WO1999002169A1 - Method for producing virus-inactivated blood plasma or blood serum in lyophilised form - Google Patents

Abstract

The invention relates to a method for producing virus-inactivated blood plasma or blood serum in lyophilised form by treating the blood plasma or blood serum with virus-inactivating substances. Said virus-inactivating substances are then removed and the virus-inactivated blood plasma or blood serum is emptied into containers which are allowed to rotate at 600 to 1000 U/min until the liquid therein takes the form of a hollow cylinder. The rotating containers are then immersed in a refrigerant to freeze the liquid. Finally, the container is removed with the frozen liquid and said liquid is lyophilised.

Description

 METHOD FOR PRODUCING VIRUS-INACTIVATED BLOOD PLASMA OR BLOOD SERUM IN LYOPHILIZED FORM

The invention relates to a method for producing 5 virus-inactivated blood plasma or serum in lyophilized form.

Blood plasma and blood serum, as well as other protein-containing solutions of components obtained from blood plasma or blood serum) are mainly used in the therapy of various diseases, as well as for emergency substitution. Since blood plasma and serum can only be obtained from blood donations, virus inactivation of liquids is necessary to prevent the transmission of viruses to the recipient of the liquid.

Virus-inactivated blood plasma is generally used to treat volume reductions in blood volume during bleeding. It continues to be used in the therapy of

₂ o Blood coagulation disorders and disorders of the inhibitors of the blood coagulation system, as well as in immunoglobulin angel. Blood plasma is also used to substitute blood coagulation factors that are contained in blood plasma, provided that such highly concentrated preparations have not yet been used.

25 are available.

Various methods are known from the prior art for the virus inactivation of blood plasma, the so-called solvent / detergent method, known in specialist circles as the S / D method.

Here, the virus inactivation with a mixture of

Tri-n-butyl phosphate as a solvent and an alkylphenyl polyethylene glycol as a detergent. Then be

35 these virus-inactivating substances with the help of plant oil and subsequent chromatographic cleaning removed.

These methods of virus inactivation are already known from the prior art. For example, EP 0 050 061 A2 describes a method for virus inactivation of biological and pharmaceutical products, in particular plasma protein products by adding Akylphenylpolyethylenglykolen (Triton-X-

100 ^® ) for virus inactivation of blood plasma.

EP 0 239 859 describes, as a further development thereof, the virus inactivation of blood plasma and other biological fluids with the aid of a solvent / detergent mixture of tri-n-butyl phosphate (TNBP) and Triton-X-100 ^® . After virus inactivation, the substances are removed by extraction with vegetable oils. Soybean oil, castor oil, cottonseed oil, corn oil, peanut oil, olive oil and other vegetable oils are used in particular for this purpose.

WO 91/14439 also describes a method for producing non-infectious blood plasma, in which the above-mentioned methods are used. The virus is inactivated by adding tri-n-butyl phosphate and

Triton-X-100 ^® . The virus-inactivating substances are then separated off by adding biologically compatible lipids such as soybean oil or castor oil. This is followed by chromatographic purification on a column with C-18 adsorbent.

A summary of the currently customary method can also be found in "Hellstern P., Sachse H., Schwinn H., Oberfrank K., Vox Sang 1992: 63: 178-185, Manufacture and in vitro Characterization of a Solvents / Detergent-Treated Human plasma ". The virus-inactivated liquids, in particular blood plasma, obtained in this way are usually filled into plastic bags for transport and use in liquid form. The filled plastic bags must then be deep-frozen to keep the blood supplies at temperatures of minus 30 ° C. In this state, in the case of blood plasma, for example, storage of up to 2 years is possible while maintaining the activity, sterility and virus activation.

