Submerged autoclaving method

Abstract

The present disclosure relates to methods for sterilizing filters and filters so-sterilized. A filter is sterilized by submerging the filter in a liquid and placing the submerged filter in a chamber of an autoclave for at least one autoclave cycle. This method is of particular use when performed on a small-scale device to emulate a change in filtration performance of a large-scale device that has been steamed in-place.

Description

P2023-4233SUBMERGED AUTOCLAVING METHODFIELD

[0001] The present invention relates to methods for sterilizing filters and filters so-sterilized. The invention is of particular use when performed on a small-scale device to emulate a change in filtration performance of a large-scale device that has been steamed in-place.BACKGROUND

[0002] Filtration is often required in bioprocessing systems, commonly during downstream processing of a biological fluid. One example is the use of virus-retentive filters during vaccine production to remove the virus from complex biological feeds, leaving the proteins or protein fragments of interest.

[0003] Such filters must usually meet minimum performance requirements, often defined in terms of filtration efficiency and flow rate through the filter. A perfect filter will remove 100% of the intended filtrate and have no limit on the flow rate of feed fluid it can handle. A real filter is never perfect and generally offers a compromise between filtration efficiency and flow rate. A designer of a system using a filter needs to know the expected performance of the filter to be able to design a system that works adequately for the specified function.

[0004] Filtration performance in bioprocessing is complicated and difficult to predict and depends, among other things, on the filter, the biological fluid, and the length of time over which the filtration is performed. It is also well known that performance of a filter will change over time during use. For example, the flow of the filter may reduce as the filter gets loaded with particulate overtime.

[0005] Bioprocessing is time sensitive, and unexpected changes in filter performance may result in a loss or reduced yield of biological end product. To avoid such wastage, it is critical that filtration performance is understood before starting a bioprocess run or batch. For this reason, filterability studies are typically performed for a specific bioprocess using small-scale test filters to predict a large-scale filter performance when designing a bioprocess. The ability to extrapolate the performance of a small-scale test filter to the equivalent large-scale filter used in a bioprocess is important in system design.

[0006] It is also often required that bioprocess systems are subject to strict requirements for sterility. The sterilization of a large bioprocess system is commonly performed in-place to avoid the need for disassembly of equipment. A common method of sterilization in-place uses steam, known as steam in-place. This sterilization process typically changes the performance characteristics of the (large-scale) filter used in the bioprocessing. The characteristics are often changed both in terms of the start-up filtration efficiency and throughput, and in terms of how those parameters change during continued use of the filter.

[0007] The change in performance characteristics of the large-scale filter can vary depending on the steam in-place parameters used, for example temperature and / or duration of steam treatment. It is desirable for the small-scale test filter used in the filterability study to emulate the performance change ofP2023-4233 a large-scale filter subjected to sterilization in-place to enable accurate modelling of large-scale filter performance. However, small-scale filters used in such filterability studies are delicate and will often break when subjected to the pressures used by steam in-place processes. Current practice is thus to sterilize small-scale test filters by gamma irradiation. However, gamma irradiation does not result in the same change in performance of a filter as a steam in-place process. This makes it difficult to predict large-scale filter performance when designing a bioprocess based on a small-scale filter.SUMMARY

[0008] The present invention provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present invention will be apparent from the description as set forth below.

[0009] A first aspect of the present invention is a method for sterilizing a filter. The method optionally comprises submerging the filter in a liquid. The submerged filter may then be placed in a chamber of an autoclave for at least one autoclave cycle.

[0010] This novel sterilization method for a filter has been found to better emulate a steam in-place sterilization process.

[0011] The filter may comprise at least one microporous polymeric membrane. The filter may be a small-scale device having a surface area of the at least one microporous polymeric membrane of less than about 20 cm2. The filter may comprise a capsule containing the at least one microporous polymeric membrane.

[0012] The liquid may be water. The water may be any one or combination of: distilled water, deionized water, ultrapure water, or clean water.

