DD141262B1 - METHOD FOR PRODUCING A PROTHROMBIN COMPLEX CONCENTRATE - Google Patents

Description

Process for producing a prothrombin complex concentrate

Field of application of the invention

The invention relates to a method for producing a concentrate of the coagulation factors II, VII, IX and X (prothrombin) from human plasma or from suitable human plasma fractions for therapeutic use in certain diseases, preferably in hemophilia B, bleeding by the use of oral anticoagulants and other diseases are due to a deficiency of one or more of these 4 coagulation factors *

Characteristic of the known technical solutions

Pactor IX belongs together with the factors II, VII and X to the so-called "prothrombin complex" or the vitamin K-dependent coagulation factor.

The methods for the isolation and accumulation of the prothrombin complex are determined by the property of the coagulation factors to be reversibly adsorbable on inorganic adsorbents or organic ion exchangers (z, B · DEAE-ZaHulose or DEAE-Sephadex) ·

The inorganic adsorbents tri-calcium phosphate or barium sulfate require the use of certain anticoagulants, whereby the blood cells can be stored and transfused in the liquid state only after addition of a special preservation solution. Blood sampling in special anticoagulants is uneconomical and complicates the most complete therapeutic use of a selected number in human plasma contained proteins ·

It is also known from barium sulfate that its use gives toxic concentrates.

To a sufficient extent, human plasma supplemented with citrated anticoagulants is available. A process is known for adsorbing the prothrombin complex of citrate-containing plasma to aluminum hydroxide. "Because of the time-consuming and labor-intensive preparation, the process, which is also less effective in terms of yield, is no longer used, and there is also a risk of increased concentrations of alurainium in the concentrate due to chelate formation.

A number of modifications of a process using DEAE zeolite as an adsorbent have been described. In this process, the human plasma used to isolate the prothrombin complex must be diluted to ensure adequate adsorption. "

This creates considerable difficulties in the further fractionation «

A process (DT - OS 2459291) is known in which tri-calcium phosphate is used as adsorbent and ion exchange plasma for the isolation of the prothrombin complex. "Preserved blood cells are not available in this process.

Von Heystek (J Heystek, HJG J Brummelhuis, H W Krijnen II The Large-scale Preparation of Prothrombin Complex, Vox Sang. 25 (1973) 113-123) describes a process which is carried out in the following stages:

adsorption

From a batch of 100 l of plasma, the prothrombin complex is adsorbed to 150 g of DEAE-Sephadex A-50 at room temperature. The adsorption time is 45 minutes. During adsorption, the gel suspension is stabilized by stirring.

Removal of the excess plasma

After a sedimentation time of 30 minutes, the supernatant plasma is first pumped into a 30 1-pest holder. After removal of the supernatant plasma is in the same

Filter holder dia DEAE-Sephadex-Plasraa-Suaрепа ion promoted.

Wash out the Sephadex Gela

Inert proteins and possible HBs antigen are removed by washing out the Sephadex gel with 3 × 10 l portions of washing liquid (0.2 mol / l NaCl, 0.01 mol / l sodium citrate, pH 7.0).

Elution of the prothrombin complex

The elution is carried out with 2 1 2.0 mol / 1 KaGl 0.01 mol / 1 sodium citrate (2 % by volume based on the plasma volume) ·

dialysis

2 l of sluate are dialyzed against 200 l of 0.01 mol / l of sodium citrate (pH 7-0). · Within 3 hours, isotonicity is reached in the eluate.

sterile filtration

Freeze-drying

After portioning (10 ml / unit), the final freeze-drying takes place.

The stages 1-6 can be carried out in a timely manner within one day ·

For process steps 5-7, a yield loss of 22 % is indicated.

H. Suomela (H. Suomela, G. Myllylä, B. Hemofilian hoi-co tekijä IX konzentraatilla, Duodecim 82 (1971) 31-38) describes a similar process.

To remove the salts used for the elution (0.45 mol / 1 NaCl, 0.05 mol / 1 phosphate) desalting is carried out by gel filtration in a column technique · The dilution occurring requires additional lyophilization · The Lyophiliaat in 0.65 % NaCl solution dissolved and re-lyophilized after sterile filtration

The total process time, including the 2 lyophilizations, is at least 3 days.

In OE - PS 312465 a method for the removal of hepatitis B causing HBs antigen is described by a fractional precipitation with polyethylene glycol.

