CS236096B1 - A method of preparing a low low molecular weight low level serum albumin solution - Google Patents

Abstract

The invention provides a method for preparing a serum albumin solution with a low content of low molecular weight compounds. The compounds accompanying the isolated albumin fraction are characterized by properties that in some cases contraindicate the possibility of application and do not have therapeutic effects. The essence of the invention is the removal of the above-mentioned compounds, present in unlimited concentration, by diafiltration on defined membranes of tubular or sheet type, intended for thickening biomacromolecule solutions. The isolated albumin fraction in an aqueous solution is diafiltered at an initial concentration of 2 to 20%wt. proteins against water for injection, added continuously at a constant volume or in specified volumes. The method is characterized by low water consumption and thus low energy consumption and high efficiency and speed, which reduces the risk of contamination.

Description

The present invention provides a process for preparing a serum albumin solution having a low content of low molecular weight compounds.

The currently used methods of isolating serum albumin for therapeutic or other purposes, such as the manufacture of cosmetic and dietetic preparations, obtain a formulation that is accompanied by low molecular weight compounds. If the preparation is to be used for therapeutic purposes, its efficacy is reduced by the presence of the compounds, and in some cases the application is even contraindicated. For this reason, some manufacturers have included purification steps as an isolation process to reduce the low molecular weight content before final albumin fraction concentration and pharmaceutical processing.

The need for the removal of low molecular weight moieties arises in all known methods for isolating the albumin fraction, whether it is a tremendous eculoid compound derived from a plasma protein source material - donor plasma, serum, dried plasma, or so-called. substitute raw materials, i.e. retroplacental serum, non-sterile plasma residues, erythrocyte masses and K-plasma, or low molecular weight compounds used in any of the following isolation methods:

Insulation techniques can be divided into several groups:

238 09β

- 2 procedures based on selective precipitation, ion exchange chromatography, gel chromatography, affinity chromatography and selective thermal denaturation, or a combination of these. The most commonly used low temperature ethanol recovery methods for albumin are based on the original work of Cohn E.J., Strong L.E., Hughes W.L., Mulford D.J., Ashworth J.N., Melin M., and Taylor H.L .: J.Amer.Chem.Soc. 68,459-475 »

1946 »on which the isolation method used for the following examples according to Α · 0 · 194957 is based - Pokorný M., Foukal Z., Pardon J .: A method of isolation of human serum albumin. The need for removal of low molecular weight compounds is also present in other methods where ammonium sulphate (Schultze H., Schonenberger M., Matheka HD: Behringwerkw Mitt. 26, 21, 1952), rivanol (Horni J, Smetana R.) is used as precipitant. Acta Med.Scand. 155,65-68, 1956), caprylic acid (Chanutin A., Curnish RR: Arch.Biochem.Biophys. 89, 218-220, 1960), polyethylene glycol (Polson A., Potgieter GM, Largier JF ,

Mears G.E.F., Joubert F.J .: Biochim. Biophys. Acta 82, 463475 (1964). Polyethylene glycol precipitation is combined with Curling ion exchange chromatography (Curling JM, Berglof J., Lindqist LO, Erikssdn S .: Vox Sang 33, 97-107, 1977), using affinity chromatography for the preparation of pure albumin is described by Harvey (Curling JM (ed.): Methods of plasma protein fractionation

Academie Press, 1980) ^ /> * «* '* Λ pfifv & utfenew pi * 6> * <** <?

-Fraction is drunk. A common feature of these procedures is the selective separation of albumin from other blood plasma proteins, but at the same time increasing the low molecular weight component by other inorganic or

-3.236 098 organic compounds, in particular sodium chloride, sodium acetate and low molecular weight peptides, formed by protein cleavage during fractionation, or contained in the starting material and accompanying albumin up to the final stage of pharmaceutical processing. These compounds make up up to 4% of the impurities of the final preparation and are:. present in free form or bound to an albumin molecule. Their presence reduces the osmotic and transport capacity of the preparation. To remove low molecular weight fractions from albumin solutions, methods based on the principle of gel chromatography on cross-linked dextran gels are used (Friedli H., Fournier E., Volk T., Kistler P, Vox Sang. 33,93-96,1977) · Štěpánek I and Uhrík J. use dialysis countercurrent to remove low-molecular moieties. Preparaate is termed salt poor albumin or low-molecular-weight albumin.

These drawbacks are overcome by the proposed method of removing ethanol and salts using membrane systems with a defined molecular separation threshold in the form of flat membranes or hollow fibers used to concentrate biopolymers. According to the present invention, the removal of low molecular weight fractions derived from starting materials and fractionation techniques used to isolate albumin consists in diluting the solution of purified albumin in redistilled water with a stepping or continuous flow of water for injection at constant volume and diafiltering. an ultrafiltration unit fitted with a membrane in the form of a flat membrane (Fiat membranes) or a set of dual fibers (Hollow fibers) with a defined cut-off limit of mole236 096

- 4 balls smaller than 10 µD. The process is carried out under aseptic conditions at temperatures in the range of 0-45 ° C, whereby its rate can be influenced due to changes in theological properties of the albumin solution, in a concentration range of 2-20% by weight, preferably in the range of 8-2% by weight. When using a starting solution which contains 5 - 10 wt. albumin and 5-10% v / v ethanol, the ethanol concentration drops below 0.5% v / v and the sodium content is below 174 mmol / l, using water for injection equivalent to five times the initial volume.