[0013] The at least one autoclave cycle may be a non-vacuum autoclave cycle. The parameters for the at least one autoclave cycle may comprise at least one of: maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles. The maximum chamber temperature may be in the range of about 115 °C to about 145 °C. The time at maximum chamber temperature may be greater than about 15 minutes per cycle. The number of cycles may be in the range of about 1 to 8.

[0014] A cumulative sterilization time for the at least one autoclave cycle may be greater than about 15 minutes, wherein the cumulative sterilization time is the number of autoclave cycles multiplied by the time at maximum chamber temperature.

[0015] The performance of the filter may be changed by the at least one autoclave cycle, such that the performance of the filter after the at least one autoclave cycle is different to the performance of the filter before the at least one autoclave cycle. The performance of the filter may be measured by at least one of: flux, water flux, water permeability, throughput, capacity (filterability), retention, forward flow, and contact angle. The change in performance of the filter may be controlled by selecting the autoclave parameters to result in a desired change in performance after the at least one autoclave cycle. The desired change in performance after the at least one autoclave cycle may be approximately the change inP2023-4233 performance of another filter after a sterilization process. The change in performance of the other filter may be due to being sterilized in-place.

[0016] The sterilization process may comprise at least one of: steam, heat, radiation, or gas.

[0017] The other filter may be a large-scale device having an effective filter area of greater than about 200 cm2.

[0018] A second aspect of the present invention is a filter that has been sterilized by the method according to the first aspect.

[0019] A third aspect of the present invention is a filter that has been sterilized by a sterilization process so that the change in performance of the filter due to the sterilization process simulates a change in performance due to a different sterilization process.

[0020] A third aspect of the present invention is a kit of parts comprising a filter and a list of parameters for sterilizing the filter by a sterilization process, wherein a change in performance of the filter due to the sterilization process using the list of parameters results in a desired change in performance of the filter.

[0021] The sterilization process of the third aspect or fourth aspect may be the method according to the first aspect.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 depicts an autoclave having a chamber with a container placed therein, the container comprising the liquid submerging a filter.

[0023] Figure 2 shows the relationships between maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles on the mean of filterability at 75% flux decay (V75) of a small-scale example filter after sterilization by submerged autoclaving.

[0024] Figure 3 shows the relationships between maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles on the mean increase in water flux of a small-scale example filter after sterilization by submerged autoclaving.

[0025] Figure 4 shows the permeability of a small-scale example filter before and after sterilization by submerged autoclaving. The permeability of another small-scale example filter is also shown before and after sterilization by a gamma radiation control method. The permeability of a large-scale filter is also shown before and after sterilization by a steam in-place method.

[0026] Figure 5 shows the capacity (filterability) of a small-scale example filter after sterilization by submerged autoclaving. The capacity (filterability) of a large-scale filter is also shown after sterilization by a steam in-place method.

[0027] Figure 6 shows the contact angle of a small-scale example filter before and after sterilization by submerged autoclaving. The contact angle of another small-scale example filter is also shown before and after sterilization by a gamma radiation control method. The contact angle of a large-scale filter is also shown before and after sterilization by a steam in-place method.P2023-4233

[0028] Figure 7 shows flux / water flux against throughput using 0.2g / L hlgG in acetate buffer solution for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, and filter membrane type.

[0029] Figure 8 shows interaction plots of fitted means for the filterability at 75% flux decay (V75) for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, and filter membrane type.

[0030] Figure 9A shows contour plots for filterability at 75% flux decay (V75) for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, with a thin membrane type.

[0031] Figure 9B shows contour plots for filterability at 75% flux decay (V75) for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, with a nominal membrane type.

[0032] Figure 10 shows interaction plots of fitted means for the percentage increase in water flow for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, and filter membrane type.

[0033] Figure 11 A shows contour plots for the percentage increase in water flow for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, with a thin membrane type.

[0034] Figure 1 IB shows contour plots for the percentage increase in water flow for a range of varying maximum chamber temperature, time at maximum chamber temperature, number of cycles, with a nominal membrane type.DETAILED DESCRIPTION

[0035] The method may involve submerging a filter in liquid and then sterilizing the filter while it is submerged in the liquid.

