SK3996A3 - Preparation method of blood albumin with reduced salts content and increased monomer content for therapeutical purposes - Google Patents

Abstract

Purification of an albumin solution by removing relatively low-molecular compounds, expressed in a molecule weight up to 67,000, and relatively high-molecular compounds, expressed in a molecule weight as from 70,000, out of albumin solutions by gelling filtration, diafiltration and ultrafiltration with respect to distilled apyrogenic water or solutions in distilled apyrogenic water with a pH value ranging from 3.0 to 9.0 and at temperature ranging from 0 to 60 degrees Celsius, such as 0.05 M of sodium chloride solution, 0.02 M of a phosphate buffer, 0.02 M of acetate buffer; the solution thus obtained is condensed by the ultrafiltration and, if desired, lyophilisation; and after adding pasteurising stabilisers it is pasteurised and sterilised by means of filtration, for example.

Description

Technical field

The invention relates to a process for the preparation of blood albumin for therapeutic purposes with reduced salt content and increased monomer content by removing relatively low molecular weight compounds up to 67% by weight and relatively high molecular weight compounds over 7ο · οοο from albumin solution using gel filtration, diafiltration and ultrafiltration relative to a solution of distilled pyrogen-free water or solutions in distilled pyrogen-free water such as 0.0 M sodium chloride solution, 0.02 M phosphate buffer, 0.02 M acetic buffer or other with a pH range of 3.0 to 9.0 and a temperature of 0 ° C to 60 ° C ·

The albumin solution was prepared from normal or hyperimmune plasma, or from normal, hypermrimeral or retroplaoentary serum · '- 2

Until now, the preparation of blood albumin has been predominantly with ethanol (Cohn E, J., Strong L. B, Hughes WL, Mulford DJ, Ashworth JN, Xelln V, Taylor ML, J.Amer.Chem.Soc, 66,459 , 1946} Taylor HL ·, Bloom 0 «0 ·, Hc Call Amer · Β ·, Hyndman LAs J Amer Amer · Chem · Soc · 76 * 1353 * 1956 I Krijnen HW, Maetenbroek GGAi Vox Sang 2,405 * 1957 i Mulford D ·, Liealay EH, Welten LDj J.Clin.Invest 34 * 963 * 1955 | Nitsohmann HS, Kistler P., Lergler Wi Helv.Chim. Aota 37 »866 * 1954 | Nitsohmann H ₀ S ,, Kistler P ·, Reufer H., Hässlg A Joss A Vox Sang 1 163 * 1956 and Kistler P. Nitsohmann HS Vox Sang 7 * 414 * 1962) The use of ethanol together with Zn ⁺² and Ba ⁺ has been described ² (Cohn E, J, Gurd FNR, Surgenor, Barnes BA, Brown RK, Derounaux G * Glllespls JM, Kahnt PW, Lever

WF, Liu CH, Mlttelman D, ltouton RF, Sobmid K, Uroma E, J.Amer.Chem.Soo, 72,465 * 1950, as well as the use of Zn ⁺² (Cohn EJ, TullIB JL, Surgenor D (M, D * Hont M: Science 114 * 479 * 1951) ·

Efforts to increase albumin yields have led to the production of albumin to introduce thermal denaturing methods (Hoeh H. * Chanutin A: Aroh.Bioohem Blophys 51 * 27 * 1954 and Lundblad JI Vox Sang · 5 * 122, 1960 Sohneider W _e , Lefevre H. * Piedler H., Mo Carty L. Jlut Blut 30, 121, 1975).

