Method for sterile filtration of albumin-containing aqueous solution

Abstract

The present invention relates to a method for the sterile filtration of an aqueous albumin-containing solution (2), where the method comprises filtering the solution (2) with at least one filter (3) to obtain a filtrate (1), where the filtrate (1) is filled in at least one container (10, 10 ') before the filtrate (1) is filled in the at least one container (10, 10'), the at least one container (10, 10 ') is filled in the at least one container (10, 10'). The at least one filter (3) is conditioned by washing the at least one filter (3) with an amount (5) of the albumin-containing aqueous solution (2) under the following conditions: i) at a differential pressure across the respective filter (3) of at most 1 bar and / or ii) by means of pressure filtration, wherein the filtrate (1) is maintained at a back pressure of at least 0.5 bar in the outlet (4) of the respective filter (3).

Description

[0001] This invention relates to a method for sterile filtration of an aqueous albumin-containing solution, and an albumin solution obtained by the method disclosed herein. The invention further discloses the use of a regulated filter to reduce the opalescence of the filtered aqueous albumin-containing solution.

[0002] Various types of albumin are important as biochemical substances, such as as components of culture media and cell culture media, and as stabilizers for diagnostic agents in biology, medicine, and pharmacy.

[0003] In the manufacture of human albumin aqueous solution, an essential step is final pasteurization of the product at 60°C for 10 hours. This heating step is introduced to inactivate infectious agents and eliminate or reduce the risk of viral transmission (such as hepatitis B virus), which can occur during various purification steps due to possible contamination of the biological raw materials. Currently, all national and international pharmacopoeias require pasteurization of the albumin solution at 60°C for 10 hours. This pasteurization must be carried out in the absolute final stage of manufacturing, i.e., in the final container, which is typically a vial made of Type I pharmaceutical borosilicate glass (also known as "neutral" glass).

[0004] Although albumin is relatively stable with respect to heat, it must be protected from gelling during pasteurization with the help of suitable stabilizers. Currently, sodium caprylate and sodium acetyltryptophan are commonly used as stabilizers, either alone or in combination.

[0005] Sodium mandelate can be used alone or in combination with sodium caprylate. The amount of stabilizer added is excessive relative to albumin, and due to their own affinity for albumin, they effectively protect it from direct heat denaturation. Insufficient stabilization leads to albumin denaturation, and thus to the gradual aggregation of albumin molecules and any other heat-sensitive impurities present, resulting in the formation of particles with high molecular weights. These particles cause the opalescent appearance of the albumin aqueous solution due to light scattering; this property is commonly referred to as opalescence. Opalescence refers to the optical effect produced by light scattering in a milky white, turbid material, characterized by the emission of colored luminescence. According to the European Pharmacopoeia, opalescence is classified as 0 to IV with 0, 3, 6, 18, or 30 scattering turbidity units (NTU) (European Pharmacopoeia 2016, EP9, Section 2.2.1: "Clarity and degree of opalescence in liquids").

[0006] However, stability studies have shown that even when using the aforementioned stabilizers, opalescence may occur during or after pasteurization when the albumin-containing aqueous solution is filtered prior to pasteurization. This opalescence is clearly visible in the Seidenader semi-automatic visual inspection system and is also visible to the naked eye when vials containing filtered albumin solution are placed in front of indirect backlighting. Opacity is observed in both the dilutest albumin solutions (4% or 5% protein content) and the most concentrated solutions (20% or 25% protein content). Furthermore, it has been observed that vials exhibiting opalescence develop white ring-shaped deposits on the inside of the vial at the liquid-air interface after several months, approximately 2-3 months of storage. Although the appearance of these white ring-shaped deposits is associated with a reduction in the opalescence of the albumin aqueous solution, this is unacceptable according to national and international pharmacopoeias.

[0007] US 5,118,794 discloses a method for stabilizing a human albumin solution for therapeutic purposes, which involves heat-treating it in a container, wherein the method includes adding a surfactant in addition to the usual stabilizing formulation. However, challenges associated with surfactants have been encountered, such as impurities, degradation, and potentially adverse immune responses.

[0008] More generally, WO 2008 / 045563 relates to a method for reducing opalescence in protein solutions and a composition of concentrated protein solutions for reducing opalescence. The method involves altering the ionic strength of the solution such that the milky appearance and / or the amount of higher molecular weight particles in the protein solution are reduced and / or eliminated, for example, by reducing the concentration of salts present in the solution. Furthermore, this document does not contain information regarding reducing opalescence in albumin-containing solutions.

