METHOD FOR STERILIZING A CHROMATOGRAPHIC MATERIAL AND THEREFORE STERILIZED CHROMATOGRAPHIC MATERIAL

Description

[0001] The present invention relates to a method for sterilizing a chromatography material which is a microporous polymer membrane.

[0002] For applications in the biopharmaceutical industry, not only sterile end products for administration are of interest, but also sterile materials used in the production of active pharmaceutical ingredients. Materials here refer to components of the individual process steps as well as media. For example, in the production of monoclonal antibodies, a sterile fermentation vessel and sterile media are mandatory even during the fermentation step to ensure adequate growth of the cell lines. After cell growth and protein biosynthesis, the antibody must be separated from cells and other unwanted components in as pure a form as possible. A variety of process steps are involved for this purpose.After cell removal by depth filtration or centrifugation, this generally includes various chromatography steps for concentrating and purifying the target molecule, virus removal procedures, and concentration and sterile filtration steps. Sterile work is also advantageous during these process steps. This can minimize the risk of contamination of the final product.

[0003] A special case is the purification of larger molecules, such as viruses or virus-like particles: Sterile filtration during processing is often not possible. Due to the size of the molecule, the pore size of the sterile filters used would retain the product, making filtration impossible. Sterile processing solutions are a decisive improvement here.

[0004] As a standard, the risk of contamination is reduced in many of the process steps mentioned by rinsing with caustic or the use of heat. However, these methods have several disadvantages: the implementation in the process is often complex. Large quantities of caustic or even superheated steam must be provided. Time-consuming cleaning must be carried out before the actual process step. Another disadvantage is often the stability of the materials used in the individual process steps. For example, superheated steam cannot be used to sterilize chromatography media. Other materials do not exhibit sufficient stability towards caustic.

[0005] In recent years, the trend toward the use of "single-use" products in the production of biopharmaceutical active ingredients has increased. Single-use and the predominant use of plastics for materials such as filtration devices have paved the way for alternative sterilization methods. In addition to autoclaving, sterilization processes such as ethylene oxide fumigation or gamma irradiation are also used. A further advantage for users is the availability of pre-sterilized products for the manufacturing process. This reduces or completely eliminates cleaning efforts for the user.

[0006] Sterilization methods can generally be divided into chemical and physical processes. In chemical processes, the use of germicidal gases is common. However, with this method, it must be ensured that all areas of the material are sufficiently exposed to the gas during sterilization. Physical methods include thermal sterilization and irradiation. The sterilizing effect of irradiation is based on the destruction of organic material by bond cleavage. The advantage of irradiation is that it can penetrate all areas of a material.

[0007] Chromatography media are typically sanitized. Sanitization is a term used, among other things, for the treatment of bacteria with alkali. A solution of up to 1 molar is often used. However, the result of this treatment is not a sterile product or a sterile process step. A major disadvantage of this treatment method is its preparation. Either the sanitization effort must be performed immediately before use in the process, or the manufacturer of the chromatographic medium must sanitize the medium in suitable chromatography devices during production and deliver it in a wet state. This not only complicates handling but also requires complex studies on stability in storage solutions. Pre-sanitized products in chromatography are known as "ready-to-use" products.

[0008] Autoclaving can also be used for sterilization. The chromatography medium must be heated to at least 121°C. However, this method is unsuitable for larger scales because the size of the devices makes it impossible to implement.

[0009] Another described method is the use of hot buffers for sterilization. This also involves heating the chromatography medium to at least 121°C. However, this requires that the chromatography system is stable against thermal stress and pressure. This method is unsuitable for disposable systems.

[0010] US 2013 / 0062267 A1 describes a sterilization method for the preparation of aseptic chromatography columns. However, the medium and the filtration device (chromatography housing) must be sterilized separately, and the chromatography medium must then be introduced into the device using sterilized equipment. Furthermore, the method is only applicable to polymer gels.