Efforts have been made to avoid the use of denaturing temperatures of about 70 ° C in technology practice. «Addition of chloroform has been found to reduce the denaturation temperature of nonalbumin impurities to a range of 40 ° C to 50 ° C (Libich M · t Cz · patent 129 936 | Libioh)

X. t AO 210 088 (Libioh M., Novotny J, Ulryoh S t t AO 239 715).

The undesirable impurities in the clinical albumin preparation include, in particular, alpha-1 lipoprotein of variable molecular weight *, which ranges from 180,000 to 350,000. The addition of chloroform was also used to pre-adipate lipoproteins from human albumin solution (Pokorny X · * Foukal Z ·, Pardon J.x

194,957) ·

The use of chloroform in isolate of albumin sulfur increases albumin recovery * but results in the binding of a portion of chloroform to albumin in amounts up to 500 + 50 mg chloroform per 1000 ml of 20% albumin * which cannot be removed by gel filtration or simple or machine dialysis. · By · · · · · · · · · · · · · · · · · · · · · · · ·

Novel knowledge about the effects of chloroform is not recommended for use in the separation of lipoproteins. Chloroform also occurs in drinking water * in an amount of 1 mg per liter * where chlorinating on water dissolved methane is formed in trace amounts and is considered to cause a massive mutagenic compound. Effect (Chen A. H * Skoohdopole J Kosk P * Cole L * Science 207 * 90 * 1980)

Although in the recent past the use of chloroform in anesthesia was · common * currently not recommended (Jones Z W W Anaestheeia 38 * 578,1983

The effect of chloroform on blood plasma proteins is very specific and affects the properties of these proteins as well as the immune system of the human and animal organisms in a wide range, while also acting as hepatotoxic and canoeogenic (Štefanovič J »,

Older Z * Uurgaä Z, Absolon 0. »J _e Hyg ₀ Epidemiol _e miorobiol. Immunol 31 * 1 * 1987 štefanovič Ja, Uurgaäová Z · * Absolonova 0 «, Žalkoviôova Bratislavl» lek »llsty 89,0.3,160 * 1988)«

The use of ethanol in blood plasma protein frakelonáoil is one of the world's most widespread method of fractionation. The effect of ethanol on blood plasma proteins is also very specific and has a negative effect on ioh biological pacidity.

For this reason, technological konoepoies have been developed to reduce the concentration of alcohol used and the prooes fracolonolole stopped after separation of the Cohn * ZZ * · ΣΙΣ * fraction and continued with ion exchanger or affinity chromatography. The development of these conjugations continued to the point where, after separation of cryopreelplate from plasma or serum, the separation of albumin for the guideline is only carried out by ion exchangers that use bound DEAE and SF groups on Sephadex basis (Curllng JH, Berglof J, Ilndquist LO, Erlksson S ·: Vox Sang · 33,97,1977) or DEAE and CM bonded groups on Sepharose basis (Curllng J «M: in the book" Methods of plasma prothns fraotionatlon p.77, Academic Press 1980 London) or the above bonded groups on any polymer carriers of defined porosity and granularity.

The thiologologic procedures, on the one hand, separate albumin from a mixture of other blood proteins, but on the other hand introduce low-molecular inorganic or organic compounds into the albumin solution. This objective fact actually exists in older precipitation methods, but also occurs in the most recent ion exchange methods » affinity ohromatography. The technological process can also enrich the concentration of some compounds with biochemical-phiological activity in solution due to their conentration in the starting material from which albumin was isolated.

A drawback of the albumin theology used hitherto is that the therapeutic preparations thus prepared may contain up to 348 mmol sodium per 1000 ml of 20% albumin, which has been implicated in reducing the clinical affection of the preparation, particularly when draining fluids from the body under pathological conditions. A further drawback is that albumin preparations contain, in addition to albumin polymers, alpha-11β-protein.

The presence of inorganic salts greatly reduces the osmotic activity and capacity of the albumin solution for injection. The presence of relatively low molecular weight inorganic and organiocyte compounds, up to a molecular weight of 67οβοο, and relatively high molecular weight compounds, expressed as molecular weights above 7o00o, in the albino solution form

- 5 prerequisites for the formation of undesirable Interactions of these compounds with albumin molecules, especially during pasteurysal and during storage as a therapeutic preparation during the expiration period.

The presence of proteins in an albumin preparation, up to a molecular weight of 67,000 and above 70,000 creates preconditions for the formation of proteinaceous associates, blended aggregates or polymers, and is highly beneficial for the quality of an albumin therapeutic preparation if these proteins are removed from the albumin solution prior to pasteurization or lyophilization.