Main disadvantages of the prior art are:

The highly pooled starting material used for adsorption can not be used for the preparation of human dry plasma because of the high risk of hepatitis · When using prothrombin complex concentrates, the recipient has a high risk of hepatitis due to the fact that between 50 and 1000 1 human plasma / batch are used in large-pool procedures. It is undisputed that the batch size is directly related to the risk of transfusion hepatitis caused by administration of the preparation. The method according to OE-PS 312465 is known to result in spontaneous activation of the coagulation factors of the prothrombin complex.

Pur this method is not yet proven that even endogenously present in traces HBs antigen or, related hepatitis in the recipient causing antigens, can be removed, which elude the usual ilachweetechnik by specific antigen-antibody reactions (z · B. · radioimmunoassay) · It is ensured that hepatitis can also be transmitted by the transfusion of HBs antigen-negative blood and blood derivatives. Another disadvantage of the prepared preparations is the risk of thxomboembolic complications or disseminated intravascular coagulation

by activated coagulation factors of the prothrombin complex present in the concentrates.

The extent of activation of the coagulation factors is most likely influenced by process parameters such as process time, duration and type of contact between the plasma and adsorbent, contact with hydrophilic surfaces and various other materials. All the factors leading to the unwanted side effects of clinical use are not yet known «

In addition, in a large-pool process, other procedural disadvantages arise such as prolonged process time, unfavorable leaching effect and difficult release of the prothrombin complex in small volumes of elution liquid.

The process steps desalting by dialysis or by gel filtration and sterile filtration, which are mandatory in all of the described processes, not only prolong the process time, but the contact with hydrophilic, sometimes even thrombogenic surfaces increases the risk of spontaneous activation of the coagulation factors in the concentrate, since the natural ones in the plasma existing inhibitors of such spontaneous activation in the isolation of the prothrombin complex are already removed to these process stages. "

In the known methods, the prothrombin complex is isolated in a relatively high dilution, due to the relatively high fluidity during the sludging. It is known that the stability of purified coagulation factors in concentrated solutions is more favorable than in dilute ones, for which the yield losses of more than 20 % in the process steps desalination, sterile filtration and lyophilization serve as proof »

Object of the invention

The aim of the invention is a method in which, compared to the prior art by simplifying the method, a substantial reduction of the processing time is achieved, the risk of undesired side effects such as thromboembolic complications and a disseminated intravascular coagulation and hepatitis risk by limiting the amount of per batch reduce the amount of human plasma used in the preparation

Such a process with reduced batch size (small-pool method) should not be inferior to a large-pool method with regard to the material and work effort, the yield and the amount of the clotting-active protein present in the single therapeutic dose and the consistency of the content from batch to batch.

The method is to be designed so that an increase in the batch size without changing the Verfahrensprinzipisn can be made if the prior art, a reduction in the risk of complications in the receiver is also possible in concentrates produced by concentrates · It should be above all the Advantage of process time reduction fully preserved ·

The process should also make it possible to isolate highly concentrated concentrates of the prothrombin complexase and, if necessary, desalt by known techniques. It is likewise to be achieved that the main amount of the protein adsorbed on the adsorbent without concentration for the production of human albumin and huraangamaglobulin is achieved by a stepwise washing process At the same time, it must be ensured that the human plasma or a suitable fraction used for the preparation of the prothrombin complex concentrate is only used as a low-pooled preparation with a total protein content of at least 45 g / l for volume substitution for clinical use due to the relatively high hepatitis risk of highly-pooled preparations

The contact of the preparation with hydrophilic surfaces or with materials which can lead to an activation of the coagulation factors of the prothrombin complex concentrate should be excluded as far as possible.

Explanation: the essence of the invention

It is characteristic of the invention that the step of adsorbing the prothrombin complex on the DEAE-Sephadex A-50 or other suitable adsorbent is spatially separated from the removal of adsorbed inert protein and elution steps by placing the former in blood canned bottles; the supernatant plasma after adsorption of the prothrombin complex separated by Centrifugation, as a low-pooled preparation with a total protein content of at least 45 g / l is used without restriction for volume substitution or for the production of human dry piasma, if no alcohol-containing starting material is used ·

It is also characteristic that the release of the prothrombin complex from the adsorbent used is controlled by the composition and amount of the elution liquid and the portion portioned without prior desalting per therapeutic dose is such that an isotonic or slightly hypertonic preparation is used after lyophilization and dissolution in the application volume sufficiently high active ingredient content is available.