[0036] The sterilizing takes place in a temperature and / or pressure-controlled chamber, such as a chamber capable of exposing an object to elevated temperature, elevated pressure, and / or steam.Preferably, the sterilizing takes place in the chamber of an autoclave, an autoclave being a machine used for sterilization of an object by subjecting the object to high pressure saturated steam. Autoclaves are typically cuboid, with a door in at least one side that is openable to accept an object or objects to be placed inside the chamber of the autoclave, and closable to seal the interior chamber of the autoclave allowing it to hold pressure for the sterilization process. In the present invention, the object placed inside the chamber of the autoclave may comprise a container holding a liquid in which the filter is submerged.

[0037] The container preferably has dimensions that allow for a filter to be held therein. The container also preferably has dimensions that allow it to be placed inside a chamber of an autoclave. The dimensions may be such that there is free space around the filter when it is placed in the container. This advantageously allows for the liquid to fully surround the filter. Having free space around the filter also allows for the filter to be easily placed in and removed from the container. The dimensions of theP2023-4233 container may include a width that is slightly larger than the width of the filter, for example in the range of about 10 mm to about 50 mm larger. Alternatively, the width of the container may be substantially larger than the width of the filter, for example more than 50 mm larger or greater than 100 mm larger. Additionally, the container preferably has dimensions that allows for a liquid contained therein to fully submerge the filter held therein. That is, the height of the container is preferably higher than the height of the filter when the filter is at rest in the container. This vertical dimension, in combination with the lateral dimensions of the container, also advantageously allow for the liquid to fully surround the filter. The height difference between the level of liquid in the container and the top of the filter in the container should be sufficient to keep the filter fully submerged in the event that some liquid evaporates from the container during the sterilization process. The height of the container may be in the range of about 100 mm to about 300 mm. However other suitable heights for the container are possible if it allows for the filter placed therein to be submerged in a liquid, for example the container may have a vertical dimension of less than about 100 mm or greater than about 300 mm.

[0038] The container may be a metal, glass or plastic container, preferably with a flat bottom such that it is stable when placed in the autoclave. The container may be rated to withstand a specific temperature that is greater than or equal to a maximum chamber temperature of the autoclave during the method. As many filters are round, the container may advantageously be cylindrical. In the case of the container being a cylinder, the width of the container may be a diameter of the cylinder. Alternatively, the container may be another geometric shape, such as a cuboid. The container preferably has an opening sized to accept the filter to be held therein. The opening may be in the top or a side of the container. The opening may be the same size as the top of the container. The container may have a lid that is attachable and detachable to cover the opening. The lid may be attached by at least one mechanical clasp or pressure fit. The lid may be hingeably attached to the container such that it can pivot to cover the opening. The lid may be attached before the sterilization process and detached after the sterilization process. It is desirable for the lid to have openings such that the high pressure saturated steam of the autoclave can enter the container. The openings of the lid additionally allow for liquid to evaporate from the container into the chamber of the autoclave. The openings of the lid may be used to remove liquid from the container while retaining the filter therein. Alternatively, there can be no lid and the opening of the container may be uncovered during sterilization so the free surface of the liquid is fully exposed to the autoclave environment.

[0039] The container optionally comprises a spout for pouring the liquid. The spout may be useful to remove liquid. The liquid may be removed before or after the sterilization process. The outer surface of the container may have measurement lines to provide information about the height of the level of the liquid or the volume of the liquid therein. The measurement lines may be useful in combination with the spout to provide the level of the liquid at a desired height before the sterilization process.

[0040] To keep the filter submerged fully it may be necessary to provide some form of downward force on the filter held within the container. If the filter is buoyant in the liquid then it will naturally otherwise float on the surface of the liquid in the container. This downward force may be applied by theP2023-4233 weight of an object that is not buoyant in the liquid, for example a glass stir rod or other such mass such as another smaller container. Alternatively, the downward force may be applied by a pusher, spring or other biasing member attached to the lid or top part of the container.