Albumin preparations for l.v. administration, made according to Stepan and Uhr (Stepanek 1.1 AO 202 181 f Stepanek I, Uhrik J. and AO 208 866 j Stepanek I »AO 249 861) have an aggression spectrum that is in the range of 77-83% monomer, 13 -22% dimer and 3-7% albumin polymer. Testing of monomer, dimer and albumin polymer content in i. in. albumin preparations were run by gel filtration on Sephaoryle S-200 Superfine with a flow registration density at 254 nm.

In addition to Cohn albumin from Fraction V, albumin also occurs in Cohn Fraction IV, which contains about 10-25% albumin, and it is advantageous and economical to utilize this albumin.

The newly proposed technology, these difficulties are not present in the thorny to a significant extent, which may be documented by the present invention was achieved by the reduction of sodium content is always below 160 mmol per l.ooo ml of 20% albumin, and _t = NOH recovery of albumin was increased by the albumin content of fraction IV which would otherwise be in the waste. The aggregation spectrum of albumin ranges from 90-96% monomer *

4-8% dimer and rarely 1-2% albumin polymer.

The worldwide tendency of the preparation of therapeutic albumin preparations is to preference for the highest possible content of albumin monomer and the lowest possible content of other impurities.

- 6 Summary of the Invention

SUMMARY OF THE INVENTION The present invention relates to a process for the preparation of blood albumin for therapeutic purposes with reduced salt content and increased albumin monomer content comprising the removal of relatively low molecular weight compounds of up to 67,000 molecular weight and relatively high molecular weight compounds of more than 70,000. by using gel filtration, lamfiltration and ultrafiltration relative to a solution of distilled pyrogen-free water or to solutions in distilled pyrogen-free water such as 9.05 M sodium chloride solution, 0.02 M phosphate buffer solution, 0.02 M acetate solution, or to other solutions with a pH range of 3.0 to 5.0 and a temperature of 0 ° C to 60 ° C ₀ Thereafter, the albumin solution is concentrated, after the addition of pasteurization stabilizers it is paeteurized, further the albumin solution is sterilized, for example by filtration, and processed to a final solution. forms of therapeutic preparation and, if necessary, undergo further processing. DETAILED DESCRIPTION OF THE INVENTION

Example # 1

The raw material may be a protein paste of fraction V, isolated, for example, by the method of Cohn et al. The proteinaceous material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to 60 ° C such that the protein concentration is in the range liter, with an optimum of 60 to loo grams per liter · Dissolution is accelerated by stirring · After dissolution, only in case of turbidity, the albumin solution becomes clear, for example by filtration ·

Under the process conditions, the above conditions are generally met when 1 kg of albumin paste fraction V is dissolved in 2,000 ml of distilled pyrogen-free water at 0 ° C to 60 ° C. The pH of the solution thus obtained is generally in the range of pH 3.0 to 9.0. The optimum pH is about 7.0 · The protein concentration in the paste-to-water ratio is almost always in the optimum · The purified albumin solution thus prepared and clarified, for example, by filtration and a relatively non-molocular compound to a molecular weight of 67 * 000, and a relatively high-molocular compound molecular weight above 7o * ooo, oa will remove for example »

A · Donkey filtration

B * Diafiltration

C * Ultrafiltration

· There are currently a number of commercial gel filtration materials based on doxtran gel, under the designation Sepha dox, polyacrylamide gels, under the designation Biogol, based on acrylamide and dextran, under the designation Sophaoryl, hydroxyalkyl methacrylate gels, under the designation Sphoron, Btyragel, polyvinyl acetate gels, Merokogel poresny silicates, Porazil, and Bioglaz pores, which have graduated sizes in the gel particles and allow a gel filtration in a relatively large molecular weight.

The molecular weight of the relatively low molecular weight compounds, up to a molecular weight of 6700, and the relatively high molecular weight compounds above the molecular weight of 7% is carried out by gel gel chromatography with respect to distilled pyrogen-free water or to solutions in distilled water such as pyrogen-free water. 0% II sodium chloride solution to 0.02 M phosphate buffer solution, 0.02 II aootate buffer solution or other solutions having a pH of 3.0 to 9.0 and a temperature of 0 ° C at 60 ° C.