[0009] The similarity between milky white solutions and aggregated protein solutions has raised concerns about the loss of protein activity in drug formulations and the potential for immunogenicity (CLELAND, JL, The Development of Stable Protein Formulations: A Close Look at Protein Aggregation, Deamidation, and Oxidation, Crit. Rev. Therapeutic Drug Carrier Systems, 1993, Vol. 10, No. 4, pp. 307-377). Since the presence of milky white and / or deposits during pasteurization indicates some degree of product denaturation and raises doubts about its tolerability and efficacy when administered to patients, the detection of milky white and / or deposits necessitates the rejection of relevant vials during final quality control checks, resulting in economic losses.

[0010] The object of this invention is to overcome these and other shortcomings of the prior art, and in particular to provide a method for sterile filtration of aqueous solutions containing albumin, especially aqueous solutions containing human serum albumin components. Another object of this invention is to provide safe and stable albumin solutions, especially albumin solutions containing human serum albumin components, wherein opalescence associated with particle formation is eliminated or at least reduced. Finally, another object of this invention is to provide a method for conditioning a filter, which can be used to reduce opalescence in filtered aqueous solutions containing albumin.

[0011] These objectives are addressed by the independent claims, wherein the dependent claims relate to preferred embodiments.

[0012] Therefore, in one aspect, the present invention is characterized by a method for sterile filtration of an aqueous solution containing albumin, particularly an aqueous solution containing a portion of human serum albumin. The method comprises filtering the solution with at least one filter to obtain a filtrate. The method is further characterized by comprising, prior to filling the filtrate into at least one container, rinsing the at least one filter with a certain amount of the albumin-containing aqueous solution, conditioning the filter under a pressure difference of up to 1 bar across the respective filter, and / or by means of pressure filtration, wherein the filtrate is maintained at a back pressure of at least 0.5 bar (0.05 MPa) at the outlet of the respective filter.

[0013] In the context of this invention, sterile filtration is a process for removing microorganisms (such as bacteria and viruses) from a liquid or gas, ensuring that the filtered medium is substantially free of microbial contaminants, for example, meeting sterility requirements at a level specified using standard test methods as defined in the current version of default standards (such as the European Pharmacopeia or the United States Pharmacopeia), and that the filtered medium is suitable for sterile applications.

[0014] Preferably, in the context of this invention, cleaning means wetting the filter, for example by rinsing.

[0015] As described in more detail below, visual inspection performed on a visual inspection device has shown that the filtered albumin solution thus obtained is clear and has at least one grade lower opalescence compared to the filtrate obtained without using the conditioned filter described herein. Specifically, the filtered albumin solution thus obtained is clear and has at most grade I opalescence, which is classified as having 3 scattering turbidity units (NTU).

[0016] To better understand this invention, certain terms are first defined. Further definitions can be found in the detailed description.

[0017] In the context of this invention, the term "albumin-containing aqueous solution" refers to any composition comprising water and at least albumin as the main protein component. More specifically, the term "albumin-containing aqueous solution" refers to any aqueous composition wherein albumin constitutes more than 80% of all proteins in the composition and is intended for clinical treatment. As used in the context of this invention, the albumin-containing aqueous solution is specifically defined in the European Pharmacopoeia as "human albumin solution".

[0018] The human albumin, the subject of this invention, is obtained, in particular, by extraction and purification from a human albumin source by any suitable method, or it can also be obtained by culturing animal or plant cell cultures, bacteria, or yeast that have been transformed to produce human albumin by genetic engineering methods known to those skilled in the art.

[0019] The at least one filter used in the methods described herein is a sterile filter. The sterile filter may be a double-layer sterile filter, which is equipped with two membranes of the same or different pore sizes. In the context of this invention, a sterile filter is a filter for removing microorganisms (such as bacteria and viruses) from a liquid or gas, ensuring that the filtered medium remains substantially free of microbial contaminants, for example, at a level that meets sterility requirements as measured using standard test methods specified in the current version of a default standard (such as the European Pharmacopoeia or the United States Pharmacopoeia), and that the filtered medium is suitable for sterile applications. Other terms for 'sterile filter' include 'sterilized filter'.

[0020] "Before filling the filtrate into at least one container" means transferring only the filtrate obtained by filtering the albumin-containing aqueous solution through a conditioned filter into one or more containers. In other words, according to the invention, the filling process begins only after conditioning at least one filter as described herein, such that the filtrate in all containers has advantageous properties in terms of long-term stability, and specifically, reduced opalescence.

