MXPA01007282A - Method for drying protein crystals - Google Patents

Abstract

The invention relates to a method for drying protein crystals contained in an aqueous protein crystal suspension. The method is characterized in that the protein crystal suspension is dried in a centrifugal dryer and that after the protein crystals have been extracted from the protein crystal suspension by filtration said protein crystals are preferably transferred into a drying medium which consists of a mixture of water and a non-aqueous solvent which can be mixed with water to any ratio and has a lower vapour pressure than water. A drying gas humidified with water is advantageously used and the protein crystals are advantageously dried in a fluidized bed.

Description

PROCESS FOR DRYING PROTEIN CRYSTALS The proteins are present in many commercially available forms of preparation. In particular, proteins are present as an active ingredient in some pharmaceutical drugs. For the preparation of these forms, it is convenient to use proteins in crystalline form. Being easier to handle, proteins in the form of dried crystals are, in particular, more stable than, for example, in dissolved form. Thus, for example, for the preparation of pharmaceutical forms containing the hormone insulin as the active ingredient, the insulin protein is used in crystalline form. The crystallized insulin is stable, for example, at a temperature of -20 ° C for some years. The crystalline forms of proteins with a molecular weight of up to several hundred thousand daltons and also peptides with a lower molecular weight are known. The amino acid sequence of the proteins can be identical to a sequence that occurs in nature or can be changed in relation to the natural form. As they contain chains of amino acids, proteins can also contain sugar radicals or other ligands as side chains. The proteins may be isolated from natural sources, or they may have been prepared by genetic manipulation or in synthetic form, or they may have been obtained by a combination of these processes. In aqueous solution, proteins have a three-dimensional structure of greater or lesser complexity that is based on a specific spatial folding of the amino acid chains. The intact structure of a protein is essential for its biological action. During the crystallization of proteins from aqueous solutions, this structure is conserved to a large extent. The crystalline structure of a protein is determined mainly by its amino acid sequence, but also by inclusions of substances of low molecular weight such as, for example, salts of metal ions and water molecules. In particular, the presence of a certain amount of intracrystalline water molecules (water of crystallization) is necessary for the stability of the crystal structure of the proteins.

Thus, for example, insulin crystals require an optimal residual water content (approximately between 1 and 7%). If the crystals are dried excessively and a very low moisture content is obtained, then the water of crystallization has already been removed from the crystals. As a result, the chemical stability of insulin is adversely affected, causing, for example, the formation of higher molecular weight compounds. It is assumed that fractions of higher molecular weight in insulin are responsible for immunological incompatibility reactions. In the extreme case, the insulin can be denatured to the extent that the crystals are no longer soluble in aqueous medium. If, on the other hand, crystals with too high a moisture content are obtained after drying, then there is too much water between the individual crystals. Insulin dissolves partially in this intercrystalline water. The stability of the dissolved insulin, however, is significantly lower than that of the solid forms. It is known that protein crystals, in particular insulin crystals, can be dried by isolating the crystals from a suspension of the crystals by filtration and drying the filter cake under reduced pressure at a temperature above 0 ° C. It is also known that the drying process can be accelerated by replacing the intercrystalline water with ethanol before drying. During drying, the crystals are distributed in a thin layer on drying sheets or shaken on the filter.

