DE2127159A1 - Process for fractionation of plasma proteins - Google Patents

Description

Elnrelc'nuno3iQrt! G to Receive forwarding

Patent Attorney 80-17.11 IP

Dipl.-Ing. R. Beetz June 1, 1971

Statens Bakteriologiska Laboratorium, 9 1 9 7 1 ζ Q

171 63 Solna, Sweden

Process for fractionation of plasma proteins

As is well known, blood consists of solid blood cells suspended in plasma. The blood corpuscles make up about half a percentage of the total volume, while the rest, the so-called blood plasma, consists of about 90% water, 9% protein, 0.9% salts and smaller amounts of organic compounds.

The blood plasma proteins occur in very different forms with widely different functions, such as enzymes, Antibodies or substances that control the balance of water-soluble salts in the organism.

Both whole blood and blood plasma are often used to treat various used for pathological phenomena. However, it was already clear that many of the proteins that are known as disease-fighting agents are used, are by no means necessary rather, they represent a burden and in certain cases are even harmful. To get the most effective ingredients in the cheapest Therefore, to be able to use them clinically, they must be isolated and enriched.

The raw material for the production of albumin and immunoglobulin preparations obtained largely by collecting retroplacental blood in maternity units and from superfluous donated blood. A limited amount of blood and plasma used to make immunoglobulin solutions with increased antibody content, which is directed against specific diseases, is obtained by vaccinating blood donors with perfectly determined antigens. Convalescent blood is used as a raw material for the same purpose. The cost of this

109850/1708

specific immunoglobulins are usually high. Jenaehdem how the blood collection is done is how the starting material is obtained either as whole blood, plasma or serum. Sometimes immunized blood donors are collected using a special procedure Plasma atmospheres in which the blood is mixed with a dextronodium citrate-citric acid buffer during collection, the prevents the blood from clotting. The blood cells can then be separated from the plasma by centrifugation. One that way obtained plasma can be injected directly and can also be used as a Use raw material for further fractionation.

Many attempts have been made to find a method which can isolate clinically important plasma proteins without loss or reduction of biological activity. One strives for a fractionation smet hodik such that the isolated proteins have the same physiological properties like in blood.

The oldest method for excreting (precipitating) plasma proteins is based on the use of neutral salts, in particular ammonium sulphate. The outdoor experience includes a system with four variables, namely salt concentration, protein concentration, pH and temperature. At the. Salt cases require large amounts of neutral salt, which must be removed by dialysis. The process is known for its high yield and was described by HE Schulze and others in the Behringwerk-Mitteilungen, volume 22, volume V) 9+ . In England a method is used in which, inter alia, ether is used as a precipitating agent, see RA Kekwik etc ... in Med. Research Council Spc. Report, Series No. 286, London, Her Majesty's Stationary Office, 195 ^. Reid's ion exchange process should also be mentioned, see AF Reid, etc. in Ind. Eng. Chen. * + 3, 197 ¹ S year 1951, as well as Cohn's alcohol process. The latter is by far the most widely used and is widely used. It has been described, inter alia, in Journal Am. Chem. Soc. 68 (19W ₅ 1 * 59, and 12 (1950) * + 63 (Cohn et al) and 71 (194-9) 5 ^ 1 (Oucley), also in American patents 2,390,07 ¹ *, 2 ^ + 37 060 and 2 5 * + 3 215 and for example in the Swedish patent I36 ¹ K) I. By doing

109850/1708

elaborated by Cohn and others in the first four decades Process - ethyl alcohol is used as a precipitant. By using the If the alcohol content changes at low ionic strength and low temperature, the plasma proteins are divided into five main fractions. If you use another organic solvent such as ether or acetone instead of ethyl alcohol, you can win more Opportunities to find suitable precipitation conditions for separating complex protein mixtures. The use of ethyl alcohol or other organic solvents instead of neutral salts has the advantage that this ^ organic solvent as a result their volatility can be easily removed by freeze vacuum drying.

The alcohol precipitation process according to Cohn, however, has the disadvantage that the various stages of precipitation have to take place at different temperatures in order to prevent denaturation of the proteins. Therefore, the plasma solution needs to 0 ⁰ C are cooled before alcohol is added, and during all fractionation temperature shall not exceed verden. However, despite the many advantages of this method, it has been shown that proteins isolated with alcohol as a precipitant give albumin and immunoglobulin aggregates. Several of the proteins that are important for clinical purposes lose their original properties on repeated contact with alcohol and appear in the immunoglobulin in an irreversibly aggregated and denatured manner. The immunoglobulin aggregate has a sedimentation constant of 9.5 to 10.1 S. Many commercial products contain up to 25 % of such protein.