[0021] “NTU” stands for “Natural Turbidity Unit”, and it is a unit used to measure the turbidity of liquids or the presence of suspended solids in a liquid. The higher the concentration of suspended solids in a liquid, the higher the turbidity. Stabilized formalin turbidity standards for opalination are available from Hach Lange GmbH (Switzerland), for example, under the trade name StablCal®. The relationship between NTU and suspended solids is as follows: 1 mg / L (ppm) corresponds to 3 NTU. Turbidity is measured by 90° light scattering.

[0022] The inventors of this invention have discovered that filtration prior to pasteurization has a decisive influence on particle formation, which results in opalescence during and / or after pasteurization, as described in Example 1 below. This is surprising because filtration studies conducted by the inventors confirmed that the size of the particles causing opalescence ranges between 450 nm and 800 nm. However, such particles should have already been separated by the sterile filter used during filtration. Even more surprisingly, particles in the 450 nm to 800 nm range were detected in both milky and non-milky samples. It has now been surprisingly discovered that it is the combination of filtration and pasteurization that causes opalescence, where aggregates causing opalescence are formed after filtration during and / or after pasteurization. By conditioning at least one sterile filter with albumin before filling the filtrate into the final container (e.g., vial), the number and size of these particles can be significantly reduced. Thus, the appearance of opalescence in the filtered albumin solution (hereinafter also referred to as "filtrate") has been suppressed or at least reduced. Furthermore, the method disclosed herein can also avoid the formation of white deposits that can be observed at the liquid-air interface after a period of time. This means that the method disclosed herein can be used to package and therefore produce filtered albumin solutions more efficiently, since fewer vials must be sorted out during quality control.

[0023] In an embodiment, at least one filter is conditioned by rinsing it with a certain amount of albumin-containing aqueous solution using pressure filtration, and maintaining the filtrate at the outlet of the respective filter at a back pressure of about 1 bar. At this back pressure, relatively rapid and complete saturation of the filter with albumin can be achieved without subjecting the filter and / or filtrate to excessive stress that could damage the filter or render the filtered albumin solution (i.e., the product (filtrate)) unusable.

[0024] In other embodiments, at least one filter is regulated by rinsing it with a certain amount of albumin-containing aqueous solution using pressure filtration, wherein the inlet pressure at the inlet of the respective filter is about 2 bar, and the back pressure of the filtrate at the outlet of the respective filter is maintained at about 1 bar.

[0025] In embodiments, the amount of albumin-containing aqueous solution used to clean at least one filter is between about 2.5 liters and about 11 liters per square meter of the filter's filtration area. In some embodiments, the amount of albumin-containing aqueous solution used to clean at least one filter is about 10 liters per square meter of the filter's filtration area. This volume ensures good cleaning and saturation of the corresponding filter by albumin.

[0026] In this embodiment, the filtrate is recycled after passing through at least one filter. In this way, the consumption of albumin-containing aqueous solutions can be reduced, and the efficiency of the method can be further improved.

[0027] In an embodiment where at least one filter is conditioned by flushing it with a certain amount of albumin-containing aqueous solution at a pressure difference of up to 1 bar across the respective filter, no back pressure is applied at the outlet of the respective filter. Under these conditions, it was surprisingly found that the albumin solution of the filtrate has particularly low opalescence, for example, opalescence grade I is classified as 3 NTU, if any.

[0028] In an embodiment where at least one filter is conditioned by rinsing it with a certain amount of albumin-containing aqueous solution at a pressure difference of up to 1 bar across the respective filter, the rinsing with the albumin-containing aqueous solution is performed at a flow rate of approximately 2 to approximately 4 liters per minute per square meter of filtration area of ​​the at least one filter. Preferably, at least one filter is rinsed with the albumin-containing aqueous solution at a flow rate of approximately 3 liters per minute per square meter of filtration surface area of ​​the at least one filter.

[0029] In an embodiment, at least one filter is rinsed with ultrapure water (infusion water) before rinsing the corresponding filter with a certain amount of albumin-containing aqueous solution. Preferably, the volume of ultrapure water used to rinse at least one filter is about 1 liter to about 11 liters per square meter of the filtration area of ​​at least one filter. Preferably, the volume of ultrapure water used to rinse at least one filter is about 1 liter to 10 liters per square meter of the filtration area of ​​at least one filter. Such an amount ensures good rinsing of the corresponding filter.