In another process, the protein crystals, for example insulin crystals, are frozen as an aqueous suspension at a temperature below 0 ° C on drying sheets and then dried by freezing under reduced pressure. In the aforementioned processes, obtaining a defined or optimum residual moisture content is not guaranteed adequately. In both processes, the drying process is controlled kinetically until the end and must be finished in good time to avoid over drying. Experience shows that it is difficult to determine the correct point in time to finish drying. The moisture content resulting from the crystals depends not only on the thickness of the layer on the drying sheet, but also on the size of the crystals, or their surface area. Since the crystallization process gives rise to insulin crystals with a size distribution of different amplitude, the crystals dried in a kinetically controlled process consists of a mixture of smaller, drier crystals and larger, more humid crystals. Another disadvantage of these processes is that it is very difficult to automate the loading and unloading of the equipment used for drying. Thus, the loading and unloading operation still requires a lot of manual labor, which harbors the risk of contamination by germs and foreign particles. Thus, for example, the pharmacopoeia with current validity requires that crystalline insulin for the preparation of pharmaceutical forms must have low germ content but are germ-free. A drying process is described below in which all the crucial process steps take place successively in a machine (centrifugal dryer): 1. The filtration of the crystals from an aqueous suspension 2. The washing of the filter cake 3. Replace the washing liquid with a drying medium 4. Drying by centrifugation of the filter cake . Separate the filter cake from the filter and convert it into a fluidized bed. 6. Dry the crystals in the fluidized bed with a humid nitrogen stream. 7. Empty the dried crystals using a nitrogen pressure change to a flanged container. Accordingly, the present invention relates to a process for drying protein crystals starting from an aqueous suspension of protein crystals, which consists of drying the suspension of protein crystals in a centrifugal dryer. The experiments described below were carried out in a commercial centrifugal dryer. For the drying process described, it is particularly convenient if the separated filter cake can be converted into a fluidized bed. Experiments have shown that if the intercrystalline water was not replaced by one of the drying media described below, it would not be possible to generate a fluidized bed; instead, a mixture of crystalline aggregates of different sizes would be formed, making uniform drying impossible. Other experiments have shown that by substituting intercrystalline water with a non-aqueous, pure drying medium, for example 100% ethanol concentration or 100% propanol, only the intracrystalline water (water of crystallization) remaining in the crystals of the protein, it is possible to produce a fluidized bed. However, during the subsequent drying phase, the drying medium could not be adequately separated even after a prolonged drying time. The dried products had a residual content of the drying medium greater than 5%. On the other hand, some of the crystallization water had already been removed during the drying time. Surprisingly, it has now been found that by replacing the intercrystalline water with a drying medium consisting of a mixture of water and a non-aqueous substance, it is possible to avoid the mentioned drawbacks. After replacing the intercrystalline water with one of the drying media described below, it was possible to produce a fluidized bed from the separated filter cake and dry the crystals satisfactorily. By using moistened drying gas, after a drying time of about 1 to 4 hours, products were obtained that were free of crystalline aggregates and with a residual content of non-aqueous substances of less than 0.1%. Accordingly, the present invention relates to a process for drying protein crystals starting from an aqueous suspension of protein crystals, wherein, in particular, the protein crystals, after they have been filtered from the crystal suspension. of proteins, are carried to a drying medium consisting of a mixture of water and a non-aqueous solvent miscible with water in any proportion and having a vapor pressure less than water. The process according to the invention for the drying of a suspension of protein crystals is advantageously carried out in a centrifugal dryer. Non-aqueous substances as constituents of the convenient drying medium have a low vapor pressure at a temperature of about 40 ° C (ie have a lower vapor pressure than water), are miscible with water in any proportion and chemically inactive at proteins in the determined conditions. Alcohols, such as, for example, methanol, ethanol, n-propanol and isopropanol, meet these conditions particularly well. Mixtures of alcohols are also convenient. The proportion of non-aqueous substances in the suitable mixtures with water in the drying medium is about 10% up to about 80% inclusive, preferably from 15% up to about 60% inclusive, more preferably from about 20% up to about 80% inclusive . In the drying process described using anhydrous nitrogen as a drying gas, the drying process is kinetically controlled until the end, meaning [sic] that the disadvantages described above may occur.