The currently The usual classification of immunoglobulins is according to Bull. World Health Organ. 30 (1964-), pages 1 + 4-7, by denoting them with characters which, on the one hand, indicate the main class and, on the other, indicate the polypeptide chains occurring in these globulinones. Y or Ig was chosen as a suitable symbol for immunoglobulin, followed by a capital letter to denote the main class, for example γ G, y M etc. or IgG, IgM etc. Next, the next type of denomination is used.

109850/1708

Aggregated immunoglobulin that is injected becomes substantial excreted from the body faster than non-aggregated- Material with the sedimentation constant 7 S. After cleaning, cause residual piasmin in the IgG fraction their protolytic effect causes fragmentation of the antibody which also causes faster excretion. These two factors influence the clinical outcome in treatment extraordinarily high degree.

Figs. 1 and 2 of the accompanying drawing each show an “ion exchanger column for the inventive method, Fig. 3-6 are absorption curves of various preparations.

By using the mutually different charge properties of the plasma proteins in connection with their solubility in a system, according to the invention, aggregate-free IgG is obtained as the end product with a high yield. A major advantage of the new process is that the coarse fractionation and final cleaning of the individual components can be carried out at room temperature and aseptic conditions. ^{Temperatures below 0} G are only required for the final work after the insulation. Fractionated material can be studied using ultracentrifugation or an appropriate type of gel filtration.

The division of the plasma proteins into coarse fractions is carried out in such a way that that the 'globulins are first precipitated at neutral pH by adding polyethylene glycol under such conditions, that the albumin fraction remains in solution. A temperature of 15 to 25 ° C and a pH of 7 does a lot of $ 13 polyethylene glycol in the reaction mixture that all Globulins can be precipitated without affecting the albumin.

The polyethylene glycol used as a precipitant has an average molecular weight of 6000 (Union Carbide, "USP grade", USA). Such Precipitations are described in English patent specification 1,006,258.

109850/1 708

Felling, with polyethylene glycol, is not only of the polyethylene glycol concentration but also depends on the ion strength, protein concentration, temperature and pH value, with changes in the pH value have the strongest effect. In contrast to ethyl alcohol, polyethylene glycol has a protein-stabilizing effect at room temperature Effect.

The precipitate obtained is preferably carried out by centrifugation (centrifugal fugieren) deposited and extracted, whereby the IgG fraction positively charged dissolves, while IgM, fibrinogen and plasminogen remain undissolved. A sodium acetate acetic acid buffer, for example, can be used to dissolve it Use at pH - 5.8. Undissolved precipitate is removed by centrifugation and the solution is with a cation exchanger at pH = 5 »8 deposited.

Carboxymethyldextran can be used with advantage as a cation exchanger use (Sephadex CM 50), and treatment with the ion exchanger can be done either in batches or in a column. In order to be able to take advantage of these two methods, namely the rapid binding of protein from large Quantities in case of a batch treatment and the easier elution in the Pillar, a special technique and equipment was worked out. In a roughly bottle or pear-shaped container 1 na £ h Fig. 1, the one below through an expansion stopper provided with a filter 5 is closed, the protein solution and the ion exchange gel Stir until equilibrium is reached. When the stirring is stopped, one forms from the ion exchanger existing gel column in the lower part of the container. This column is at the top through an upper expansion plug provided with a filter 2 separated, which can be moved with a rod 3 and is designed so that it passes through the unbound protein layer can be moved up to the upper level of the gel column and there a tight seal between the gel and unbound protein solution forms. The expansion plugs 5 and 2, which are provided with filters are connected via hoses with a four-way valve 7 to the solutions in different ways and from different containers

109850/1708

to be able to circulate and direct it to and from the column h , for example when eluting from below, washing from above, etc. .

After the treatment with the cation exchanger, the solution contains, among other things, β-lipoproteins, ctp-macroglobulins, Lipids, residues of precipitated albumin and a smaller proportion of IgG.

Then the ion exchanger is eluted using an eluting buffer, whose pH or ion concentration is increased. As can be seen from Figs. 1 and 2, the elution can be carried out in a countercurrent or cocurrent process take place. The latter is essential, especially when the eluent has a higher density than that eluted Protein solution. Sodium chloride solution with sodium phosphate buffer is used as the eluent.