[0030] In an embodiment, the method further includes a pasteurization step of the filtrate. Specifically, the filtrate is pasteurized at 60°C for 10 hours after filtration. Under these conditions, despite upstream safety precautions, bacteria and viruses that have entered or are present in the filtered albumin solution are reliably inactivated and rendered harmless.

[0031] Preferably, the filtrate is pasteurized in at least one container. This eliminates the risk of recontamination of the filtered albumin solution due to retransfer.

[0032] In one embodiment, the filtrate is continuously filled into sterile vials, which are then sealed with stoppers.

[0033] In an embodiment, the method further includes the steps of pasteurizing the filtrate as described herein and incubating the pasteurized filtrate to obtain an incubated filtrate. Specifically, the step of incubating the pasteurized filtrate is performed for a period of approximately 10 to 20 days, preferably for approximately 15 to 20 days. Preferably, the step of incubating the pasteurized filtrate is performed for 15 or 16 days. The incubated filtrate can be stored. For example, the storage can last for a predetermined period of time.

[0034] In one embodiment, the step of incubating the pasteurized filtrate is carried out at a temperature of about 25°C to about 35°C, preferably at a temperature of about 29°C to about 32°C.

[0035] In an embodiment where at least one filter is regulated by means of a certain amount of albumin-containing aqueous solution being washed with pressure filtration and the filtrate at the outlet of the respective filter being maintained at a back pressure of at least 0.5 bar, with the back pressure being monitored at least during the regulation of the respective filter.

[0036] In an embodiment where at least one filter is conditioned by rinsing it with a certain amount of albumin-containing aqueous solution via pressure filtration and the filtrate in the outlet of the respective filter is maintained at a back pressure of at least 0.5 bar, the rinsing of the respective filter with the certain amount of albumin-containing aqueous solution is carried out at a flow rate of about 1 to about 3 liters per minute per square meter of filtration area of ​​at least one filter of the respective filter.

[0037] Preferably, the corresponding filter is cleaned with a certain amount of albumin-containing aqueous solution at a flow rate of approximately 2 liters per minute per square meter of filter surface area of ​​at least one of the corresponding filters.

[0038] In the examples, albumin is selected from human serum albumin, bovine serum albumin, ovalbumin and recombinant human serum albumin.

[0039] In this embodiment, the albumin concentration in the albumin-containing aqueous solution and / or filtrate is between 19% and 30% based on the total weight of the albumin-containing aqueous solution and / or filtrate. This is achieved before and / or after filtration. Alternatively, the albumin concentration in the albumin-containing aqueous solution and / or filtrate is between 3% and 6% based on the total weight of the albumin-containing aqueous solution and / or filtrate. Again, this is achieved before and / or after filtration.

[0040] In an embodiment, the method further includes the steps of pasteurizing the filtrate to obtain a pasteurized filtrate and evaluating the opalescence of the pasteurized filtrate. Preferably, the opalescence of the filtrate is evaluated by measuring turbidity or assessing changes in the formation of higher molecular weight particles.

[0041] The term "higher molecular weight particle" refers to the association of at least two molecules. In some embodiments, the molecules are lipids and / or proteins, such as albumin, where protein association results in the formation of higher-order aggregates of the monomeric protein. Association can occur through non-covalent (e.g., electrostatic, van der Waals) protein-protein interactions. The proteins can be the same or different. The molecular weight of higher molecular weight particles is typically around 10. 4 Da or higher, typically around 10 6 Da or higher. The weight-average molecular weight of aggregated molecules in solution can be detected, for example, by one or more of the following methods: light scattering techniques, such as static light scattering and / or dynamic light scattering, or asymmetric flow field flux fractionation. Details of the above measurement methods will be described in more detail below.

[0042] In addition to evaluating the opalescence of the pasteurized filtrate, the opalescence of the pasteurized and incubated sample can also be determined. Therefore, in the embodiments, the method further includes, in particular, the steps of pasteurizing the filtrate to obtain a pasteurized filtrate, incubating the pasteurized filtrate to obtain an incubated filtrate, and evaluating the opalescence of the incubated filtrate.

[0043] Specifically, the luminescence can be assessed immediately after pasteurization and / or incubation. However, the assessment of the luminescence of the pasteurized and optionally incubated filtrate can also be performed after the corresponding filtrate has been stored for approximately one day to several years, particularly approximately five years. Preferably, the assessment of the luminescence of the pasteurized and optionally incubated filtrate is performed approximately 1 to 6 months after storage of the corresponding filtrate, and more preferably approximately 1 to 3 months after storage of the corresponding filtrate.