The aforementioned materials should be used in optimum grain size, which will ensure flow in the thiologiological conditions and can be supplied dry or in a preservative solution. * Sephaoryl materials are typically supplied in a preservative solution, for example at 0 »02 (m / m). )% aqueous solution of sodium asld, which must be quantitatively removed by washing before use ·

A column packed with swollen equilibrated gel material, such as Sephacyyl S 200 HR, is allowed to aspirate an albumin solution, at a concentration of up to 160 g per liter, at a rate such that the flow through the bottom of the column is between 0.01 ml and p.

3.0 ml per milliliter per 1 cm area · The amount of albumin solution applied to the column should not exceed 25 to 30% by volume of gel column counts · Separation efficiency can be monitored according to the degree of separation of ethanol, sodium and ninhydrin positive substances g albumin rostoku ·

Separation of the relatively low molecular weight compounds is performed until the ethanol concentration drops to about 0.1 (w / w)% and the sodium concentration value to about 0.01 (w / w) X at 5% or 20% albumin concentration before adding pasteurization stabilizers ·

During the technological process of separation of relatively high molecular weight compounds, it is necessary to monitor the content and localization of albumin monomer, albumin dimer and albumin polymer in individual gel-endangering fractions which can be determined by gel filtration by HPLC for example on SephaOPyle 8-200 Superfine with flow registration density at 254 nm or other wavelength ·

It should be noted that a very small portion of ethyl alcohol ea binds to albumin · If complete separation of either low or high molecular weight compounds from the albumin solution has not been achieved, it is necessary to reduce the protein content and volume of the albumin graft to the knee and slow the flow. the occurrence of an albumin monomer which is further processed.

B. Preparation of Albumin Monomer It is also possible to carry out diafiltration. The dlafiltration can be carried out in the form of hollow threads, for example, whereby the albumin solution flows under mlex pressure to? within the yarn membrane and through the holes in the yarn diaphragm, the molecular weight compounds pass up to 67 οοο into the outer side of the overflowing distilled pyrogen-free water solution or into the solutions in distilled pyrogen-free water, e.g. 0.05 M sodium chloride solution, 02 M phosphate buffer solution, 0.02 µl acetate buffer solution or other solutions having a pH of 3.0 to 9.0 and a temperature of 0 ° C to 60 ° G, which carry compounds to a molecular weight of 67 ° C from the yarn membrane to waste that we do not collect.

The filtering can also be carried out in the form of a cassette system, in which a replaceable membrane or a plastic plate with a defined tongue size is or is replaceable in a planar arrangement. For example, the albumin solution under gentle printing flows up to 5 ° C downstream of the mismatched membrane and the pores in the planar membrane wall or porous plate penetrate very slowly, and the compound flows to a molecular weight of 67,000 into a flowing solution of pyrogen-free distilled soil or water, for example, to 0.05 sodium chloride solution, 0.02 H phosphate buffer solution, 0.02 U acetate solution, or other solutions having a pH of 3.0 to 9.0 and a temperature of 0 to 60 ° C, and which flow, for example, under a plate or membrane and thus wash away relatively low molecular weight compounds, to a molecular weight of 67 * 000, into waste, which is also not collected. After separation of the compounds to a molecular weight of 67,000, the separation of relatively high molecular weight compounds above 70,000 is made from the albumin solution; .

The vulcanization of compounds having a molecular weight of up to 67% from the albumin solution can be carried out, for example, by means of a H1OP5O-2O diafiltration ultrafiltration cartridge arranged in the form of a hollow

The 10 yarns having a molecular weight exclusion limit of 5e · ooo * The separation of the compounds with a molecular weight above 7 ο οοο from the albumin graft can be carried out using a diaphiltration ultrafiltration cartridge of H10P100-20 arranged deformed hollow yarn having a leaching molecular weight. ooo. The abovementioned diafiltration, ultrafiltration cartridge, the company's products Amioon and can be used, for example, the device Amioon DC-30 or other aarladenlaoh for this purpose amateur made · The solution of compounds with a molecular weight of loo * ooo * which is entrained in the waste rejected _to because it's albumin monomer solution and further processed *