However, the virus-inactivated liquids can also be manufactured, stored and sold as lyophilized products. The advantage of lyophilization (freeze drying) is that considerably less energy is required to store these products. For example, lyophilized biological fluids, for example lyophilized blood plasma, can be stored at cow's cabinet temperatures of 2 to 8 ° C for 2 years while maintaining the activity. For various reasons, however, lyophilization of blood plasma or blood serum has not yet been implemented. Most of the blood plasma is generally produced in plastic bags and deep-frozen and accordingly has to be thawed in a complex manner before use.

When freeze-drying biological liquids, the conventional procedure, in which the liquid product is filled into bottles, the bottles are subsequently deep-frozen to at least minus 30 ° C. and then subjected to the freeze-drying process, often has problems since the lyophilisate obtained shows only poor solubility when solving. Furthermore, the lyophilized preparation has a lower stability, since a proportion of residual water is present in the lyophilisate, which freeze drying process can not be removed. In the freeze-drying process, too, it often happens that the frozen product migrates out of the bottle when the water is drawn off due to the migration of the solvent crystals in the frozen solution. This must be prevented by expensive measures.

WO 96/29556 describes a device and a method for the lyophilization of biological fluids, the containers rotating about a horizontal axis of rotation and being filled with the biological fluid. The liquid is then frozen by injecting a liquid or gaseous coolant into the liquid. However, this method has the disadvantage that the horizontal rotation of the containers can lead to the leakage of the liquid or to contamination of the outer wall of the container. Furthermore, there is also the risk of introducing pollutants by injecting the coolant into the liquid.

The technical object of the invention is therefore to provide a process for the production of blood plasma or serum in lyophilized form, in which the above-mentioned disadvantages of the freeze-drying processes do not occur, a more soluble lyophilisate is obtained and because of a smaller amount of residual water a height ^¬ re stability.

This technical problem is solved by a method for producing virus-inactivated blood plasma or serum in lyophilized form by treating the blood plasma or blood serum with virus-inactivating substances, removing the virus-inactivating substances, the virus-inactivated biological fluid being filled into containers, which are then filled filling at 600 to 1000 rpm as long can rotate until the liquid in the container has taken the form of a hollow cylinder, immersing the rotating container in a cooling medium so that the liquid therein freezes, removing the container with the frozen liquid and lyophilizing the frozen liquid.

A solvent / detergent mixture is used as the virus-inactivating substance, which preferably contains TNBP (tri-n-butyl phosphate) as the solvent and Triton-X- ^100® (alkylphenylpolyethylene glycols) as the detergent. To remove the virus-inactivating substances, use is preferably made of vegetable oils and / or chromatographic cleaning processes on C-18 column materials.

In a further preferred embodiment, the virus-inactivated biological liquid is subjected to sterile filtration before it is filled.

Round containers are preferably used, with glass bottles being particularly preferred. The containers are immersed in the cooling medium for 1 to 40 minutes, depending on the cooling medium used. The cooling medium is particularly preferably selected from the group consisting of ethanol, liquid nitrogen or other liquid gases or gas mixtures.

The use of liquid nitrogen or other liquid gases or gas mixtures as the cooling medium is particularly preferred. Immersion times of 1 to 10 minutes, preferably 1 to 5 minutes, are achieved here.

In the following, the production of biological fluids will be described in more detail using the example of blood plasma. The virus-inactivated blood plasma is produced separately according to blood groups 0. A, B and AB. It is free of cells and cell components, but contains, for example, the coagulation factors and their inhibitors, among other components. Frozen fresh plasma from medically controlled donors is used for the production, which was obtained, tested, certified and released according to the applicable guidelines. The blood plasma is processed on a production scale of approximately 100-300 kg to lyophilized blood plasma. This is done in designated rooms of cleanliness classes 100 and 10,000 (US Federal Standard 209 E). The blood plasma is produced under defined conditions using the solvent / detergent method. The process of starting raw material, frozen fresh plasma, goes through the following stages.