[0044] The opalescence of the incubated filtrate can be evaluated at the incubation temperature, specifically at a temperature of about 29°C to about 31°C. Alternatively or additionally, the opalescence can also be evaluated at the storage temperature of the incubated filtrate. This temperature can vary depending on the product in question and can, for example, be between about 2°C and about 8°C or ambient temperature. It is also conceivable that the opalescence of the incubated filtrate can be evaluated neither at the incubation temperature nor the storage temperature, but at room temperature in a laboratory setting. The term "room temperature" is as understood by those skilled in the art and can specifically mean a temperature of about 20°C.

[0045] Therefore, in embodiments where the method further includes a step of evaluating the opalescence of the incubated filtrate, the opalescence of the incubated filtrate is evaluated at a temperature of ≤ 35°C. Preferably, the opalescence of the incubated filtrate is evaluated at a temperature of ≤ 30°C, more preferably at a temperature of ≤ 25°C.

[0046] In some cases, the opalescence of the incubated filtrate is evaluated at a temperature of approximately 20°C.

[0047] In the embodiments, the opalescence of the incubated filtrate was evaluated at a temperature of about 2°C to about 8°C.

[0048] In the embodiments, the opalescence of the filtrate and / or the incubated filtrate is evaluated by one or more of the following methods: visual inspection, dynamic light scattering, and tunable resistive pulse sensing (TRPS).

[0049] In embodiments where the method further includes a step of evaluating the opalescence of the filtrate and / or the incubated filtrate, the opalescence of the filtrate and / or the incubated filtrate is reduced by at least one grade compared to the filtrate of the same albumin-containing aqueous solution that has not yet been filtered through a conditioned filter. Preferably, the pasteurized filtrate and / or the incubated filtrate has at most Class I opalescence, which is classified as 3 NTU. Even more preferably, when evaluated 2 months after filtration, the filtrate and / or the incubated filtrate exhibits at most Class I opalescence, which is classified as 3 NTU.

[0050] In one embodiment, an aqueous solution containing albumin is applied to at least one filter at a pressure of approximately 1 bar. At this pressure, good throughput can be achieved without overloading the filter or the aqueous solution containing albumin.

[0051] In this embodiment, at least one filter has a filtration area of ​​approximately 0.01 m². 2 up to approximately 0.8 m 2 Preferably, at least one filter has a filtration area of ​​approximately 0.4 m². 2 Utilizing this filtration surface area, relatively large volumes, such as those encountered in the production of filtered albumin solutions in the pharmaceutical industry, can be filtered within an acceptable timeframe.

[0052] Preferably, at least one filter has a pore size of approximately 0.22 µm or less. If at least one filter is a dual-layer filter equipped with two membranes, the membrane first arranged in the flow direction of the filter can have a larger pore size than the membrane arranged downstream in the flow direction (i.e., the membrane of the sterile filter). Specifically, the pore size of the first membrane arranged in the flow direction of the dual-layer filter can be approximately 0.45 µm or less, and the pore size of the other membrane arranged downstream in the flow direction of the dual-layer filter (i.e., the membrane of the sterile filter) can be approximately 0.22 µm or less. Filters or membranes with this pore size are generally readily available on the market. Specifically, the pore size of the filter can be 0.22 µm or 0.20 µm. Membranes with this pore size can achieve the desired sterilization effect without unduly restricting the flow rate through the filter.

[0053] In this embodiment, at least one filter is a polyethersulfone (PES) membrane filter or a nylon filter. Such filters are suitable for use in the pharmaceutical industry and are generally readily available.

[0054] In an embodiment, prior to filtering the albumin-containing aqueous solution with at least one filter, the method further includes the step of filtering the albumin-containing aqueous solution with a pre-filter, wherein the pre-filter has a pore size of about 0.45 µm or less.

[0055] The amount of stabilizer added to the albumin-containing aqueous solution is proportional to the albumin concentration of the solution in question. In an example, the albumin-containing aqueous solution is stabilized with at least one stabilizer selected from sodium N-acetyltryptophan and sodium octanoate at approximately 0.08 mM per gram of albumin. This amount effectively protects albumin molecules from heat denaturation during pasteurization and reduces aggregate formation, thereby achieving stability over a desired period of time (specifically within the shelf life of the final albumin product, e.g., at least one month, preferably at least three months, more preferably at least six months, most preferably one year or longer).