[0041] It is envisaged that more than one fdter can be submerged in a liquid and placed in a chamber of an autoclave for sterilization at the same time. For example, two fdters, three fdters, four fdters or more than four fdters could be placed in separate containers, or the same container, to be submerged in the liquid. In the case of more than one fdter being submerged in liquid in the same container, the dimensions of the container may be such that there is free space around the fdters when they are placed in the container. For example, there may be in the range of about 10 mm to about 50 mm of free space between the fdters in the container. Alternatively, there could be less than about 10 mm or more than about 50 mm of free space between the fdters. A benefit of sterilizing more than one fdter concurrently is that the fdters being sterilized would be subjected to identical autoclave parameters. Another benefit of sterilizing more than one fdter concurrently is an increased efficiency in time providing a desired number of fdters so-sterilized.

[0042] The fdter may comprise at least one microporous polymeric membrane. The fdter may comprise a plurality of microporous polymeric membranes, for example two membranes or three membranes, in layers. The at least one microporous membrane may have a first microporous surface and a second microporous surface, with a microporous bulk between the first surface and the second surface giving the membrane a thickness.

[0043] The surface area of the first microporous surface may be less than about 20 cm2. The surface area of the second microporous surface may be less than about 20 cm2. The fluid to be filtered is introduced to the first surface and the filtered fluid exits from the second surface. A fdter having a plurality of microporous membranes, for example a first microporous membrane and second microporous membrane, will pass the fluid from the second surface of the first microporous membrane to the first surface of the second microporous membrane. The effective filtration surface area of each microporous membrane may therefore be less than about 20 cm2.

[0044] The at least one microporous membrane may be contained within a capsule to form the fdter. The capsule may be made of any suitable material for the purpose, for example plastic or metal. The capsule preferably allows the fluid to be filtered to be passed in one side to the at least one microporous membrane and the filtered fluid to be passed out of a second side. The first side and the second side of the capsule should only be fluidly connected through the at least one microporous membrane.

[0045] The liquid that the fdter is submerged in may be water, preferably clean water. It is desirable for the water to have low levels of contaminant types to prevent contamination of the fdter. The contaminant types to minimise include particulates, bacteria, viruses, organic compounds, inorganic compounds, dissolved matter, and dissolved gases. The clean water may have been obtained by any combination of disinfection, purification, filtration, distillation, reverse osmosis, and / or demineralization to remove said contaminants. The clean water may be any one or combination of distilled water, deionized water or ultrapure water.P2023-4233

[0046] Alternatively, the liquid may be any one or combination of solvents such as pentane, hexane, benzene, heptane, toluene, 1,4-dioxane, diethyl ether, tetrahydrofuran (THF), chloroform, dichloromethane, ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), nitromethane, propylene carbonate, ammonia, formic acid, w-Butanol, isopropyl alcohol, w-propanol. ethanol, methanol, acetic acid, or water.

[0047] It is preferable that the liquid does not evaporate or boil over during the sterilization process. Whether the liquid evaporates or boils over during the sterilization process is at least partly dependent on the parameters for the at least one autoclave cycle. The method may thus involve setting parameters for the autoclave cycle that substantially prevent or minimise liquid evaporation or boil-over.

[0048] An autoclave cycle typically contains a purge phase, an exposure phase, and a drying phase. In the purge phase, air is removed from the chamber of the autoclave through a vacuum pump (in vacuum cycles) or by steam displacement (in gravity or non-vacuum cycles). In the exposure phase, the chamber is fdled with steam, and the temperature and pressure are maintained at the required levels for a specified period. The exposure phase is where the actual sterilization occurs. In the exhaust phase, the steam is released and the pressure is brought back to normal. In some cycles, a vacuum may be applied in the exhaust phase to help dry the object being sterilized.

[0049] Typically, an object having lumens, cavities or pores such as a microporous filter is subjected to a vacuum autoclave cycle, which involves using a vacuum pump to remove all the air from the autoclave chamber before introducing the steam as the sterilization medium. This removal of air may be crucial to effective sterilization of solid objects because the latent air around the object can act as a barrier, preventing the steam from effectively penetrating and contacting all surfaces inside the object, thus reducing sterilization efficacy.