[0011] While sterile disposable fermentation containers and filtration devices have already become established in the field of filtration and fermentation, ready-to-use sterilized products have not yet become established in chromatographic applications. One main reason for this is the stability of the materials.

[0012] Both the support matrix and the ligand can be damaged by exposure to heat, gamma irradiation, or other sterilization methods, or they can be difficult to access. Support matrices used in chromatography include polymer gels, membranes, nonwovens, films, or fibers. Ligands can include proteins, substrates, carboxymethyl, sulfonates, quaternary amines, aminoethyl, metal chelates, or hydrophobic groups. Irradiation of the materials can, for example, damage or detach the side chains of polymers. Treatment with oxidative media such as reactive ethylene oxide can also lead to a change in the ligands. State-of-the-art sterilization processes have been shown to alter material properties, as can be directly observed, for example, in discoloration of the chromatography material.Although such discolorations do not necessarily have to be negative in terms of product contamination in the process, they can nevertheless be undesirable, as described, for example, in US 2011 / 0064608 A1.

[0013] WO 2004 / 009143 A1 describes a method for sterilizing a biological material. WO 2015 / 109146 A1 describes sterile chromatography and manufacturing methods. WO 03 / 030949 A1 describes methods for sterilizing biological materials. WO 2015 / 109246 A1 describes the sterilization of chromatography columns. WO 2011 / 076386 A1 describes containers for chromatography media.

[0014] The present invention is therefore based on the object of providing a method for sterilizing a chromatography material which is a microporous polymer membrane, wherein the claimed method does not change the material properties of the chromatography material, so that the chromatography material can be used for sterile chromatography.

[0015] This object is achieved by the embodiments of the present invention characterized in the claims.

[0016] The invention is defined by the subject matter of claim 1, i.e. by a method for sterilizing a chromatography material, comprising the steps: (a) treating the chromatography material with an aqueous, basic buffered salt solution; and (c) irradiating the chromatography material treated in step (a) with gamma radiation; wherein the chromatography material is a microporous polymer membrane. According to a preferred embodiment, the process according to the invention further comprises step (b) between steps (a) and (c) and / or step (d) after step (c): (b) drying the chromatography material treated in step (a); (d) drying the chromatography material irradiated in step (c), wherein in step (c) the chromatography material dried in step (b) is irradiated with gamma radiation when step (b) is carried out.

[0017] For the purposes of the present invention, the term "chromatography material" refers to any material that can be used as a stationary phase in chromatographic processes. In the chromatographic process, a mixture of substances is separated by the stationary and mobile phases due to interactions between the individual components of the mixture.

[0018] The chromatography material in the process according to the invention is a microporous polymer membrane. The process according to the invention is particularly suitable for microporous polymer membranes whose outer and / or inner surface(s) have been modified by ligands, such as proteins, substrates, carboxymethyl, sulfonates, quaternary amines, aminoethyl, metal chelates, or hydrophobic groups, since the process according to the invention allows the chromatography material to be sterilized without changing the material properties with respect to the support matrix and ligand.

[0019] According to the present invention, the chromatography material to be sterilized is a (surface-modified) microporous polymer membrane. According to the invention, the term "microporous polymer membrane" refers to a polymer membrane with a pore size of 0.01 µm to 10 µm. According to the present invention, the membrane can comprise cellulose, cellulose derivatives, polyester, polyethylene (PE), polyamide, polyethersulfone (PES), polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene (PP), and polysulfone as structural components, whereby these materials can be used individually or in appropriate combinations.

[0020] Microporous polymer membranes for use in chromatographic applications are particularly suitable for incorporation into filtration devices (chromatography housings), for example, by forming a chromatographic bed from several serially arranged polymer membrane layers, as described in DE 197 11 168 A1. In such devices, also referred to as membrane adsorbers, at least one layer of a polymer membrane is present as a web material, with the at least one polymer membrane typically having a thickness of 50 µm to 350 µm. Typically, the at least one polymer membrane has an internal porosity of 50% to 90%.