[0056] In another aspect, the present invention relates to a filtered albumin solution obtained by the aseptic filtration method disclosed herein, and more particularly to a pharmaceutical composition comprising the filtered albumin solution. This albumin solution is characterized by lower opalescence compared to similar albumin solutions that have been filtered through one or more filters but have not been conditioned as described herein.

[0057] In another aspect, the present invention relates to the use of a filter that has been conditioned in order to reduce the opalescence of a filtered albumin-containing aqueous solution by rinsing with a certain amount of albumin-containing aqueous solution at a pressure difference of up to 1 bar across the filter or by pressure filtration, and by maintaining the filtrate at the filter outlet at a back pressure of at least 0.5 bar.

[0058] Some embodiments of the invention are described in more detail with reference to the accompanying drawings, wherein the same reference numerals are used to denote the same or corresponding elements. The various views and illustrations of the embodiments shown in the drawings are schematic representations:

[0059] Figure 1 : Schematic diagram of a visual inspection box used for testing opalescence;

[0060] Figure 2 : Schematic diagram of a sterile filtration device;

[0061] Figure 3 Summary of the results of the visual inspection in Examples 1-3;

[0062] Figure 4Images of the opalescence from vials of Examples 1-3 are shown;

[0063] Figure 5 Images of white deposition rings from vials of Examples 1-3 are shown.

[0064] Figure 6 Summary of the optical density results in Examples 1-3;

[0065] Figure 7 Summary of turbidity results in Examples 1-3;

[0066] Figure 8 : A schematic diagram of filtration using applied back pressure;

[0067] Figure 9 : A schematic diagram of filtration using pressure reduction.

[0068] Instrumental Methods

[0069] The opalescence of the samples is examined and the degree of opalescence is determined using a vision control box 20 with a black inner wall 21 and a white light source 22 mounted on the top of the box. Sample containers 10 and 10' are placed in front of the slit 23 in the box, exposing them to indirect light, such as... Figure 1 As illustrated in the diagram. According to the European Pharmacopoeia 11.2, 2023, “2.2.1 Transparency and opalescence of liquids”, opalescence is classified into classes 0-IV (corresponding to 0, 3, 6, 18 and 30 NTU, respectively).

[0070] Dynamic light scattering (DLS) was measured using Malvern Zetasizer nano zs to determine the particle size distribution (non-quantitative), mean Z-Ave, and optical density (mean derived count rate (DCR), in kcps) in the size range of 0.4–7000 nm. Z-Ave is the harmonic mean of the intensity-weighted hydrodynamic diameter, previously used as a reference value for evaluating albumin opalescence. The mean derived count rate (DCR) provides an indication of the number of particles. Undiluted, milky, and transparent filtered albumin solutions were measured at 20 °C in forward (12.8) and backward (173) scattering modes.

[0071] Turbidity was determined by 90° light scattering. The results are given in scattering turbidity units (NTU).

[0072] Production of white starting material

[0073] To provide starting material under controlled conditions leading to significant opalescence and the formation of white precipitate rings following aseptic filtration as known in the art, 20% albumin permeate prepared by Kistler / Nitschmann precipitate C was concentrated and reconstituted with stabilizers tryptophan and caprylic acid (20 mM each) to a 25% albumin solution. This large volume of albumin solution was aerated by blowing compressed air directly into the solution through a nozzle to create small bubbles. The flow rate was adjusted to ensure continuous bubble formation. To avoid microbial contamination, a 0.22 µm filter was connected to the inlet of the container holding the large volume of albumin solution. Aeration was performed for a period of 6 hours. The starting material obtained in this manner was used as an aqueous albumin-containing solution in comparative experiments.

[0074] Example 1

[0075] The reference sample was prepared by filtering the starting material as described above without adjusting the filter. Figure 2 A schematic diagram of a sterile filtration apparatus is shown. An aqueous solution containing albumin 2 is filtered using a pump (not shown), which provides a pressure of 2 bar without applying back pressure to the outlet 4 of the filter 3. The filter 3 used has a pore size of 0.2 µm and a sterile filtration area of ​​0.015 m². 2 The disposable cartridge contains the same material and the same pleated filter membrane as a 10-inch filter typically used on a production scale. The filter 3 is first rinsed with 260 mL of ultrapure water (not shown), and then washed with 150 mL of an albumin-containing aqueous solution 2. The filtrate 1 of the albumin solution is continuously collected in 20 50 mL vials 10, and the vials are immediately sealed with stoppers (not shown). The vials 10 are then pasteurized at 60°C for 10 hours and incubated at 30°C for 15 days. The resulting 20 samples are used as a reference, and samples obtained according to the improved method of the present invention are compared with the reference.