[0050] It has been found by the present inventors that sterilization of an (unsubmerged) filter using a vacuum autoclave typically has a negative impact on the performance of the filter membrane, due to the high pressure changes involved and the propensity of the vacuum to dry out the membrane. This can sometimes result in a rupturing of the filter membrane rendering the filter not suitable for use. A similar issue can occur when performing a steam in-place sterilization method on a fragile filter - the membrane can be ruptured by the pressure of the steam, rendering the filter not suitable for use. The submerging of the filter during sterilization (e.g. by autoclaving) keeps the filter wet and helps to prevent the filter from being damaged thereby rendering the filter more able to withstand the pressures introduced in the sterilization process.

[0051] It has also been found by the present inventors that the application of a vacuum at the end of the autoclave cycle would dry out the filter membrane. If the filter membrane dries out then then the performance of the filter membrane may be negatively affected.

[0052] When performing submerged autoclaving, as considered in the present invention, it is not necessary to remove the air from the chamber of the autoclave. The filter, being submerged in a liquid, is instead fully surrounded by the liquid which contacts all surfaces inside and outside of the filter.Advantageously the filter membrane will not dry out when it is fully submerged in liquid as in the presentP2023-4233 invention. Without wishing to be bound by theory, it is thought that the steam acts as a medium to heat up and / or pressurise the liquid submerging the fdter, and the heated and / or pressurised liquid acts to sterilize the submerged fdter. Therefore, there is no requirement to remove air from the chamber and a non-vacuum autoclave cycle may be used. Further, the submerged fdter is exposed to increased pressure similar to in the vacuum cycle, but it is applied uniformly around the fdter by the liquid, thus reducing the chance of damage to the fdter membrane. The non-vacuum autoclave cycle is simpler to use without extensive training, and non-vacuum cycle capable only autoclaves tend to be less expensive to purchase and maintain, and they have fewer moving parts and are thus more reliable with lower maintenance requirements, compared to autoclaves capable of vacuum cycles. Another advantage is that non-vacuum cycle capable only autoclaves tend to be smaller in dimension compared to autoclaves capable of vacuum cycles, allowing them to more easily be placed and used in a variety of different environments. For example, non-vacuum cycle capable only benchtop autoclaves are widespread, allowing the present invention to be carried out in conventional laboratories or workshop without the need for additional or specialised equipment.

[0053] Care has to be taken to avoid the ‘boil-over effect’, which occurs when the pressure in the autoclave chamber is released too quickly during the exhaust phase. Rapid depressurization could cause the liquid in the container to boil suddenly and spill over. To prevent the ‘boil-over effect’ it is desirable to control the decrease of pressure to occur gradually, maintaining the integrity of the liquid and ensuring effective sterilization without boiling over. Non-vacuum autoclave cycles tend to release the pressure more gradually than vacuum autoclave cycles using vacuum pumps, thus have a reduced likelihood of experiencing the ‘boil-over’ effect.

[0054] The maximum chamber temperature and time at maximum chamber temperature for the autoclave cycle are chosen by the user dependent on the circumstances. Additionally, the user may select more than one autoclave cycle to be run consecutively. The consecutive autoclave cycles may be run with the same or different objects, and at the same or different autoclave parameters. Running multiple consecutive autoclave cycles can advantageously provide a more thorough sterilization of the object to adhere with certain regulations or protocols which guarantee high levels of sterilization. It can also allow for objects requiring different sterilization parameters. Running consecutive cycles with adjusted parameters can accommodate various items in a single batch, optionally removing or adding objects to the chamber between cycles. The number of consecutive cycles may be in the range of about 1 to 8.

[0055] The pressure in the autoclave chamber is typically driven by the chosen temperature. An amount of steam contained within the constant volume of the autoclave chamber increases in pressure directly proportional to the increase in temperature, as dictated by the ideal gas law for example.