Separation of compounds with a molecular weight of up to 67 * 000 from the albumin solution can also be carried out, for example, using Pellicon series f T, µL or PHSA diafiltration ultrafiltration membranes with an exclusion limit of up to 30 ° C, for example on a COT-50 T7P. It is also possible to successfully use the SK 12133-10Ox 1C0T * nitrooellulose membrane manufactured by Sartorius with a cut-off of up to a molecular weight of 30 * 000, for example on an amateur-made apparatus ·

Separation of compounds having a molecular weight above 70 * 000 from the albumin solution * after separation of relatively low molecular weight compounds * to a molecular weight of 67 * ooo * can be effected, for example, by diaphiltration ultrafiltration membranes of the Pellicon series PT, PL or PHSA with the molecular weight exclusion limit. on Millipore CUF «-50 TST or on equipment for this purpose made by amateur * Compound solution and molecular weight up to loo * oo9 * which is entrained into waste * because it is an albumin monomer solution and further processed *

The dilution and concentration process is carried out until then * the separation of relatively low-molecular compounds * into the molecular 67 * ooe * has not reached the desired concentration range, especially in

- 11 ethanol, sodium and ninhydrin positive substances · If nol are relatively separated, high molecular weight compounds, and molecular weights above 70 ° C, such as alpha-11β-protein or albumin dimer, should be continued in the separation process.

Melt may also be used to prepare the albumin monomer solution. A vampire spiral diafiltration ultr & filter pavers launched by Amioon. Under these conditions, it is suitable to use spiral rotary cartridges 810X30 or 4040X30 with a molecular weight cut-off of 30 ° C eo to separate relatively low molecular weight compounds up to a molecular weight of 6,000. For separating relatively high molecular weight compounds, b with a molecular weight above 10000, it is suitable to use a S10X100 or S40X100 spiral cartridge having a molecular weight cutoff of 10000.

Sterilization. The diafiltration device is carried out in the same manner as described for sterilization of the ultrafiltration apparatus.

The preparation of the albumin monomer can also be carried out by means of an ultr < The ultrafiltrate can be referred to as pressure dialysis and can be carried out in the form of hollow threads, whereby the aibuaine solution flows under gentle pressure into the thio 100, for example, inside the yarn membrane and passes through the pores in the yarn membrane wall. We did not detect.

The ultrafiltration can also be carried out in the form of a cassette system, where a planar arrangement contains or a replaceable membrane or plastic plate & defined pore size. The Albu minin solution under a gentle pressure of up to 600 kP flows for example above the membrane and through the pores in the planar wall. membranes or porous plates pass through very slowly the compound's flow to a molecular weight of 67 «boo to waste * which we do not catch ·

The albumin for ultrafiltration separation may be in a distilled pyrogen-free genus or in a non-thyroid-like pyrogen-free genus * for example r 0 * 05 M sodium hydroxide year * r 0 * 02 II phosphate buffer * r 0 * 02 M year aootate or other With a pH of 3 * 0 to 9 * 0 and a temperature of 0 * 0 to 60 * 0 ·

After separating the relatively low molecular weight alonoson * to a molecular weight of 67 · οοο *, the ooparalol is transferred to a relatively high molecular weight alonoson * above a molecular weight of 7 · · a * and albumin roots ·

The alosonin soparol having a molecular weight of up to 67% and albumin grade can be rolled out, for example, by using ultrafiltration hollow thread cartridges arranged in the form of hollow threads which have a 5o _c ooo molecular weight separation. After the separation of alonosin having molecular weight up to 67%, it is possible, for example, to use a H10P100-20 hollow fiber filter cartridge arranged in the form of hollow yarns * which has a melt-weighted medium of about 10% by weight. and can be used, for example, on the Amioon DC-30 or on other equipment for amateurish use. · Hoatok alúSonin with a molecular weight up to 1οο · οοο aaytyt * finger eye yeah to the year of albumin monomer and treat it · ·