This implies time and cost efficiency over the above methods, which require costly materials for gel chromatography, respectively. 200 times the amount of redistilled water for dialysis. The diafiltrate obtained can be concentrated to a concentration by conventional means, i.e. by lyophilization or evaporation, or preferably by the same ultrafiltration device, in which the albumin content is increased to a concentration of more than 20%, and after sterile filtration and stabilizer addition is converted i.e. to a solution containing 5 or 20 wt. albumin.

EXAMPLES

Example 1.

Sediment fraction In a production batch of 23 kg total weight, it was suspended in redistilled water at + 4 ° C in a ratio of 2 L of water per kg of sediment. The solution after the addition of activated carbon in an amount of 50 g per kg of sediment of fraction V and mixing was filtered through asitz-cellulose filter layers of Seitz K5 and

- 5 hollow fiber type HP 26.5-43-PM1O company area of 2.5 m ² , separating molecules up to 10 µD, volume in increments of 8 1 pyrogen-free water for injection,

236 09S

EKS2. 68 L of filtrate at pH 6.40 contained 8.3% by volume of ethanol and 8% by weight of protein, of which 93% was albumin. The solution was diafiltered on an Alfa Laval UPP apparatus fitted with a Romicon ultrafiltration tray. The total consumption was always 138 l of water. After diafiltration, the solution was concentrated by ultrafiltration to a volume of 24.5 L at a concentration of 17.5% albumin. The concentration was adjusted to 20% by the addition of 500 g of lyophilized albumin, and after stabilization (0.02 M sodium caprylate and 0.02 M N-acetyld, 1-tryptophan) added, the solution was pasteurized in a conventional manner, sterile filtered, expelled and subjected to prescribed chemical, biological and clinical trials on the basis of which he was released and dispatched.

98.7 wt.

19.1% wt.

7.03

11, 13, 19 Units 11, 14, 21 Units 0.045 mg / 1g albumin

52.2 mmol / l

0,6 0,8 1,0 ° C no reaction in clinical trial purity of elfo protein pH appearance heat stability hernia content content Na sterile harmless pyrogen-free

- 6 Example 2. 23β axis

The albumin solution prepared analogously to Example 1 contained 7.6 wt.% Protein, of which 91.8 wt.% Albumin, and 8.1 wt.% Atanol in 70 L. Pellicon was used for diafiltration. Millipore equipped with 10 cassettes o

PTGC flat membranes with a total area of 46 m, separating molecules up to 10 µD, volume was replenished with water for injection in 35 doses of 35 L · After diafiltration, the solution was concentrated to 22.15 L with a concentration of 23.4% albumin to the same device. After adjusting the concentration to 20 wt. and the addition of the stabilizer, the solution was pasteurized in a conventional manner, sterile filtered, filled and subjected to prescribed chemical, biological and clinical tests on the basis of which it was released and shipped, the purity of the elfo protein PH appearance beat. stability content of the contents of the package contents of the contents For sterile harmless pyrogen-free

97 »9 wt.

19.7 wt.

6.68

12, 14, 17 inf. Units 11, 12, 13 inf. units of 0.047 mg / 1g albumin 100 ml

56.55 mmal / l

0.4 0.2 0.6 ° C without clinical reaction

-7 Example # 3. _23β08β

The albumin solution prepared analogously to Example 1 contained 7.8% by volume ethanol and 8.3% by weight. Proteins, 92.3% albumin, in 87 l. Diaellic filtration was performed by Pellicon of Millipore equipped with 10 cassettes of flat membranes type PTGC with a total area of 46 m, separating molecules up to 10D, volume was filled with water for injection in five doses after 1. After diafiltration, the solution was lyophilized. 6640 g of the lyophilisate was dissolved in 33 l of a 20% solution, and after the addition of the stabilizer, the solution was pasteurized in a conventional manner, sterile filtered, filled and subjected to the prescribed chemical, biological and clinical tests for release and shipping.

97.7 wt.

9.7 wt.

6.71

10, 12, 15 units 18, 22, 24 inf. units 0.055 mmol / l

102 ml

56.55 mmol / l

0,4 0,3 0,1 ° C no reaction in clinical trial purity elfo protein pH appearance heat stability hernia content package contents content sterile harmless pyrogen-free

Claims (1)

Process for preparing a serum albumin solution having a low content of low molecular weight compounds expressed as sodium content of less than 174 mmol / l for 20% by weight. albumin solution, characterized in that the aqueous solution of purified albumin is diluted after filtration with a solution having a sodium content of less than 174 mmol / l and subjected to diafiltration on flat membranes or ultrafiltration cartridges with a cut-off limit of 10 µm to 10 µm at least four times by dilution and the solution thus obtained is concentrated to the desired concentration of 5% by weight of 20% by weight. albumin.