Our experiments using wet nitrogen as the drying gas have shown that the removal of water from the crystals during the drying phase is stopped and the residual water content achieved in the dried crystals is determined by the water content in the drying gas, is At the end of the drying phase, a thermodynamic equilibrium is established between the water content in the drying gas and the water content in the crystals. Therefore, the residual water content in dehydrated crystals is independent of their size (or surface area). In addition, experiments using anhydrous nitrogen as a drying gas have shown that the non-aqueous constituent of the drying medium could not be removed in a sufficient amount, even after a prolonged drying period. The dried products had a residual non-aqueous constituent content greater than 1%. Surprisingly, it has been found that in a process for drying protein crystals using a drying gas, the residual content of the non-aqueous constituent of the drying medium could be eliminated except for less than 0.1% if a drying gas containing Water. The drying process preferably takes place in a centrifugal dryer. In the process for drying protein crystals, it is particularly convenient if the protein crystals, after they have been filtered from the protein crystal suspension, are taken to a drying medium consisting of a mixture of water and a non-aqueous solvent miscible with water in any proportion and that has a vapor pressure less than water. In the process, it is particularly convenient if the protein crystals, after they have been filtered from the protein crystal suspension, are converted into a fluidized bed for the purpose of drying. Our experiments with the described drying process have shown that if a sterile glass suspension without extraneous particles is used, and the wash water, the drying medium and the drying gas are sterilized by filtration, and the centrifugal dryer will be cleaned and sterilized conveniently , you would get an anhydrous product without germs and without foreign particles.

The process of the present invention is particularly convenient for drying animal insulin crystals, human insulin or an analog thereof. The insulin analogs referred to herein are derivatives of insulins that occur in nature, namely, human insulin or animal insulin, which differ from the insulin that occurs in the otherwise identical, corresponding nature by virtue of the substitution at least one amino acid radical that occurs in nature and / or the addition of at least one amino acid radical or an organic radical, or both. The process according to the invention is illustrated in more detail below, in particular with reference to the examples.

Construction and mode of operation of the centrifugal dryer The centrifugal dryer consists of a centrifugal drum arranged in the horizontal direction with a compact wall. Inside the drum is a cylindrical screen vessel firmly connected to the drum. The space between the porous surface of the screen container and the compact surface of the drum wall is radially divided into a plurality of chambers. The surface of the rear end of the drum has holes, so that during the rotation of the drum it is possible to introduce a gas (for example, the drying gas) into the chambers from the outside, or a liquid (for example, the mother liquor of the suspension). of crystals) can be removed from the cameras outwards. On the front, the drum is sealed with a piston or piston that rotates with the drum. When the drum is at rest, the piston can be moved in an axial direction. When the piston moves backward, a fixed circular discharge path is opened which is then accessible from the inside of the centrifugal drum. After the drum has been opened, the drum can turn slowly again (for the purpose of discharging the anhydrous product).

In the centrifugal dryer that was used in the experiments, the centrifugal drum had a diameter of 400 mm and a length of the cylindrical wall of 200 mm, the sieve container had a filter area of 0.25 m and a pore size of 10 μ . The centrifuge was loaded with product (glass suspension) axially through the hollow drive shaft when the drum was rotating (475 rpm), the filter cake (crystals) formed a layer on the screen, and the filtrate was compressed through the filter. sieve towards the chambers and from there through the base of the drum towards the fixed outer space. The filtrate (mother liquor of the crystallization) leaves the centrifuge through the discharge port and is discarded. The filter cake is then washed with water in the same manner. Then, the wash water is replaced in the same way by one of the drying means provided in the Examples. After the filter cake has been dried by centrifugation at a relatively high speed (1200 rmp), the filter cake is separated from the screen by blowing nitrogen at a relatively low speed (6 rpm), and the separated product becomes a fluidized bed and dried. For this, a pulsating stream of nitrogen is passed from outside (through the holes in the bottom of the drum) through the lower chambers of the drum and through the lower section of the screen into the interior space of the drum. The drying gas flows through the rotating fluidized bed of the product and exits the drum through the upper section of the screen and the upper chamber of the drum. After the drying phase is completed, the piston moves backward, thereby opening the circular discharge path. At a low speed and pulsating pressure changes, the nitrogen throws the dehydrated product into the discharge path. At the end of the discharge path, the nitrogen is separated into a cyclone and the dehydrated product is collected in a container for storage. The drying gas (nitrogen) is moistened with sterile water vapor before entering the centrifugal dryer in a separate apparatus and heated to the drying temperature. The temperature and water content in the nitrogen stream is controlled.