Separation of alonosin * with a molecular weight of up to 67% and albumin rococoa can also be carried out, for example, by the use of a PTFE / PHSA series Pollloen ultrafiltration membrane and a secreted modal of up to 30 * 000 per aarladon Xlll / TOT 50 8M 12133-160x1602 * ultra-cellulosic membrane * Sartorlus product can be used up to a molecular weight of 5ο · οοο for example on an amateur ahetone apparatus ·

- 13 βeparéol of a compound having a molecular weight of over 7ο · οοο albumin rostoc, after separation of the relatively low-molecular weight compounds, to a molecular weight of 67 · ooo, can be rolled using diaphiltrated ultrafiltration membranes of the Pellloon type PT, PL or PHSA molar mass. For example, on a Millipore CUP-5O TIP, or on a device for this purpose, an amateur shot. Also, a 811 14669-160x1660T polysulfone membrane can be used with success; a Sartoriue e product excreted with a molecular weight cut-off _and, for example, an amateur shot. We bind the compound of e-molecular weight to loo.ooo because it is an albumin monomer plant and continue to process it ·

For the preparation of rosin eye albumin monomer, it is also possible to use TS-Ampler-spun diafiltration ultrafiltration cartridges.

In the thiologologic condensate, a relatively low-molecular-weight compound on aeparole is preferred, with a molecular weight of about 70 to about 67 MW and a spinning cartridge of 810X30 or 840X30 e being used. it is advisable to use Sprayer 810X100 or 840X100 e cartridges with a molecular weight exclusion loo.ooo.

Prooee bending and sono-concentration of albumin rostoku. until the separation of the relatively low molecular weight compounds · to the molecular weight of 67 · oco, did not reach the desired concentration range especially for ethanol · sodium and ninhydrin-positive substances · The nlo separation is separated by relatively high molecular weight compounds above the required molecular weight over 7 for example alpha-11lproprotein or albumin length · it is necessary to continue the process of division ·

- 14 For sterilization of the ultrafiltration device, it is always necessary to follow the theological instructions by producing the device. to 60 ° 0.

Determine the hydrohydrin positive substances according to the method of Ruheman (Ruheman S J t Chem Chem S 97 * 1438 »1910) · Determination of ethanol aa is carried out by Agulhoh Reaco (Agulhon H * and Ruli · Soo Chlm.Pranoe / 4 /» 9 »881 »1911) 81 already in the original arrangement or in disbelieving appeals according to Kellet (Kellet 1 * 0. I Analyat 62,728,1937) or Vebba C Vebb D * A * t Sol.Preo.Roy Dublin Soo · 21» 281 »1936) · Sodium content aa determined by flame fetometer * Alpha-1 content of 11popretein aa determined by immuno-hemoglobin teohnlkaml such as nofalome sprays, turbidlmetrlocs or diffuse *

Removal of the relatively low-lobe compounds, to a molecular weight of 67% and a relatively high molecular weight compound, expressed as a molecular weight of more than 70%, may advantageously make it possible to carry out the combination of the palladiltrole, especially in the first stage of production and ultrafiltrole. the albumin solution obtained can be processed to the final product by a freeze-dried process and a freeze-dried process.

Z · The method beside the lyoflllzáele ·

For separating relatively low molecular weight and relatively high molecular weight compounds with albumin in the solution, the albumin solution is encapsulated, for example with ultralum triturate on conoentral 26 + 2% of protein, and added so concentrated concentrate to the concentrate to produce 20% albumin. n-aacetyl-d, l-tryptefane was 0.04 M (9.64 g - 15 ** β.βοο ml of 20% albumin) or by adding concentrated roster of exchangeable ethers to give a final conententral for example n-acetyl-d , 1-tryptophan was 0.02 M (4 * 92 g in 1.000 ml of 20% albumin) and caprylic acid for example to be 0.02 M (2 * 68 g in 1.000 ml of 20% albumin) ·

In the production of, for example, 5% albumin, an amount of conjugated stabilizer roster is added such that the final conontrocess of, for example, methyl ethyl d-1-tryptophan is 0 * 01 U (2.46 gy 1.00 ml ml of 5% albumin rostok) or by adding concentrated concentrate of saline stabilizers so that, for example, the final conjugate of n-acetyl-1,1-tryptophan was 0.005 V (1 * 23 g in 1.000 ml of 5% albumin) and caprylic acid 0.005 MC 0.72 g / l. After addition of the stabilizers, the albumin solution is paotcurls, for example at 60 ° C ± 1 ° C for 1 hour, then sterilized, for example by filtration, filtered and, if necessary, subjected to further processing.