It is known that to release the prothrombin complex from the DEAE-Sephadex A-50 gel, a sodium chloride concentration of 0.5 mol / l is required, and that when performing gel chromatographic separations by the batch technique, the property of gel shrinkage depends on the ionic strength of the solution can be advantageous for certain applications

It has been found that hydrophobization of the glass surface of the preserved blood bottles achieved by siliconization lowers the gel losses upon single vessel adsorption. It has been found that by rotating agitation the adsorption of the prothrombin complex is gentle, without any foaming associated with the danger of denaturation, on agitators in the blood bottles can be dispensed with.

According to the invention, the release of the prothrombin complex is controlled with a minimal amount of correspondingly composed elution liquid (0.15-0.20% based on the volume of human plasma used to isolate the prothrombin complex) such that, taking into account the liquid leakage occurring during the shrinkage of the gel a sufficiently high concentration of ions required for release is achieved. "Expediently, the release of the prothrombin complex from the gel can additionally be assisted by the temperature-dependent shrinkage of the gel.

The efficiency of the process determined by the yield of isolated clotting-active protein, based on the amount of clot-active protein present in the volume of human plasma used at the beginning of the process, depends on the reproducibly constant amount of trapped and non-separated washing liquid before the elution step and on the quantity the separation of the eluates between the individual steps of the elution, if this ever has to be carried out stepwise. It has been found that in contrast to the usual practice it is expedient to begin the elution with a concentrated electrolyte solution and to continue the elution with liquids which contain only so much salts that after their addition to the gel a re-adsorption of the prothrombin complex can not take place , As a result, the electrolyte concentration in the eluate and the amount of eluate can be reduced to a minimum space

 It was found that with gradual elution and use

In the elution stage, the coagulation-active protein is not additionally released from the gel, but these liquids only wash out the coagulation-active protein already released in the first elution step.

It was found that as completely as possible separation of the eluates from the DEAE Sephadex gel with eluate amounts between 0.5 and 0.8 % of the plasma volume used they contain the coagulation factors II, VII, IX and X in such a high concentration that at a portioned eluate amount between 25 and 37 % by volume of the application volume of the therapeutic single dose a sufficiently high active ingredient content is achieved. Upon dissolution of the lyophilized preparations in the application volume, the coagulation factors are contained in a solution of conical to slightly hypertonic acid, so that desaturation of the eluates prior to their lyophilization is dispensed with according to the invention *

With the known technique of overpressure filtration, the eluates can be separated with sufficient effectiveness if the therapeutically employable concentrate volume is 0.6-0.3 % of the volume of the starting material used for the isolation of the prothrombin complex.

It has surprisingly been found that separation of the elution liquids from the gel by means of sentrifugal filtration is possible if the dependence of the mechanical stability of the DEAE Sephadex A-50 gel on the ionic strength of the elution liquids is taken into account by selecting the appropriate conditions for the centrifuging. In this technique, the concentrate volume used therapeutically can be reduced to 0.4 to 0.5 % of the volume of human plasma used for isolation * The efficiency of the technique of separation of the elution liquids by centrifugal filtration is higher than in the technique of overpressure filtration * The separation of the elution liquid can be carried out by the technique of centrifugal filtration in single - stage elution with a higher yield of clotting - active protein than in the overpressure technique described in

usually requires a multi-stage elution or washing out of the released prothrombin complex. Sin technical problem of the method according to the invention is the use of a suitable separation device for the separation of granulated gels of process liquids. A suitable device is described in patent 132,850. When using a hose made of a suitable mesh with support device, a separation device is available in which the process steps leaching of the DEAE Sephadex gel and elution of the prothrombin in a closed arrangement under aseptic conditions are possible. The location-independent vessel allows rapid adjustment of adsorption and desorption equilibria on granulated gels and the separation of process fluids from the adsorbents optionally by decantation, overpressure, vacuum or centrifugal filtration. It is particularly advantageous for the centrifugal filtration that the separating device is not arranged as usual across, but in the direction of acceleration. The manner of attachment of the separator to the vessel eliminates the need for support devices for the separator inserted into or attached to the vessel.

All process steps are carried out according to the invention in a closed arrangement under aseptic conditions, so that eliminates a sterile filtration in the preparation of Prothrombinkomplexkonzentrates.