First example In a crystallization vessel adapted with an agitator, 1000 g of porcine insulin were crystallized from an aqueous solution at pH 5.5 with the addition of zinc ions. When the crystallization was complete, the suspension (approximately 100 1) contained rhombohedral crystals with an average particle size of 25 μ. As described in the general section, the agitated crystal suspension was placed in the centrifugal dryer for about 30 minutes. The filter cake was washed with approximately 50 1 of water and then the wash water was replaced with 70% aqueous ethanol (drying medium). After the filter cake had been separated from the screen, the crystals were dried in a fluidized bed. During the drying time, the drying gas was preheated to a temperature of 40 ° C and adjusted to a water content of 4 g of water per 1 kg of nitrogen. After 120 minutes, the drying was complete and the product was emptied into a container for storage. The analysis of the dried crystals showed a residual water humidity of 5% and a residual ethanol content of less than 0.1%.

Second example In a container for crystallization adapted with a stirrer, 1500 g of human insulin were crystallized under sterile conditions from an aqueous solution at pH 5.5 with the addition of zinc ions. When the crystallization was complete, the suspension (approximately 150 1) contained rhombohedral crystals with an average particle size of 20 μ. The centrifugal dryer was washed before use successively with a 10% sodium hydroxide solution, particle-free water, 10% acetic acid and again with particle-free water. The centrifugal dryer was then sterilized with particle-free steam. As described in the general section, the agitated crystal suspension was transferred to a centrifugal dryer for about 45 minutes. The filter cake was washed with approximately 50 1 of water and then the wash water was replaced by 50% aqueous ethanol (drying medium). Both liquids had been sterilized by filtration beforehand. After the filter cake had been separated from the screen, the crystals were dried in a fluidized bed. During the drying time, the sterile filtered drying gas was preheated to a temperature of 40 ° C and adjusted to a water content of 3 g of water per 1 kg of nitrogen. After 150 minutes, the drying was completed and the product, as already described, was emptied into a container for storage. The analysis of the anhydrous crystals gave a residual water humidity of 4% and a residual ethanol content of less than 0.1%. The product was free of germs and foreign particles.

Third example In a container for crystallization adapted with stirrer, 1000 g of human insulin were crystallized from an aqueous solution at pH 5.5 with the addition of zinc ions. When the crystallization was complete, the suspension (approximately 100 1) contained rhombohedral crystals with an average particle size of 25 μ. As described in the general section, the agitated crystal suspension was transferred to a centrifugal dryer for about 30 minutes. The filter cake was washed with approximately 50 1 of water, the water of the wash was replaced by aqueous ethanol at 30% concentration (drying medium). After the filter cake had been separated from the screen, the product was dried in a fluidized bed. During the drying time, the drying gas was preheated to a temperature of 40 ° C and adjusted to a water content of 5 g of water per 1 kg of nitrogen. After 120 minutes, drying was completed and the product was emptied into a storage container. Analysis of the anhydrous crystals showed a residual water content of 6% and a residual ethanol content of less than 0.1%.

Example 4 In a crystallization vessel adapted with stirrer, 1000 g of porcine insulin were crystallized from an aqueous solution at pH 5.5 with the addition of zinc ions. When the crystallization was complete, the suspension (approximately 100 1) contained rhombohedral crystals with an average particle size of 30 μ. As described in the general section, the agitated crystal suspension was transferred to the centrifugal dryer for approximately 30 minutes. The filter cake was washed with approximately 50 1 of water and the wash water was replaced by aqueous propanol at 72% concentration (drying medium). After the filter cake had been separated from the screen, the product was dried in a fluidized bed. During the drying time, the drying gas was preheated to a temperature of 40 ° C and adjusted to a water content of 4 g of water per 1 kg of nitrogen. After 140 minutes, the drying was completed and the product was emptied into a container for storage. Analysis of the anhydrous crystals showed a residual water humidity of 5% and a residual propanol content of less than 0.1%.