[0021] In step (a) of the process according to the invention, the chromatography material to be sterilized is treated with an aqueous, basic-buffered saline solution to preserve the material for the irradiation step (c) of the process according to the invention. The chromatography material can be treated either immediately after its production, i.e., after production of the material and any surface modification during which appropriate ligands are bound to the matrix, or only after storage. The term "treatment" is not subject to any particular restriction according to the invention. For example, the chromatography material to be stabilized is mixed with an aqueous, basic-buffered saline solution and shaken, for example, for a period of approximately 10 minutes, to achieve complete wetting or complete exchange with the solution present in the pores.The treatment according to the present invention can be carried out at room or ambient temperature. According to the invention, the chromatography material to be treated is, for example, placed in the above-mentioned solution in a suitable vessel, which is then placed on a shaker that is rotated at 30 to 150 rpm. Alternatively, the treatment of the chromatography material according to the invention can also be carried out by stirring the solution in which the chromatography material is located. In a further embodiment, the chromatography material according to the present invention can also be passed through a tank in which the solution is optionally circulated.

[0022] Step (a) of the method according to the invention can be carried out both as described above with an isolated chromatography material, i.e. the chromatography material is not located in a (chromatography) housing, and with a chromatography material that is already installed in a (chromatography) housing, i.e. is in an installed state. According to the invention, the aqueous, basic-buffered salt solution is introduced into the housing as the treatment solution in such a way that the treatment objectives described above are achieved. For example, the treatment solution can flow through the housing with the installed chromatography material. Further explanations for the case in which the chromatography material is already installed in a housing are given below.

[0023] The aqueous, basic buffered saline solution used in step (a) is not subject to any particular restriction according to the invention.

[0024] Buffer solutions contain a mixture of a weak acid and its conjugate or corresponding base (or the respective salt), or a weak base and its conjugate or corresponding acid. Ampholytes (bifunctional molecules) can also serve as buffers. The factor determining the pH is the ratio or protolysis equilibrium of the buffer pair. According to the present invention, acid-base pairs with a pKa of greater than 4 are generally suitable.

[0025] The salt or buffer solution according to the present invention may comprise one or more salts, as long as the buffer solution has a pH greater than 7, preferably greater than 8.

[0026] According to the invention, water will be used as the solvent for the basic buffered saline solution. Mixtures are also possible, such as a mixture of water and glycerol. In principle, solvents with which corresponding salt or buffer solutions can be prepared according to the present invention are suitable, while the chromatography material to be treated is inert towards such solvents. According to a preferred embodiment, the saline solution in step (a) comprises at least one salt selected from the group consisting of sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen citrate, disodium hydrogen citrate, and trisodium citrate. According to a preferred embodiment of the present invention, the saline solution in step (a) comprises disodium hydrogen phosphate.The salt concentration of the salt solution in step (a) is preferably from 0.01 to 1 mol / L, particularly preferably from 0.02 to 0.1 mol / L. At a salt concentration of less than 0.01 mol / L, it cannot be ruled out that the treated chromatography material may be altered due to the effect of the radiation during subsequent irradiation. At a salt concentration of more than 1 mol / L, it also cannot be ruled out that the chromatography material may be damaged or subjected to undesired changes by the treatment solution itself.

[0027] According to a preferred embodiment, the aqueous, base-buffered saline solution in step (a) further comprises 10 to 40 wt.% glycerol. This is particularly advantageous when the chromatography material is a porous polymer membrane, as in the present invention, since in this case the glycerol protects the fine porous membrane structure during drying steps (b) and (d).