The frozen fresh plasma is first thawed at 10 to 15 ° C. The plasma is pooled and then conditioned by adjusting the temperature, pH and adding predetermined amounts of sodium dihydrogen phosphate and glycine. The plasma is then clarified. As a virus-inactivating substance, tri-n-butyl phosphate as a solvent and Triton-X-100 ^® as a detergent are added to the blood plasma at 26 to 28 ° C. and pH 7, so that a final concentration of 1% is achieved in each case. Virus inactivation takes place at 29 ° C for four hours. The solvent / detergent reagents are then separated from the virus-inactivated plasma by means of castor oil extraction at 15 to 18 ° C. There is a phase separation. The blood plasma is filtered through filters, e.g. B. the sizes 0.45 - 1.0 μm. The remaining solvent / detergent reagents and the residues of the castor oil are then removed from the virus-inactivated blood plasma by chromatographic purification using C-18 column materials. The pH from 6 to 7 is adjusted by adding citric acid. Then sterile filtration is carried out over 0.45 and 0.2 μm filters. The virus-inactivated sterile-filtered blood plasma is either filled into transfer bags or bottled for lyophilization. The transfer bags are then frozen. There is an intermediate storage until the final packaging at at least minus 30 ° C. The bottles intended for lyophilization are provided with GT stoppers.

Then the filled glass bottles with their liquid contents are set in rotation in an apparatus around their longitudinal axis at 600 to 1000 rpm. The axis of rotation is vertical. As a result, the liquid in the container rises on the walls and releases the bottom of the container. The liquid takes the form of a hollow cylinder in the container. This changes the ratio of surface area to thickness of the liquid in the container in favor of the surface, ie. H. the surface becomes larger, the thickness less. This change in the ratio of surface to thickness is important for the subsequent freeze drying, in which solid solvent crystals have to migrate through the entire frozen column of ice in the container in order to be removed from the frozen solution.

The container can be rotated before the freezing process outside or inside the cooling medium. It only has to be ensured that the freezing process does not start until the liquid is evenly distributed on the vertical walls of the container and has taken the form of a hollow cylinder.

For example, ethanol, liquid nitrogen or other liquid gases or gas mixtures can serve as the cooling medium for the temperatures, which must be minus 35 ° C or lower. It is important that the cooling medium can be removed without residue or evaporates independently to prevent the user from coming into contact with the container Side effects are caused. The freezing process is complete when the entire contents of the container are frozen. The time required for this is between 1 and 40 minutes, depending on the cooling medium.

The use of liquid nitrogen as the cooling medium is particularly preferred. When using liquid nitrogen or other liquid gases or gas mixtures, the low temperatures result in considerable advantages over the use of ethanol. This means that the contents of the bottle freeze faster after only 1 to 5 minutes. This has numerous advantages over the ethanol previously used as a cooling medium. Ethanol had to be kept at temperatures of around minus 40 ° C. When several containers were immersed, it took about 30 minutes to freeze the liquids contained in the container. Furthermore, it was often not possible to freeze several batches in a row, since the ethanol heated up too much and therefore breaks had to be taken between the individual batches.

Due to the use of nitrogen as the cooling medium and the much lower cooling temperature, such disadvantages no longer exist. Rather, the freezing process is now also suitable for an industrial continuous process on a larger scale, since faster freezing is possible and there are no longer any breaks between the individual batches.

The process of bottling and freeze-drying will be described in more detail below. After sterile filtration has been carried out, a connection to an ultra-pure area (RK 100), in which a filling station is located, is established via an autoclaved hose. For filling the glass bottles, one becomes specific for this purpose installed glass bottle filling system used. Bottles with volumes of 100 or 250 ml of glass type II according to the DAB are used as glass bottles. The glass bottles are only washed in an industrial bottle washer immediately before use, they leave the dishwasher on a conveyor belt under laminar flow, that is, under clean room conditions, and from there they are immediately led into a hot air sterilization tunnel on a conveyor belt. The bottles are transported through the tunnel on a conveyor belt, simultaneously sterilized at an air temperature of 360 to 370 ° C and then cooled to room temperature by sterile-filtered cold air. After leaving the hot air tunnel, the bottles are transported to the bottle filling machine via a turntable, where they are kept ready for subsequent filling.