Example 5 In a crystallization vessel adapted with stirrer, 1500 g of di-Arg-insulin were crystallized from an aqueous solution at pH 6.3 with the addition of zinc ions. After crystallization was complete, the suspension (approximately 100 1) contained crystals with an average particle size of 20 μ. As described in the general section, the agitated crystal suspension was transferred to a centrifugal dryer for about 30 minutes. The filter cake was washed with approximately 50 1 of water and the wash water was replaced by 25% aqueous propanol (drying medium). After the filter cake had been separated from the screen, the product was dried in a fluidized bed. During the drying time, the drying gas was preheated to a temperature of 40 ° C and adjusted to a water content of 3 g of water per 1 kg of nitrogen. After 160 minutes, the drying was complete and the product was emptied into a container for storage. Analysis of the anhydrous crystals showed a residual water moisture content of 6% and a residual propanol content of less than 0.1%.

Example 6 In a vessel for crystallization adapted with an agitator, 1000 g of human insulin were crystallized from an aqueous solution at pH 5.5 with the addition of zinc ions. When the crystallization was complete, the suspension (approximately 100 1) contained rhombohedral crystals with an average particle size of 25 μ. As described in the general section, the agitated crystal suspension was transferred to the centrifugal dryer for approximately 30 minutes. The filter cake was washed with approximately 50 1 of water, and the wash water was replaced by aqueous n-propanol at 15% concentration (drying medium). After the filter cake had been separated from the screen, the product was dried in a fluidized bed. During the drying time, the drying gas was preheated to a temperature of 40 ° C. The water content in the drying gas at the start of the drying time was adjusted to 15% and reduced to 8% during the drying time. After 120 minutes, drying was completed and the product was emptied into a container for storage. Analysis of the anhydrous crystals showed a residual water moisture content of 6% and a residual propanol content of less than 0.1%.

Claims (13)

1. A process for drying protein crystals starting from an aqueous suspension of protein crystals, which consists of drying the suspension of protein crystals in a centrifugal dryer.

2. A process for drying protein crystals starting from an aqueous suspension of protein crystals, preferably as claimed in claim 1, wherein the protein crystals, after they have been filtered from the protein crystal suspension, they are taken to a drying medium consisting of a mixture of water and a non-aqueous solvent miscible with water in any proportion and having a vapor pressure lower than water.

3. The process as claimed in claim 2, wherein the proportion of the solvent in the drying medium is from 10 to 80% inclusive.

4. The process as claimed in claim 3, wherein the proportion of the solvent in the drying medium is from 15 to 60% inclusive.

5. The process as claimed in one or more of claims 2 to 4, wherein the solvent is an alcohol or a mixture of alcohols.

6. The process as claimed in claim 5, wherein the solvent is methanol.

7. The process as claimed in claim 5 or 6, wherein the solvent is ethanol.

8. The process as claimed in one or more of claims 5 to 7, wherein the solvent is n-propanol.

9. The process as claimed in one or more of claims 5 to 8, wherein the solvent is isopropanol.

10. A process for drying protein crystals, preferably as claimed in one or more of claims 1 to 9, wherein the drying gas that is used has been moistened with water prior to the drying process.

11. The process as claimed in claim 10, wherein the drying gas is nitrogen.

12. A process for drying protein crystals starting from an aqueous suspension of protein crystals, preferably as claimed in one or more of claims 1 to 11, wherein the protein crystals, after they have been filtered from the suspension of protein crystals, becomes a fluidized bed for the purpose of drying.

13. The process as claimed in one or more of claims 1 to 12, wherein the protein crystals are animal insulin crystals, human insulin or an analog thereof.