[0028] In step (c) of the process according to the invention, the chromatography material treated in step (a) is irradiated with gamma radiation in order to sterilize the chromatography material. Advantageously, the material properties of the chromatography material do not change due to the preservation step (a) of the process according to the invention as a result of the gamma irradiation, as is the case when untreated chromatography materials known in the art are irradiated, i.e. materials that have not undergone preservation step (a). According to the present invention, the irradiation is carried out, for example, using a cobalt source or with electron beams with an energy of 1 to 12 MeV. The material to be irradiated is transported around the radiation source, for example, on conveyor belts. The number of passes depends on the desired minimum dose, which is usually monitored.According to the invention, such irradiation can be carried out in ambient air or under a protective atmosphere. According to a preferred embodiment, irradiation in step (c) is carried out with a gamma radiation dose of 1 kGy to 100 kGy. At a radiation dose of less than 1 kGy, there is a risk that the chromatography material will not be sufficiently sterilized. At a radiation dose of more than 100 kGy, the irradiated materials may be damaged, in particular they may lose their strength and become brittle. Particularly preferably, irradiation in step (c) is carried out with a radiation dose of 15 kGy to 85 kGy, in particular 30 kGy to 70 kGy.

[0029] As already stated above, the process according to the invention further preferably comprises step (b) between steps (a) and (c) and / or step (d) after step (c): (b) drying the chromatography material treated in step (a); (d) drying the chromatography material irradiated in step (c), wherein in step (c) the chromatography material dried in step (b) is irradiated with gamma radiation when step (b) is carried out.

[0030] In step (b) and / or step (d) of the process according to the invention, the chromatography material treated in step (a) or the chromatography material irradiated in step (c) is dried. The term "drying" is not subject to any particular restriction according to the invention. For example, drying in step (b) and / or step (d) can take place at room temperature or at elevated temperature, for example, in an oven. Additionally or instead, drying according to the present invention can be carried out by blowing dry inert gases onto or through the chromatography materials treated in step (a) and / or the chromatography materials irradiated in step (c). According to a preferred embodiment of the present invention, the chromatography material is dried in step (b) and / or step (d) at a temperature of 70°C to 90°C for a period of 5 to 20 minutes.

[0031] If the aqueous, base-buffered saline solution comprises glycerol as described above in a preferred embodiment, this can cause residual moisture after drying. According to the invention, this can remain in the treated chromatography material. Furthermore, according to the present invention, such residual moisture can be further reduced or removed by carrying out both step (b) and step (d).

[0032] According to one embodiment of the process according to the invention, the chromatography material is enclosed in a sterile, irradiable package after step (a) or after step (b) and then irradiated with gamma radiation in this package. This allows the chromatography material sterilized by gamma irradiation to be advantageously stored and transported in a sterile manner without the risk of subsequent contamination after irradiation. Such sterile, irradiable packages are known to those skilled in the art and are commercially available, for example, under the registered trademark Tyvek® from DuPont.

[0033] For example, a final product, e.g., a closed system with sterile connectors, is placed in appropriate packaging, such as a bag and cardboard box, and then irradiated in this package. According to the present invention, both individual cardboard boxes and their arrangements on pallets can be subjected to irradiation.

[0034] According to a further embodiment of the present invention, the chromatography material is already installed in a housing, at least when performing step (c) of the method. According to a preferred embodiment of the invention, the housing can already be incorporated into an irradiable package.

[0035] Furthermore, according to the invention, the chromatography material can also be installed in a housing during the entire process, i.e., during the implementation of steps (a) (as already described above), optionally (b), (c), and optionally (d). Either drying of the chromatography material is not necessary for its final application, or the design of the housing allows for steps (b) and / or (d) to be carried out, for example, by applying heat and / or blowing or blowing through appropriate gases.

[0036] Furthermore, a chromatography material sterilized by the method according to the invention is described here. The chromatography material sterilized according to the invention can advantageously be used for the chromatographic removal of contaminants from a fluid under sterile conditions. Such chromatographic removal methods are known to those skilled in the art.

