DK141523B - PROCEDURE FOR INSULATING ALBUMIN FROM BLOOD BLOOD PRODUCTS AND PLACENT BLOOD - Google Patents

Description

(W) \ * a / (11) SUBMISSION SITES 1 i | 15Z3 r \ AM IWI ADY (51) Intel. · A 61 K 37/02 // A 61 K 35/14 DENMAK a 61 k 35/50 f (21 ) Application No. 1252/75 (22) Submitted to death 25 · <nai *. 1975 (23) Race day 25 · Mar. 1975 (44) The application presented and.

the publication notice published on the 1 ^ · · »9®0 DIRECTORATE OF THE PATENT AND TRADEMARKETS Required from the

Mar 28 1974, 2415079s DE

(71) PLASMESCO AG, Haenibuehl 8, 6300 Zug, CH.

(72) Inventor: Waldemar Schneider, 58 Hagen, Felthetr. 180, DE: Christian Froehlich, 58 Hagen, Feithstr. 180, DE: Harald Fiedler, 44 Mueneter, Sperlichstr. 15, DE: Hans Lefevre, 44 Mueneter, Sperlichstr. 15 »DE.

(74) Plenipotentiary in the proceedings:

The engineering company Giersing & Stallinger ._'_ "(B4) Process for isolating albumin from blood, blood products and placental blood.

The invention relates to a method for isolating albumin suitable for therapeutic and diagnostic use in humans, from human blood, blood products and platelet blood, comprising the steps of a) separating plasma from the solid components (blood cells and platelets) and, if b) complete precipitation and separation of globulins, heating the separated liquid to 60-75 ° in the presence of an albumin-stabilizing agent, for example, in the presence of an albumin-stabilizing agent, e.g. c) enrichment of the albumin, preferably by precipitation and separation.

Blood is a liquid which consists of solid and liquid constituents.

The solid constituents include red and white blood cells as well as the platelets. The plasma or liquid part of the blood contains approx. $ 90 water and $ 10 solids. Among the substances dissolved in the plasma are among other things the albumin, which as the only egg white component of the plasma is heat resistant to more than approx. 60 ° C. It is known per se to separate the blood plasma from the red and white blood cells as well as from the platelets. Furthermore, it is known to remove -globulins as well as solidification promoters such as e.g. fibrinogen from the plasma. For therapeutic or diagnostic purposes, it is desirable to obtain, as far as possible, pure albumin solution of albumin paste which, as far as possible, should no longer contain additional egg white constituents of the blood plasma.

In particular, for the isolation of the albumin from the blood, a method known by the name "COHN method" (J0Am0ChemSoc.68 (1946), page 459) is employed as in a yield of approx. $ 70 enables a purity of the final product of only $ 95-98. This method assumes a plasma formed from different blood samples, and the process is in principle a fractional precipitate under different conditions. In the first step, at -3 ° C and a pH of 7 * 2, during an alcohol addition of $ 8, the first sediment, predominantly fibrinogen, is first separated.

The remaining liquid is released in three further operations, all at temperatures below 0 ° C and increasing alcohol content of the liquid, gradually for / {- globulins as well as and p> globulins. Then a so-called crude albumin paste is obtained, which is either converted to a 5 $ albumin solution or thinned a further purification, at the end of which a 20 $ albumin solution is obtained. The entire fractionated separation by the known COHN method requires about 6 - 8 days on normal 8-hour working days. Furthermore, this method has a disadvantage that numerous operations must be performed at precisely set temperatures below 0 ° C. This either requires that the entire laboratory be kept at this temperature or that the reaction vessels be cooled at great expense. Both are a disadvantage and especially felt by the lab staff as very uncomfortable.

Furthermore, from GB patent 739 and a method for isolating albumin from mammalian blood by selective denaturation and precipitation of the globulins from an aqueous solution of the so-called albumin at a temperature of 45 - 75 ° C in the presence of caprylation is known. By this known method yields of up to 76 $ of the initially available albumin amount and a purity of the product of 9h - 98 $ can be obtained.

The invention has for its object to provide a process of the kind mentioned in the beginning with which albumin products can be obtained having a purity of practically $ 100 and in a substantially higher yield than the known methods, while also disclosing ., practical disadvantages of the COHN method are avoided.