[0076] Visual inspection of the reference sample vials in the visual inspection kit confirmed the opalescence (Class III) of each vial after pasteurization and incubation, as shown in Table 1 and Figure 3 The results summarized in the paper show that statistical significance was calculated using a two-tailed t-test with Sigma Plot 12.0 software. Figure 4 The image also shows an example of a vial with opalescence (Class III) (middle image; Example 1).

[0077] Furthermore, after two months of incubation, a visual evaluation was performed on the white ring on the inside of the vial containing the reference sample at the liquid-air interface, and the white ring was indeed observed in the reference sample vial. Figure 5 The white arrow in the image indicates this (middle image; Example 1).

[0078] like Figure 6 As shown, the optical density measured by dynamic light scattering (DLS) is highest in the reference sample. This indicates that the particle size is relatively large and the particle content is high. Therefore, the reference sample is also the most turbid, as... Figure 7 As shown in the figure. Similarly, a two-tailed t-test was calculated using Sigma Plot 12.0 software. Figure 6 and 7 The statistical significance of the data.

[0079] Example 2

[0080] In one embodiment of the improved method for reducing the opalescence of filtered albumin solutions described herein, 20 samples are prepared as described in Example 1, the only difference being that a back pressure of 1 bar is applied to the filter during rinsing with water and albumin, wherein the flow direction through filter 3 is indicated by arrows, as shown. Figure 8 As illustrated in the diagram. The circled numbers represent the pressure values ​​before and after filter 3, in bar.

[0081] Visual examination of vials obtained by saturating the filter with back pressure during water and albumin rinsing revealed that these samples showed a reduction in opalescence from Class III (reference) to Class (II), classified according to the European Pharmacopoeia 2016 (EP9, Section 2.2.1: Transparency and opalescence of liquids), as shown in Table 1 and Figure 3 The results summarized in the paper show that statistical significance was calculated using a two-tailed t-test with Sigma Plot 12.0 software. Figure 4 An exemplary vial with opalescence (Class II) is also shown (left; Example 2). Figure 5 As shown, two months after incubation, none of the vials from Example 2 showed a white ring during visual inspection (left image; Example 2).

[0082] In addition, such as Figure 6 and 7 As shown, compared to the reference sample, back pressure saturation of the sterile filter during rinsing also reduced optical density and turbidity. Similarly, statistical significance was calculated using a two-tailed t-test with Sigma Plot 12.0 software.

[0083] Example 3

[0084] In another embodiment of the improved method for reducing the opalescence of filtered albumin solutions described herein, 20 samples were obtained as described in Example 1, the only difference being that they were washed with an albumin-containing aqueous solution under reduced pressure of 1 bar (instead of 2 bar), as... Figure 9As shown schematically, the flow direction through filter 3 is indicated by arrows, and the pressure values ​​before and after filter 3 are indicated by circled numbers in bar.

[0085] Visual examination of vials obtained by saturating the filters with depressurized or slower albumin rinsing showed that the opalescence exhibited by these samples decreased from Class III (reference) to Class I or Class 0 (with 5 exceptions), as shown in Table 1 and Figure 3 The results summarized in the paper show that statistical significance was calculated using a two-tailed t-test with Sigma Plot 12.0 software. Figure 4 The illustration also shows an exemplary vial exhibiting only Class I opalescence, which does not correspond to opalescence according to European Pharmacopoeia 2.2.1 "Transparency and opalescence of liquids" (right image; Example 3). Like the sample container from Example 2, none of the vials from Example 3 showed a white ring upon visual inspection after two months of incubation. Figure 5 As shown in the image on the right; Example 3.

[0086] like Figure 6 and 7 As shown, saturating the sterile filter with reduced pressure (i.e., slower flow rate during rinsing) also resulted in decreased optical density and turbidity compared to the reference sample. Similarly, statistical significance was calculated using a two-tailed t-test with Sigma Plot 12.0 software.