[0037] Furthermore, a closed chromatographic filtration system is described herein, comprising a sterile filtration device containing a chromatography material sterilized according to the invention, assembled sterile tubing, and sterile connections. Such filtration systems without the chromatography material sterilized according to the invention are known to those skilled in the art.

[0038] The method of the present invention advantageously enables the sterilization of a chromatography material, which is a (surface-modified) microporous polymer membrane, without altering the material properties of the chromatography material. The chromatography material sterilized according to the invention can advantageously be stored after sterilization without discoloration. Since the adsorption capacity of the chromatography material is advantageously not altered by the sterilization according to the invention, the chromatography material sterilized according to the invention can be used in particular for the chromatographic removal of contaminants from a fluid under sterile conditions.

[0039] The present invention is further illustrated by the following non-limiting examples. Examples Comparison example:

[0040] Water-wetted membrane adsorbers of types 1 to 3 were treated with an aqueous glycerol solution containing 30 wt% glycerol while shaking for 10 min at room temperature and then dried for 10 minutes at 80°C in a drying oven. Type 1:MA Sartobind ®< S from Sartorius Stedim Biotech GmbH, commercially available Type 2:MA Sartobind ®< HIC Phenyl from Sartorius Stedim Biotech GmbH, commercially available Type 3:Membrane, manufactured according to DE 10 2013 017 014 from Sartorius Stedim Biotech GmbH Sterilization method according to the invention:

[0041] The water-wetted membrane adsorbers of types 1 to 3 were treated as in the comparative example, whereby the glycerol solution with a proportion of 30 wt.% glycerol additionally contained 1 wt.% of a salt (sodium acetate trihydrate or disodium hydrogen phosphate dihydrate or trisodium citrate trihydrate). Example 1: Discoloration during sterilization of membrane adsorbers

[0042] To determine the discoloration after gamma irradiation with a dose of at least 50 kGy (minimum measured surface dose 50.7 kGy, maximum measured surface dose 55.8 kGy), rectangular membrane adsorber blanks were subjected to a stability study. For this purpose, membrane blanks from membranes treated with an aqueous, alkaline-buffered saline solution (designated in the figures: "acetate," "phosphate," "sodium citrate") and from membranes not subjected to the inventive treatment step (designated in the figures: "standard") were subjected to accelerated aging after irradiation under controlled conditions (40°C ± 2°C, 75% ± 5% relative humidity). Experience has shown that 100 days of storage under the selected accelerated aging conditions corresponds to approximately 1.5 years of storage under normal conditions.Under the same accelerated aging conditions, a membrane blank was also aged which was first sterilized using ethylene oxide (designation in the figures: "EtO"), ie first treated with ethylene oxide and then exposed to the gamma irradiation described above.

[0043] After rinsing, the cut pieces were sealed in gamma-stable plastic bags and stored. Membrane samples were taken and analyzed at defined intervals (0, 100, and 200 days).

[0044] Figure 1 shows the optical appearance of the respective membranes after 0, 100 and 200 days.

[0045] The aging study shows that over a period of at least 200 days, the optical appearance of the membrane adsorbers sterilized according to the invention advantageously remains stable, in contrast to a sterilized membrane adsorber without prior application of the treatment step (a) according to the invention, ie no changes in the material properties in the form of discoloration have occurred. Example 2: Influence on the adsorption capacity

[0046] Using the method according to the invention, a cation exchange membrane based on stabilized cellulose of the type MA Sartobind ®< S from Sartorius Stedim Biotech GmbH, commercially available, was rinsed and sterilized under the conditions listed above (see "Sterilization Method According to the Invention" and "Exemplary Example 1").

[0047] The binding capacity was then determined as follows: 1 g / L lysozyme was dissolved in phosphate buffer (buffer components potassium hydrogen phosphate and dipotassium hydrogen phosphate) with a molarity of 10 mM and a pH of 7.0. Three 5 cm² layers of the membrane adsorber were installed in a filtration housing and connected to a chromatography system (Äkta Explorer, GE Healthcare). Before loading with the protein solution, the adsorber was equilibrated with 20 mL of a 10 mM phosphate buffer (pH 7) at 10 mL / min.