By reprecipitating the eluate with polyethylene glycol, all globulins are precipitated, with the exception of a minor one Amount of transferrin. The precipitate is centrifuged and redissolved in a phosphate buffer at pH 6.6, centrifuged until clear and stirred with an anion exchanger. All Protein residues with the exception of IgG are then bound to the anion exchanger diethylaminoethyldextran (Sephadex DEAE A 50). The IgG solution is then cooled to -0.3 ° C and with chilled ethanol of a concentration of $ 25 with lowering of the Temperature was added to -7 C, the IgG being completely precipitated out of the solution. The precipitate is collected by centrifugation deposited, slurried in chilled 25 percent ethanol and washed at -7 ° C. The washed-out precipitate is dissolved in ice-cold glycine solution and filtered sterile and after freeze-drying to prepare a 16 to 16.5 percent IgG solution in 0.3 M glycine used.

109850/1708

With the method according to the invention a fast one is achieved Fractionation when working at room temperature and under aseptic conditions Circumstances. The process can be used in laboratories as well as industrially and allows numerous separation options » The reproducibility of the process is very good and independent of the initial quantity. The dilution that becomes liquid low, and the fractionation amount is always less than 150 $ of the initial amount throughout the process. The efficiency the "ion exchanger is used to the highest degree"

example

Immunoglobulin (IgG) is isolated from human plasma.

The plasma is stored frozen at -20 ⁰ C, at +! + Thawed ⁰ C and then centrifuged to resign to unresolved remnants of fibrinogen, cell fragments and blood body. The AHF factor is obtained from this lack of solution.

One part by volume of centrifuged clear solution is precipitated at a temperature of 15-25 ° C., with half a part by volume of 39 percent polyethyne glycol solution in 0.05 M sodium citrate solution with citric acid to pH 7j0. The final concentration of 13 $ polyethylene glycol in the reaction mixture precipitates almost all globulins, which are separated off by centrifugation for 10 minutes at 300 ^x g. The solution obtained during centrifugation mainly contains albumin, which is then extracted from it.

The precipitate deposited by centrifugation is dissolved in a volume of 0.03 M sodium acetate buffer with pH 5-8, the proteins with the exception of fibrogen, IgG and plasminogen being dissolved. The undissolved substances just mentioned are removed by centrifugation at 1500 xg for 15 minutes. The remaining clear centrifuged globulin solution is stirred at room temperature for hO minutes in the pear-shaped container (Fig. 1), namely with your cation exchanger (Sephadex CM 50)

109850/1 708

in equilibrium with 0.05 M sodium acetate buffer at pH 5j8 in an amount corresponding to 6 g dry weight of the ion exchanger per kg of starting plasma.

The ion exchanger with the bound protein is allowed to settle and separated using the expansion stopper provided with a filter 2 depends on the unbound protein. The bound protein is then upstream with 0.05 M sodium phosphate buffer eluted, this buffer containing 0.2 M sodium chloride at pH 7-8. The eluted globulin has a pH of approximately 7 After dilution with distilled water to a part of the volume (the initial volume), the solution is precipitated again with 39 $ polyethylene glycol solution in 0.05 M sodium phosphate buffer, pH = 7 »at a final concentration of 13 $ PEG. Here will be all globulins like, with the exception of a smaller amount of transferrin.

The globulin precipitate is centrifuged for 10 minutes at 300 × g and dissolved in a volume of 0.02 M sodium phosphate buffer with pH 6.6. The dissolved precipitate is centrifuged for 15 minutes at 1500 xg until clarification and stirred with the anion exchanger (Sephadex DEAE A 50) at room temperature for 20 minutes, the amount of the ion exchanger corresponding to 6 g of dry matter per kilogram of starting mass, and equilibrium with 0.02- M sodium phosphate buffer at pH 6 _f 6 prevails. The unbound IgG solution is then cooled to -0.3 ° C and, with constant temperature reduction and vigorous stirring with simultaneous temperature-controlled supply, cooled 75% ethanol is added so that the heat of the solution is quickly distributed and dissipated. At a final concentration of 25% ethyl alcohol and -7 ° C, the immunoglobulin is completely precipitated from the solution. The precipitate is deposited xg by 15 minutes centrifugation at -10 ⁰ C and 1500, slurried at pH 7.0 and -7 ° C in 1/6 space part of 25 $ ethanol in 0.005 M sodium phosphate buffer and centrifuged again.