II. Method using lyophilisate

After oeparation of the relatively low-alcoholic and relatively high-hemocoloculum of the albumin-containing compound, the albumin solution is saccharinated, e.g.

In the manufacture of 20% albumin and lyophilized albumin, it is fused in such a conjugate of n-acetyl-d, 1-tryptefane in distilled water.

It is pyrogen-free that the final coneontraola of n-azetyl-1,1-tryptefan in 100 ml of 20? Albumin solution, for example, 0.04 U, or in the estrogen of the stabilizers in distilled apyrogenic water, for example, H and the final conjugate of n-acetyl-1,1-tryptophan to be 0.02 M in 1.000 ml of 20 ml of albumin.

In the production of 5% by weight of albumin and lyophilized albumin, it dissolves, for example, in such kenoentral n-α-ethyl-d, 1-tryptophan in distilled pyrogen-free water so that the final con-nentral n-azethyl-d, l-tryptophan is present in 1. Ml of a 56 M albumin solution of 0.01 M or in a solution of stabilizers in pyrogen-free distilled water For example, the n-azoethyl-d, l-tryptophan hen kenoentration was 0.00 V and the caprylic acid also was 0.005 M in l.ooo ml 5% album 'min ·

Dissolving the lyophilized albumin in an aqueous solution of the stabilizer or stabilizers does not increase the volume of the resulting solution about half a volume dealing with the object of lyophilized solubilized albumin. After the stabilizers have been added, the albumin solution pasteurizes, for example at 60 ° C ± 1 * 0. o sterilizes, for example by filtration, is refilled if necessary · subjected to further treatment ·

The thiologologic variant, the preparation of a therapeutic albumin preparation using lyophilizate is, compared to the variant beside lyophilized, in a cough, energy and investment disadvantageous.

Example 2

The albumin paste or albumin solution may have been isolated by other methods. The albumin paste of albumin or albumin solution is treated as in Example 5.1, except that the criterion for removal of low molecular weight sio * compounds will still be related to sodium content but will not be sodium content. be related, for example, to the presence of ethanol, but to the concentration of the compound or compounds used on precleation, equilibrium, immobilization or the elution of albumin from a gel-filtering ion exchange or affinity column.

The content of relatively low-spherical compounds, up to a molecular weight of 67 eoe, and a relative high molecular weight above the molecular weight of 7 ° ooe, should be analyzed analytically and the process of separation can be monitored because the qualitative and quantitative aspect of occurrence will be related to the fractionation technique used.

Industrial usability

A method for preparing blood albumin for therapeutic purposes with reduced salt content and increased monomer content is a manufacturing process which has my industrial applicability and provided that the source of evil raw or normal or hyper-plasma or normal, hyper-lunar or retroplaoentem serum conforms to valid veterinary or human standards valid in the Slovak Republic for the convenience of raw material for the production of therapeutical blood derivatives.

Pastoral content and other qualitative and quantitative parameters of Si already at 20%, 5% or otherwise reconstituted albumin solution must comply with applicable veterinary and human standards as well as national or European pharmacopoeial criteria for albumin preparation for therapeutic or diagnostic use. .

Claims (5)