[0048] Dynamic capacity was determined by measuring absorbance at 280 nm using a UV-VIS photometer on the Äkta Explorer chromatography system from GE Healthcare. The concentration was calculated from the measured absorbance using the Beer-Lambert law. The extinction coefficient for lysozyme was determined to be 2.47*10 -3 < L / (g*cm).

[0049] The binding capacity was determined for the time point at which 10% of the concentration of the starting solution was measured at the outlet of the filtration housing. Kap 10 % = V 10 % − V tot * E AL ε * d * A Membran Cap 10% :Dynamic capacity 10% [mg / cm 2 ] V 10% :Loaded volume of the starting solution at breakthrough 10% of the concentration of the starting solution [L] V tot :Dead volume of the chromatography system [L] E(AL):Extinction of the starting solution ε:Specific extinction coefficient = 2.47 * 10 -3 [L / (mg*cm)] d:Path thickness UV measurement = 1 [cm] A Membran :Membrane area [cm 2 ]

[0050] Figure 2 shows the effect of the process steps according to the invention on the relevant adsorption properties of the membrane.

[0051] As is clear from Figure 2As can be seen, the process according to the invention (irradiation with a dose of at least 50 kGy after the rinsing step according to the invention) advantageously does not significantly impair the adsorption capacity of the membrane compared to non-sterilized membrane adsorbers, i.e., the sterilization process according to the invention advantageously does not alter the material properties of the chromatography material. Membranes irradiated without the rinsing step according to the invention, in contrast, show a decrease in adsorption capacity due to changes in the material properties.

[0052] The figures show: Figure 1: Visual appearance of membranes with and without the inventive sterilization process after defined aging periods (0, 100, and 200 days). Figure 2: Comparison of the adsorption properties of membranes with and without the inventive sterilization process.

Claims

1. Method for sterilizing a chromatography material, comprising the steps: (a) treating the chromatography material with an aqueous, alkaline-buffered salt solution; and (c) irradiating the chromatography material treated in step (a) with gamma radiation; wherein the chromatography material is a microporous polymer membrane.

2. Method according to claim 1, further comprising step (b) between steps (a) and (c) and / or step (d) after step (c): (b) drying the chromatography material treated in step (a); (d) drying the chromatography material irradiated in step (c) ; wherein in step (c) the chromatography material dried in step (b) is irradiated with gamma radiation.

3. Method according to claim 1 or 2, wherein the microporous polymer membrane has a thickness of 50 to 350 µm.

4. Method according to any one of claims 1 to 3, wherein the aqueous, alkaline-buffered salt solution in step (a) comprises at least one salt selected from the group consisting of sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen citrate, disodium hydrogen citrate, and trisodium citrate.

5. Method according to any one of claims 1 to 4, wherein the salt concentration of the salt solution in step (a) is from 0.01 to 1 mol / l.

6. Method according to any one of claims 1 to 5, wherein the aqueous, alkaline-buffered salt solution in step (a) further comprises 10 to 40 wt% glycerol.

7. Method according to any one of claims 2 to 6, wherein the chromatography material in step (b) and / or in step (d) is dried in a temperature range of 70°C to 90°C for a duration of 5 min to 20 min.

8. Method according to any one of claims 1 to 7, wherein in step (c) irradiation takes place using a gamma radiation dose of 1 kGy to 100 kGy.

9. Method according to any one of claims 1 to 8, wherein the chromatography material is situated in an integrated state in a housing when carrying out the method.

10. Method according to any one of claims 1 to 8, wherein the chromatography material in step (c) is situated in an irradiatable packaging.

11. Method according to claim 9, wherein the housing containing the integrated chromatography material in step (c) is situated in an irradiatable packaging.