The precipitated precipitate washed in this way is in

109850/1 708

1/20 volume part of ice-cold 0.1 M glycine solution through an asbestos filter sterile filtered, frozen in thin layers and freeze-dried. Then you can get a 16 to 16.5 percent Prepare immunoglobulin solution in 0.3 M glycine for clinical use.

The immunoglobulin solution obtained does not contain any aggregates, has uniform molecular weight and retained antigen neutralizing properties. Any pyrogenic material in the starting material is removed by the cleaning process, and the same applies to so-called Australian antigen (au antigen), a Protein found in the blood of people infected with the hepatitis virus (Jaundice) are infected. - The yield is high and the globulin is durable.

Since the precipitated material or material bound to an ion exchanger always contains precipitated or unbound material, the final purification is therefore carried out according to the method in such a way that the isolated material during precipitation in the remaining liquid, the supernatant, and in the unbound material after treatment with the ion exchanger is available. Because that part of the component mixture is bound that has the lowest concentration, the capacity of the «ion exchanger? can be used as a maximum with respect to the total amount of components. <■

In order to examine the products obtained, gel filtering curves of preparations produced by different methods were made * Here, sample solutions of around 16 percent IgG were subjected to gel filtering in agaros columns (Biogel 1.5 A). The dimensions of the column were 8 × 750 mm, and the treatment was carried out in 0.1 M tria-HCl buffer at pH 8.0 with 0.5 M NaCl and 2% n-butanol with 50 μl of sample applied. Measurements were made at 280 and 260 nm in a flow-through cuvette with a layer thickness of 1 mm.

109850/1708

* The curve for 280 nm is fully drawn, while the curve for 260 nm is dashed.

Fig. 3 is the absorption curve of IgG, which was produced according to Schultze's ammonium sulfate process. Fig. H and 5 are absorption curves of IgG, which was produced by two variants of Cohn's method. Fig. 6 shows the absorption curve for an IgG preparation produced according to the invention.

One can easily see that an aggregate formation, namely the first peak of the curves in Fig. 3-5? in the inventive pre- ready according to Fig. 6 is missing, and that no decomposition has taken place due to incomplete separation of plasmin, since in Fig. 6 the last tip is missing. The symmetry of the absorption curve Fig. Of the preparation produced according to the invention is a sign of the uniform distribution of molecules in this material.

109850/1708

Claims (2)

■ • ■ tit ·? ··· * - :.) ** ·: - - - .. uöfi · ^ claims 1. Fractionation process. Sound plasma proteins, characterized! a) that blood cells and cell fragments are removed from the plasma of blood, b) that the globulins in the plasma with polyethylene glycol with a concentration Clay 10 to 15 percent by weight and at a pH value between 6.5 and 8.0, Goals at 7.0, be felled, c) that the precipitate is separated out by centrifugation and that this enables albumin to be obtained from the remaining solution, d) that the precipitation is dissolved at around pH 5.8 in around 0.05 M sodium acetate acetic acid buffer and undissolved fibrinogen and J-H immunoglobulin through Centrifugation is removed, e) that the globulins through cation exchangers such as carboxymethyldextran around pH 5.8 are adsorbed and eluted with 0.2 M sodium chloride in 0.05 M phosphate buffer at pH 7.5-8.0, preferably 7 »8, f) that with polyethylene glycol at pH 6.5-8.0, preferably 7.0, precipitated and the precipitate is centrifuged and dissolved in 0.02 M phosphate buffer at pH 6.6, g) that by means of an anion exchanger such as diethylaminoethyldextran all globulins except J- G immunoglobulin are adsorbed at pH 6.6, h) that the i ^ Q immunoglobulin solution is cooled to -0.3 ° C and J- G- Immunoglobulin with Xthanol, the concentration of which is 25 percent by volume between -5 and -10 C, preferably at -7 C, is precipitated, i) that the precipitate is separated, slurried and with 25 percent by volume » Ethanol at pH 7.0 between -5 and -1O ° C, preferably at -7 C, washed and centrifuging again, j) and dissolved in glycine and filtered sterile and freeze-drying is subjected k) and that a 16 to 16.5 percent J * G immunoglobulin solution in 0.3 M Glycine is prepared as an injection agent (injection agent). 109850 / Ί 708 2. The method according to claim 1, characterized in that that the precipitation of the globulins according to process step b) boi a polyethylene glycol concentration of 13 percent by weight is made 3 · method according to claim 1 or 2, characterized in that g ο k e η η, that the cases with Polyäthylcnglykol in the process ens step b) at a temperature of 10 to 25 ° C., preferably at 15 to 18 ° C. 109850/1708