PATENT 'CLAIMS A process for the preparation of blood albumin for therapeutic use, characterized in that it has a reduced salt content and an increased albumin monomer content, characterized in that it removes relatively low molecular weight compounds of up to 67,000 molecular weight and relatively high molecular weight compounds from blood albumin solution e. Molecular weight> 7,000,000, by gel filtration, diafiltration and ultrafiltration relative to distilled pyrogen-free water or to solutions in distilled pyrogen-free water having a pH of 3.0 to 9.0 and a temperature of 0 ° C to 60 ° C, then the albumin solution is concentrated, after the addition of pessurizing stabilizers, it is pasteurized, further processed into the final form of the therapeutic preparation and, if necessary, subjected to a black treatment. 2. The process according to claim 1, wherein, for example, Gohn's albumin fraction V protein material or Cohn, e albumin paste obtained by purification of Fraction IV, dissolves in such an amount of distilled pyrogen-free water at a temperature of 0 ° C to 60 ° C. so that the protein concentration in the albumin solution is in the range of lo to 150 gn liter at a pH in the range of 3.0 to 9.0 and is treated with? so that the albumin solution becomes clear, free of relatively low molecular weight compounds, to a molecular weight of 67%, by gel filtration, diafiltration or utrafiltration until the ethanol concentration decreases to 0.1% and the sodium concentration value to 0.01 So at 5% or 20% concentration of albumin prior to the addition of pasteurization stabilizers, provided that if the albumin paste or albumin solution has been isolated by other precipitation methods or 19 ion exchange or affinity chromatography, in addition to the concentration of hydrogen, the criterion for removal of relatively low molecular weight compounds will be related to the concentration of the compound or compounds used to precipitate, immobilize, equilibrate or elute albumin from a gel filtration, ion exchange or affinity column. The method of clause 1, characterized in that, after separation of the relatively low molecular weight compounds, to a molecular weight of 67,000, eo, the relatively high molecular weight compounds, above the molecular weight of 70,000, are separated from the albumin solution by gel filtration, diafiltration and ultrafiltration. as long as the concentration of the albumin monomer in the solution solution rises and the concentration of the undesirable relatively high molecular weight compounds decreases. . The method of claim 1, wherein the protein solution of albumin, after removal of relatively low molecular weight compounds, to a molecular weight of 67,000, and that after separation of relatively high molecular weight compounds above a molecular weight of 70,000, is not filtered but concentrated by, for example, ultrafiltration. The +2 protein solution is then diluted with a concentrated aqueous solution of a pasteurizing stabilizer or a mixed concentrated aqueous solution of a pasteurizing stabilizer, wherein the sodium content is less than 40 mmol per 100 ml of 5% albumin where ca uses for example 0, 01 M concentration of n-ethyl-d, 1-tryptophan or mixed stabilizer where, for example, 0.005 M concentration of caprylic acid and 0.005 M concentration of n-acetyl-d, 1-tryptophan are used, further the albumin solution is pasteurized, e.g. 60 ° C + 1 ° C, then sterilized, for example, by filtration, filler it shall be subjected, if necessary, to further processing. 5. The method of claim 1 wherein the protein solution of albumin, after removal of relatively low molecular weight compounds, has a molecular weight of 67% and relatively high molecular weight. of the compounds, above the molecular weight of 7.000, is ultimately filtered, for example by ultrafiltration to a concentration of 11 + 5% of proteins, filled and subjected to freeze-drying after freeze-drying, then lyophilized albumin is dissolved in aqueous pasteurizing stabilizer solution or mixed aqueous solution of pasteurized stabilizers. sodium is less than 160 mmol per 1000 ml of 20% albumin containing, for example, a 0.04 M concentration of n-acetyl-d, 1-tryptophan or, for example, a mixed stabilizer of 0.02 M concentration of caprylic acid and 0.02 M concentration of n-acetyl-1,1-tryptophan, the albumin solution is further pasteurized, for example for 10 hours at 60 ° C + 1 ° G, then sterilized, for example by filtration, filled and, if necessary, subjected to further processing * 6. The method of claim 1, wherein the separation of the relatively low molecular weight compounds and the relatively high molecular weight compounds from the albumin solution is carried out by gel filtration elution and by diafiltration and aultrafiltration by repeated dilution and re-concentration relative to distilled pyrogen-free water or solutions in distilled pyrogen-free. a pH of 3.0 to 9.0 and a temperature of 0 ° C to 60 ° C. 7. The method of claim 1, wherein the material from which the albumin paste or albumin solution is isolated is normal or hyperimmune plasma, or normal, retroplacental or hyperimmune serum, all with defined antiviral, antitoxic or antibacterial antibody activity, wherein: the starting material must comply in full with applicable saccharine or human standards for starting raw material for the manufacture of therapeutic blood derivatives.