In one method of the kind mentioned initially, this is achieved according to the invention by further precipitating the globulins in the presence of more than 4 $ alcohol with the composition CH 2 -n (CH 2) - 0H (n = 0, 1,2) at a pH value above 6.0.

Blood, blood products or placental blood may be used as starting products for the process according to the invention. is understood as intermediates derived from the CQHN framework.

Most important in the process of the invention is the precipitation of globulins (operation b) by heating the albumin-containing liquid in the presence of alcohol. Below, the albumin - unlike all other plasma proteins - is protected against precipitation due to the addition of stabilizing substances. Protein or albumin stabilizing substances are understood to mean those which even by heating pure albumin solutions to above 60 ° C prevent any visible change of the albumin. Works by Ballou et al. (J. Clinical Xnveet. 23 (1944), page 454, J. Brol. Chem. 153 (1944), page 5 ^ 9) refers to propionate, butyrate, valerate, caprylate, phenylacetate, phenylbutyrate, tryptophan, etc. the desired stabilizing properties.

The process of the invention provides a significantly greater yield and a higher purity of the final product than both the CHON method and the method of GB Patent Specification No. 739 and 24, enabling the achievement of yields above 90 ° at a purity of 10%, cf. Schneider, Fefåvre, Fiedler, and McCarty * "An Alternative Method of Large Scale Plasma Fractionation for the Isolation of Serum Albuminum *, published in the journal" Blut ", Band 30, pages 121-134 (1975), and especially pages 124, Table 2 * Relation of plasma temperature and pH to albumin recovery, and page 129, third section with accompanying Figure 1. At the same time, the process of the invention is substantially simpler than the COHN set method.

In order to obtain the above-mentioned high yield and high purity, according to the invention, it is desirable that in the hot precipitate about 9 $ ethyl alcohol is used in the separated liquid, on the basis of the total amount. For the same purpose, about 0.004 moles of sodium chloride caprylate can also be used as an albumin stabilizer.

It should be noted that non-albuminous plasma components which have not been recovered in the first process operation can remain in the albumin-containing liquid without disturbing the globule precipitation.

141523 h

In particular, to enrich the albumin from the albumin-containing liquid, maleic acid and polyethylene glycol, in particular, have proved to be suitable precipitation media. However, it is also possible for this concentration operation to use other polymerized aliphatic polyhydric alcohols, organic acids, certain salts, monomeric alcohols, or other agents suitable for egg white enrichment.

In order to convert the precipitated albumin paste to a solution of the desired concentration, the paste is dissolved in a preferably buffered liquid in connection with said operations. The albumin solution can then preferably be heat ester without a stabilizer added.

Variants of the method as well as additional features and advantages of the method of the invention are explained in more detail below with reference to the drawing, in which Fig. 1a illustrates the precipitation process by the so-called COHN method, Fig. 1b similarly illustrates the method of the invention and Figs. . Figures 2a to 2h show immuno-electrophoresis (iEP) diagrams for various albumin preparations prepared by known methods and by the method of the invention, respectively.

Fig. 1a shows schematically the different steps of the so-called COHN method. A mixed plasma is added to which 8 $ ethanol is added and precipitated with a pH of 7 »2 at -3 ° C. Here, fraction I. is separated from the remaining liquid at -5 ° C and with a pH of 5 »8 with 19 $ ethanol. Thereby fractions II and III, consisting mainly of β-globulins, are secreted. The remaining liquid with higher alcohol content, a pH of 5 »8 and a temperature of -7 ° G are treated. This yields fraction IV, consisting mainly of .jC and H-globulins. The remaining liquid is treated again at a pH of k, 8 and a temperature of -7 ° C (efchanol content 4θ $). Thereby, the so-called crude albumin is excreted as sediment. Discard the remaining liquid »· After freeze drying to remove the alcohol, the crude albumin can be converted into a $ 5 solution. However, it is also possible to agitate the crude albumin paste again and convert it into a purified albumin paste in two additional operations (as shown in the diagram).