The sterile filter output is z. B. directly connected to the bottle filling machine via a milk pipe thread. Due to the nitrogen overpressure present on the vessel with the virus-inactivated plasma, the virus-inactivated blood plasma is conveyed to the bottle filling machine after opening the bottom valve and the nitrogen overpressure line through the sterile filter. The bottle filling stops automatically as soon as the desired amount of blood plasma has passed the flow pulse generator and the flow pulse generator closes the flow valve. The filled bottles are sealed under clean room conditions with a siliconized, autoclaved rubber stopper (GT stopper) and then frozen in an apparatus with rotation. After freezing, the bottles are removed from the freezer, placed in transport containers and stored at minus 30 ° C until freeze-drying. The filled glass bottles are only removed from the minus 30 ° C intermediate storage immediately before freeze-drying. The glass bottles are made under clean room conditions opened by lifting the freeze-drying plug and the freeze-drying program, which lasts several days, is started.

After freeze-drying has ended, the bottles are automatically closed under vacuum in the freeze-drying chamber by lifting the chamber panels and pressing the rubber stoppers into the glass bottles. To remove the glass bottles, the lyophilization chamber is automatically slowly ventilated, the bottles with the freeze-dried blood plasma are removed and a test device is used to check whether there is negative pressure in the bottles. The bottles are then bound and assembled.

The following examples are intended to explain the invention in more detail.

EXAMPLES

example 1

Rotational freezing rr. t ethanol as cooling medium

A 250 ml glass bottle, which is filled with 200 ml of liquid virus-activated blood plasma, is closed with a rubber stopper and rotated at room temperature at 800 U / mm. The rotating bottle is then immersed in an ethanol bath set at a temperature of minus 40 ° C. The mixture is rotated for 30 minutes. After the freezing process, the bottle is temporarily stored in a deep-freeze store at at least minus 30 ° C and then subjected to freeze-drying. Example 2

Rotational freezing with liquid nitrogen as the cooling medium

The procedure is carried out as in Example 1, but with the difference that the bottles rotate at 800 U / mm before being immersed in a cooling medium. This process is continued until the liquid in the bottle takes the form of a hollow cylinder. Only then is it immersed in the cooling medium of liquid nitrogen. The process is finished in less than 5 minutes. The bottles are removed and stored at least minus 30 ° C until freeze-drying.

Claims

claims 1. Process for the production of virus-inactivated blood plasma or blood serum in lyophilized form by treating the blood plasma or blood serum with virus-inactivating substances, removal of the virus-inactivating substances, the virus-inactivated blood plasma or blood serum being filled into containers which are filled after Let it rotate at 600 to 1000 U / mm until the liquid in the container has taken the form of a hollow cylinder, immerse the rotating container in the cooling medium so that the liquid poor in liquid freezes, remove the container with the frozen liquid and Lyophilization of the liquid. 2. The method according to claim 1, characterized in that a solvent / detergent mixture is used as virus-inactivating substance. 3. Process according to claims 1 and 2, characterized in that tri-n-butyl phosphate (TNBP) as solvent and alkylphenylpolyethylene glycols (Tπton-X-100 ^® ) are used as detergent as virus-inactivating substance. 4. Process according to claims 1 to 3, characterized in that vegetable oils and / or chromatographic cleaning processes on C-18 column materials are used to remove the virus-inactivating substances. 5. The method according to claims 1 to 4, characterized in that the virus-inactivated blood plasma or Blood serum is subjected to sterile filtration before filling. 6. The method according to claims 1 to 5, characterized in that glass bottles are used as round containers. 7. The method according to claims 1 to 6, characterized in that the immersion of the container is carried out for 1 to 40 minutes, depending on the cooling medium used. 8. The method according to claims 1 to 7, characterized in that the cooling medium is selected from the group ethanol, liquid nitrogen or other liquid gases or gas mixtures.