying as specified in the examples, it is also possible to perform an ultra-filtration or a careful evaporation of the water of the solution by rotational evaporation.

If the concentration of the supernatant liquid produced after the globulin separation is effected by other 4 2 4 6 3 methods than by albumin precipitation (e.g. by ultrafiltration, rotation-evaporation), the desired osmolality, the salt composition and the hydrogen ion concentration must be adjusted by dialysis with a corresponding liquid.

In addition to blood plasma or blood serum it is possible to fractionate, in this way, all albumin-containing products, regardless of what process these fractions have undergone beforehand. It is also possible to subject to this process other body fluids, extracts from organs and tissues (placental). For example, one of the intermediate fractions of the Cohn method could be subjected to the present process.

The yield amounts according to experiments, to more than 90% of the originally present albumin, whereas in the conventional methods, in spite of lower degrees of purity, a yield of at best from 60 to 70% can be expected.

Claims (12)

1. CLAIMS;1. A process of isolating albumin from a medium comprising blood, blood products, other body fluids and/or tissue extracts, including the steps of a) separating plasma from the solid constituents of the said medium, b) precipitating the globulins by heating the separated plasma in the presence of more than a 4% concentration of an alcohol having the formula CH 3 - (CtI 2 ) n -OH, wherein n is 0, X or 2, and in the presence of one or more albumin stabilisers (as hereinbefore defined) to a temperature of from 60 to 75°C., and separating off the fluid from globulins; and c) recovering the albumin contained in the residual fluid.

2. A process according to Claim 1, wherein the dissolved non-albumin constituents are recovered from the plasma, prior to the precipitation of the globulins.

3. A process according to Claim 1 or 2, wherein the alcohol is ethyl alcohol.

4. A process according to any one of Claims 1 to 3, wherein the precipitation of the globulins is performed in the presence of sodium caprylate as the albumin stabiliser and ethyl alcohol at a temperature of from 60 to 75°C.

5. A process according to any preceding Claim, wherein the albumin is recovered by precipitation with a protein precipitating agent.

6. A process according to Claim 5, wherein said agent 5 is from 20 to 30% of polyethylene glycol.

7. A process according to Claim 5, wherein said agent is from 15 to 20% of maleic acid.

8. A process according to any one of Claims 1 to 4, wherein the albumin is recovered by freeze-drying.

9. 10 9. A process according to any one of claims 1 to 4, wherein die albumin is recovered by ultra-filtration. 10. A process according to any preceding claim, wherein the recovered albumin is dissolved in a buffered, liquid and adjusted to a desired concentration. 15

10. 11. A process according to claim 10, wherein the resulting albumin solution is heat sterilized without the addition of a stabilizer.

11.

12. A process for isolating albumin from blood, blood products, other liquids and tissue extracts in 20 accordance with claim 1 and substantially as hereinbefore described in any one of the foregoing Examples.