In general, the preparation of the purified paste is needed 6-8 days if working with a normal 8-hour day. Furthermore, very careful cooling must be done, which requires a correspondingly high supply of process-technical equipment.

5 k. ' In contrast, the process of the invention can be performed substantially simpler, see FIG. Ib. Here, too, one assumes mixed plasma or other liquids containing both albumin and globulins. · The liquid is subjected - in a similar manner to the process according to GB Patent No. 739 and 24 - to a heat treatment at a temperature of 68 ° C, but in the presence of aliphatic, low molecular weight alcohols and stabilizing sttft punches. As mentioned, as mentioned stabilizing substances are suitable aliphatic carboxylic acids or their salts, such as sodium caprylate and other compounds listed in Table III of Ballou (J.Brol.Chem. 153 (1944) p. 599) and Table I (J. Chera.Invest. * 23 (1944) page 45¾) ¾ 1

In this first operation, soya is carried out at a pH of 6 5 and an ethanol content of 9 $, all globulins are denatured and precipitated.

After cooling, the remaining liquid is acidified to cool H and mixed with a precipitation medium such as ca. 22 $ p o lye thyl engly c o 1 (pEG) or maleic acid. A very pure albtt & érøasta is excreted, the sum converted into a 5 - 20 $ solution. * ~ '1 ^

As is already apparent from the principle descriptions, the process according to the invention requires a considerably shorter time and a considerably smaller process technical tender than the wet COHN method, In addition, however, the recovered albumin is also significantly cleaner than which are extracted by the methods well known in the art. FIG. 2a to 2h show itmoun electrophoresis diagrams (IEP) *, which shows the following!

FIG. 2a and 2c show in EP diagrams for a natural plasma ve in which the albumin seal is formed on the left. The thinner seals then to the right are derived from globulins and other plasma constituents which, by a "pure" albumin solution, must be regarded as contaminants. Albumin solutions prepared by the COHN method are shown in FIG. 2g and 2h, cf. the aforementioned journal article. It is clearly seen that some of the contaminants have been removed, without, however, removing all unwanted genuine white constituents. FIG. Figures 2b and 2d show albino thread solutions prepared by the process of the invention. Almost $ 100 purity can be achieved here. FIG. Figure 2f shows the residual liquid Bom produced after the last operation of the process (precipitation of the albumin). It will be seen here that this wash practically no longer contains J albumin. It follows that, in the first process operation (heating to 68 ° C in the presence of *. Alcohol and sodium caprylate), virtually complete separation of albumins and other egg white substances in the plasma has been achieved.

The individual operations of the method according to the invention are explained in 6 U1523

Example Xt Plasma separation

For the plasma separation, a plasma is used from which the clotting factors are removed. The clotting factor VIII and the fibrinogen were removed by cryoethanol sedimentation. The prothrombiric complex is removed by DEAE cellulose adsorption. The plasma used is Hepatitis B antigen negative, has normal transaminase values and contains no visible hemoglobin. To the plasma used, sodium caprylate is added until a concentration of 0.004 mol is obtained. The mixture containing approx. 9 $ ethanol, heated at a pH of 6.5. The pH value is obtained by 0.5 n HClo. The temperature is brought over approx. 3 hours under uniform heat supply up to approx. 68 ° C. The liquid is then cooled. The pH is reduced by HCl to pH 4.8. The acidified plasma is then left overnight at ca. 10 ° C.

Separation of the fraction

The already hot fractionated plasma is pumped through silicone tubing into continuous flow centrifuges. The egg white secretions secreted by the plasma accumulate in the rotors. The albumin remains in the remaining liquid. The egg white separates in the rotors still contain albumin which can be recovered by resuspending and centrifugation and added. the liquid obtained at first centrifugation prior to the concentration operation.

Concentration of the albumin while simultaneously reducing the salt content (precipitation of the albumin)

The remaining liquid is filtered through a so-called SSK filter to remove residual lipids and altered proteins. Then, the precipitating agent, in this case maleic acid, is added at a concentration of $ 18 at room temperature. Within a few seconds, the albumin is precipitated. The albumin suspension is centrifuged again. Including the maleic acid and salts in the protein-free liquid. The albumin accumulates in the rotors as paste. It is stirred in a container in distilled water and converted into an $ 8 solution. After clarification filtration, the albumin can be directly converted to a 4-5 $ solution of use wherein the osmolality is adjusted with glucose or other suitable substances. Then the product is sterile filtered, bottled and pasteurized for at least 10 hours at 60 ° C.