By dispensing with the method according to the invention, which limits the process steps of desalting and sterile filtration, which limit the batch size, a rational process flow is possible even with a batch size of between 8 and 111 starting material. By limiting the batch size, the known high risk of Poattransfuaionahepatitia prothrombin complex concentrates can be lowered. This is of particular importance for the therapy of acquired deficiencies of the prothrombin complex. With the omission according to the invention of the two process steps of desalination and sterile filtration, the

Process time shortened, contact with not; blood-neutral surfaces, which promote the activation of the factors of the prothroobic-corplex, and prevent bacterial contamination and the introduction of pyrogens by working in a closed arrangement under aseptic conditions. This reduces the risk of thromboembolic complications and pyrogenic reactions in the clinical use of the preparations is exemplified with the adsorbent DEAE-Sephadex A-50 (Pharmacia Uppsala, grain size 40-120 μ) , but it is not limited to this. Other adsorbents (eg QAE-Sephadex) can also be used in the process according to the invention *

embodiment

1, adsorption of the prothrombin complex

In 18 individual supernatants (or a multiple thereof) of the human plasma fraction cryoprecipitate or equivalent alcoholic supernatants with a volume between 440 and 480 ml in siliconized 500 ml Blutkonaenen bottles are at a temperature of 2 to. 4 ⁰ C in the closed system by means of a timed dosing pump 25 ml of DEAE-Sephadex A-50 suspension (9.0 g / 480 ml 0.075 mol / 1 HaGl) promoted. The adsorption takes place at room temperature. Sufficient stabilization of the Gelsuspenaionen and the intended as complete as possible adsorption of the prothrombin complex is achieved by placing the blood bank bottles on a device on which the P'laschen rotate at 4-12 U / min. For adsorption it is also possible to use shakers which allow foam-free mixing «

2. Separation of the adsorbent DEAE-SepJaadex A-50 The DEAE Sephadex gel is separated from the plasma by decantation, but preferably by single-vessel centrifugation (10 min, 1600 rpm). The plasma supernatant is in the closed system z. B. by means of overpressure by a gaseous

Medium (nitrogen) removed · In the case of the use of alcohol-containing supernatants, the plasma supernatant may, for. Can be used for the production of human albumin or human mammal globulin * supernatants of the human plasma fraction cryoprecipitate can be used with a total protein content of on average 50 g / l for the production of human dry plasma *

3 "Remove inert protein by washing out the adsorbent DEAE-Sephadex A-50

The DEAE-Sephadex gel remaining in the individual bottles after centrifugation or after decanting is washed in 25 ml of washing liquid (eg 0.20 mol / 1 ЖаСІ / 0.01 mol / l sodium citrate / pH 7.0). The washing liquid is added by means of an automatic metering device in a closed arrangement. In another embodiment, the supernatant plasma is removed after centrifuging to a remainder of about 25 ml / container and the addition of washing liquid at this time is omitted The individual DEAE Sephadex Suspens ions are collected in two siliconized 500 ml blood bottles in a closed arrangement by means of the overpressure technique and centrifuged again (10 min, IOOOO / min) or directly into a suitable separator z «B. after 7 / P DDR 132 850 promoted. The initially separable protein solution (total protein content 24 to 50 g / l) contains about 85 % of the adsorbed on the DEAE Sephadex A-50 gel inert protein and can be used without concentration for fractionation purposes for the production of human albumin and gamma globulin * Thereafter, in the Separator washed gel with another 3 1 washing liquid (eg * 0,20 mol / 1 UaCl / 0.01 mol / 1 sodium citrate / pH 7.0) continuously under preferential application of the overpressure technique washed in a closed arrangement until the gel the first has lost blue coloring. The separator used can be shaken to accelerate the desorption in a suitable device.

4. Elution (release) of the prothroinbin complex 4.1 «Overprint tachnik

ITaGh cessation of leaching are by means of overpressure (0.15 H? A, 20 min), the remainder of the leachate removed to such an extent that in the subsequent elution after addition of z. B. 15 ml of Sluting Fluid I (4.0 mol / 1 IJaCl / 0.05 mol / 1 sodium citrate / pH 7.0) required for the price reduction of the prothrombin complex of DEAE-Sephadex gel liatriumchloridkonzentration of not more than 0.55 - 0.60 The separation of the eluate I takes place by overpressure filtration (0.15 MPa, 10 min). After addition of z. B. 30 ml of elution liquid II (0.50 mol / 1 UaCl / 0.02 mol / 1 sodium citrate / pH 7.0) and separation of the same by overpressure filtration a total of 60 - 65 ml of the concentrate of coagulation factors II, VII, IX and X receive. By means of an automatic dosing device 2.5 to 3 »5 ml of the concentrate are portioned per therapeutic single dose immediately after its separation and finally lyophilized. In an application volume of 10 ml / therapeutic single dose, about 200 to 250 units of factor II, IX and X are present in an isotonic Ьіз low-grade hypertonic solution. This makes it possible to dispense with desalination of the concentrates by gel filtration or dialysis before lyophilization. Yields of 55-60 % and a 25-30 fold accumulation of clotting factors and an up to 120-fold protein-related enrichment are achieved. The total process time is 4 to 4.5 hours.