[0087] Table 1

[0088]

[0089]

Claims

1. A method for sterile filtration of an aqueous solution (2) containing albumin, wherein the method comprises filtering the solution (2) with at least one filter (3) to obtain a filtrate (1), characterized in that, Before filling the filtrate (1) into at least one container (10, 10'), the at least one filter (3) is conditioned by washing the at least one filter (3) with a certain amount (5) of the albumin-containing aqueous solution (2) under the following conditions: i) Under a pressure difference of no more than 1 bar across the corresponding filter (3); and / or ii) by means of pressure filtration, wherein the filtrate (1) is maintained at a back pressure of at least 0.5 bar at the outlet (4) of the respective filter (3).

2. The method according to claim 1, wherein the back pressure at the outlet (4) of the respective filter (3) is about 1 bar.

3. The method according to claim 1 or 2, wherein the amount (5) of the albumin-containing aqueous solution (2) used for cleaning the at least one filter (3) is about 2.5 liters to about 11 liters per square meter of the filtration area of ​​the at least one filter (3), preferably about 10 liters per square meter of the filtration area of ​​the at least one filter (3).

4. The method according to any one of the preceding claims, characterized in that, Before cleaning the corresponding filter (3) with the albumin-containing aqueous solution (2) of the specified amount (5), the at least one filter (3) is rinsed with ultrapure water (6).

5. The method according to claim 4, wherein the volume of ultrapure water (6) used to rinse the at least one filter (3) is about 1 to 10 liters per square meter of the filtration area of ​​the at least one filter (3).

6. The method according to any one of the preceding claims, wherein before and / or after filtration, the albumin concentration in the albumin-containing aqueous solution (2) and / or the filtrate (1) is 19% to 30% based on the total weight of the albumin-containing aqueous solution (2) and / or the filtrate (1), respectively.

7. The method according to any one of claims 1 to 5, wherein before and / or after filtration, the albumin concentration in the albumin-containing aqueous solution (2) and / or the filtrate (1) is 3% to 6% based on the total weight of the albumin-containing aqueous solution (2) and / or the filtrate (1), respectively.

8. The method according to any one of the preceding claims, wherein the method further comprises the steps of pasteurizing the filtrate (1) to obtain pasteurized filtrate (7) and evaluating the opalescence of the pasteurized filtrate (7).

9. The method according to any one of the preceding claims, wherein the method further comprises the steps of pasteurizing the filtrate (1) to obtain pasteurized filtrate (7), incubating the pasteurized filtrate (7) to obtain incubated filtrate (8), and evaluating the opalescence of the incubated filtrate (8).

10. The method according to claim 8 or 9, wherein the opalescence of the pasteurized filtrate (7) or the incubated filtrate (8) is evaluated at a temperature of < 35°C, preferably at a temperature of < 30°C, and even more preferably at a temperature of < 25°C.

11. The method of claim 10, wherein the opalescence of the incubated filtrate (8) is evaluated at a temperature of about 20°C.

12. The method of claim 10, wherein the opalescence of the incubated filtrate (8) is evaluated at a temperature of about 2°C to 8°C.

13. The method according to any one of claims 8 to 12, wherein the opalescence of the pasteurized filtrate (7) and / or the opalescence of the incubated filtrate (8) is evaluated by one or more of visual inspection, dynamic light scattering, and tunable resistance pulse sensing.

14. The method according to any one of claims 8 to 13, wherein the opalescence of the pasteurized filtrate (7) and / or the incubated filtrate (8) is reduced by at least one grade compared to the filtrate of the same albumin-containing aqueous solution (2) that has not been filtered through a conditioned filter, preferably the pasteurized filtrate (7) and / or the incubated filtrate (8) has at most grade I opalescence, the grade I opalescence being classified as having 3 NTU.

15. The method according to any one of the preceding claims, characterized in that... The albumin-containing aqueous solution (2) is applied to the at least one filter (3) at a pressure of about 1 bar.

16. The method according to any one of the preceding claims, wherein the filter area of ​​the at least one filter (3) is about 0.01 m². 2 To approximately 0.8 m 2 Preferably, the filtration area of ​​the at least one filter (3) is about 0.4 m². 2 .

17. The method according to any one of the preceding claims, wherein the at least one filter (3) has a pore size of about 0.22 µm or less, preferably about 0.22 µm or about 0.20 µm.

18. The method according to any one of the preceding claims, wherein, prior to filtering the albumin-containing aqueous solution (2) with the at least one filter (3), the method further comprises the step of filtering the albumin-containing aqueous solution (2) with a pre-filter (9), wherein the pre-filter (9) has a pore size of about 0.45 µm or less.

Images