Example 2s

Preparation of a $ 20 Albumin Solution.

The "plasma separation" and "separation of the fractions" operations are the same as in Example 1. There is precipitation of the albumin and preparation of a 20 $ albumin solution.

7 141523

The remaining liquid is filtered through SSK filter to remove residual lipids and altered proteins. Then polyethylene glycol having a molecular weight of approx. 4000 to 6000 at a concentration of $ 22 at room temperature. Within 30 minutes the albumin is precipitated. The albumin suspension is centrifuged again. Including the polyethylene glycol and salts in the protein-free liquid. The albumin gathers 1 rotors as paste. It is stirred in a container in distilled water and converted into an 8 $ solution. This solution is freeze-dried after a clarification filtration "The dried powder is stirred in distilled water, whereby the osanolality - as already explained in Example 1 - may be adjusted"

Example 3:

Preliminary remarks:

It is known that two fractions obtained and discarded by the COHN method contain albumin in an amount which it pays to recover. For this, a simple extraction method is required. These are fractions IV-1 and fraction IV-2.

Fractions IV-1 are those fractions recovered from the remaining liquid II and IH by the addition of 19 $ alcohol at a temperature of -7 ° C and a pH of 5 »2. This operation is not shown in FIG. la. The yield is approx. 20-25 g of moist paste per liter of starting plasma containing approx. 30 $ protein. Fraction IV-2 is understood to mean the fraction recovered from the remaining liquid II to III in two operations.

These two operations are first the recovery of the fraction IV-1 sera above. Then, alcohol is added up to an amount of $ 40, while maintaining the temperature of -6 ° C and a pH of 6.5. Thereby, fraction IV-2 is precipitated. The precipitate is removed by centrifugation. The result is approx. 30 to 35 S pasta per liters of plasma. The pasta is made up of $ 30 for protein.

For the recovery, the following procedure is proposed: 1.0 kg of fraction IV-1 (paste) is suspended in 2.0 liters of water containing 0.004 mole of sodium caprylate as a stabilizing substance. With sodium hydroxide, the pH is brought to 7 to 7 ° 5 * The suspension is stirred slightly at room temperature. The Ph value is checked and regulated periodically. Most of the paste dissolves in 1 hour. The crude solution obtained contains about, $ 10 protein and 4 to 5 $ alcohol. The solution is set to $ 9 alcohol.

Then a heating to 68 ° C follows. The precipitate, which mainly contains unwanted globulins, is centrifuged and washed to remove the albumin still contained. The remaining liquid is clearly filtered and precipitated in the manner described (Examples 1 and 2) with polyethylene glycol or maleic acid or other precipitation media.

Example k In a similar manner to Example 3, 1.0 kg of fraction IV-2 (paste) can be suspended in 3 * 0 1 water containing 0.004 mole of sodium caprylate as a stabilizing substance. The solution contains approx.

7.5 # protein and approx. 7 # alcohol.

The albumin is recovered from the solution in the manner described in Example 3. To recover the albumin from the solution, it should be set to 9 # alcohol.

It has been found experimentally that approx. 20% of the protein in fraction IV-1 is albumin. 1 to 2 g of albumin can be recovered further. liter of output plasma. In the same order of magnitude, the additional albumin gain is at fraction IV-2.

Instead of the enrichment of the albumin mentioned in the examples by PEG precipitation and / or freeze drying, ultrafiltration or gentle evaporation of the solution water can also be done by r o t ation evaporation.

If the concentration of the liquid remaining after the globulin separation occurs by methods other than albumin precipitation (eg by ultrafiltration, rotary evaporation), the desired osmolality, the salt composition and the hydrogen ion concentration must be adjusted by dialysis against a corresponding liquid.

The yield is, according to experimental experience, above 90% of the initially albumin present, while in the known methods mentioned above, only a yield of no more than 76% can be counted, despite the lower purity.