4.2. Centrifugal filtration I

The elution takes place with the elution liquids I and II. At the end of the washing process, a reproducibly constant liquid content is achieved in the DEAE Sephadex gel by centrifuging the separating device. Uach addition of 15 ml Elutionsflüsaigkeit I, the separator for 15 minutes in a temperature-controlled bath at ⁰ ° C. shaken and then centrifuged in a refrigerated centrifuge at ⁰ ° C for 20 to 25 minutes at 800 to 2000 rev / min.

In the first elution, yields of 60-65 % are already achieved in a concentrate volume of 45-50 ml.

Further processing of the concentrate takes place as described under 4.1 ·. By a 2, elution with 30 ml of elution liquid II, the yield can be increased by about 5 % *

4 · 3 · centrifugal filtration II

After completion of the leaching, excess washing liquid is removed by overpressure filtration (0.15 MPa, 10 min). In the elution, first 15 ml of elution liquid I, then 30 ml of elution liquid II are transferred to the separator and shaken for 20 minutes on a suitable device at room temperature. The majority of the eluate (about 70-75 %) is purified by overpressure filtration (0.15 MPa, 10 min) away. Subsequently, by centrifugal filtration (30 min, 2000 rpm / min, 10 ⁰ C), the remaining Sluat separated and combined with the product obtained by pressure filtration "

An average of 83 + 3 ml prothrombin complex is obtained. Sterile filtration according to 4.1. up to 4.3 · separated Sluate is omitted, since all process steps are carried out in a closed arrangement under aseptic conditions · each therapeutic single dose is portioned 3.0-3.5 al eluate. In an application volume of 10 ml / single therapeutic dose, 200-250 units of Factor II, Ei and X are present in a slightly hypertonic solution, so that desalting of the eluate prior to portioning is not necessary.

Depending on the amount of elution liquid used, it is possible to prepare concentrates containing 90-100 units of factor II, IX and X per ml.

Claims (5)

Srfindungsanspruch 1. A method for producing a Prothrombinkomplexkonzentratea from human plasma by adsorption on organic ion exchangers characterized in that - used for adsorption of human plasma with a volume of 44.0 - 480 ml, adsorption takes place in 3-siliconized blood vials rotating at a speed of 4 to 12 rpm, at room temperature, after the adsorption of the plasma excess is separated by centrifugation, the inert protein is removed in a stepwise washing process by means of 0.2 mol / l NaGl / 0.01 mol / l sodium citrate washing liquid, - then a HaC! concentration of not more than 0.55 - 0.60 mol / 1 is set by the addition of 4.0 mol / 1 NaCl / 0 _f 05 mol / 1! Tatriumzitrat elution liquid after separation of sluatos I, the elution is continued with 0.30 mmol / 'HaCl / 0.02 mol / l of IT sodium citrate-solvent and both eluates are combined, the amount of eluate portioned per single dose is 25-37 % of its therapeutic application volume, - All process steps are carried out under aseptic conditions in a closed arrangement and by final Lyopniliaation the concentrate of coagulation factors II, VII, IX and Z is won.- 2. Method according to item 1, characterized in that the separation of the prothrorabine complex concentrate from the adsorbent DEAE-Sephadex A-50 preferably takes place by centrifugal filtration. 3. The method according to item 1 and 2, characterized in that the price etching of the prothrombin complex one-step and the separation of the Prothrombinkoraplezkonzentrates as a Bluat by overpressure filtration and subsequent Zentrifugalftitration. 4. The method according to item 1, characterized in that for the isolation of the Prothrombinkompiexkonzentrates preferably the plasma supernatants of the human plasma fraction Kgz? О-precipitate are used 5. Method nacn item 1, characterized in that for the isolation of Prothrombinkomplexkonzentrats the Plasraa supernatants of the human plasma fraction Cohn I can be used.