[0056] The maximum chamber temperature may be in the range of about 115 °C to about 145 °C, preferably in the range of about 120 °C to about 135 °C, more preferably about 121 °C to about 130 °C. The time at maximum chamber temperature per autoclave cycle may be greater than or equal to about 15 minutes, optionally greater than or equal to about 20 minutes, optionally greater than or equal to about 25 minutes, optionally greater than or equal to about 30 minutes, optionally greater than or equal to about 45P2023-4233 minutes, optionally greater than or equal to about 60 minutes. The liquid in the container would preferably reach the maximum chamber temperature for the time at maximum chamber temperature per cycle.

[0057] A cumulative sterilization time can be defined as the number of autoclave cycles multiplied by the time at maximum chamber temperature. For example, if the time at maximum chamber temperature is 15 minutes and the number of autoclave cycles is 4, then the cumulative sterilization time is 60 minutes. For a further example, if the time at maximum chamber temperature is 30 minutes and the number of autoclave cycles is 8, then the cumulative sterilization time is 240 minutes. The cumulative sterilization time defines the equivalent of one longer autoclave cycle split into respective cycles at shorter times at maximum chamber temperature. The maximum chamber temperature may vary or stay the same for each of the respective autoclave cycles making up the cumulative sterilization time.

[0058] A filter sterilized while submerged in a liquid may have its performance changed by the autoclave cycle. In other words, the filtration performance of the filter before the sterilization process (comprising the submerged autoclaving) may be different to the filtration performance of the same filter after the sterilization process. The submerged autoclaving sterilization is thought to affect the pore size and pore structure of the microporous membrane which alters the filter performance.

[0059] The performance of a filter sterilized by any method will thus typically change from before the sterilization method to after the sterilization method. Another method of sterilization is gamma sterilization, which uses gamma rays, typically from a radioactive isotope like Cobalt-60, to kill microorganisms. The effect of gamma sterilization on the performance of a filter is not the same as the effect of submerged autoclaving sterilization on filter performance.

[0060] The performance of the filter and / or the filter membrane may be measured by at least one of flux, water flux, water permeability, throughput, capacity, retention, forward flow, and contact angle.

[0061] The flux of the filter is a measure of how much fluid passes through the filter per unit area over a given time. The flux of a given filter depends on the microporous structure of the polymeric membrane and the pressure difference exerted across said membrane, and characteristics of the fluid being filtered. Having sufficient flux performance characteristics is desirable in viral filtration to ensure efficient virus removal while allowing the desired product to pass through. Over time, the flux of a filter membrane typically decreases. This reduction is primarily due to fouling, which is the accumulation of particles, biomolecules, and other contaminants on the membrane surface and within its pores. If the fluid being measured passing through the filter is water, then this is called the water flux. Water flux is often used as a baseline or reference point because water is a standard, readily available, consistent fluid. It helps in comparing the performance of different filters or assessing the filter’s condition overtime.

[0062] Water permeability is a measure of a filter’s propensity to allow water to pass through, measured as a flow or flux per unit pressure. This is a critical feature for ensuring that the filter effectively removes filtrate, for example virus particles, from water passing therethrough without significantly impeding the flow of water.P2023-4233

[0063] The throughput of a filter is a measure of the volume of fluid that can be processed through the filter over a given period, often normalized per unit area of filter. This can vary significantly depending on the type of filter, the specific application, and the conditions under which it is used. For example, some filters achieve throughputs of 1,800 litres per square meter over approximately two days.

[0064] The capacity of a filter is a measure of the amount of fluid it can process before it becomes clogged or its performance significantly declines so it is no longer usable. This is also called filterability and can be determined with reference to a percentage of the starting flux. For example, filterability at 75% flux decay (V75) is a measure in litres per square meter at which the flux has decreased to 75% of the starting flux. In other words, the flow rate of the filter has decreased by 75% during use of the filter, potentially due to fouling.

[0065] The retention of a filter is a measure of its ability to capture and remove a substance (e.g. filtrate) from a fluid, typically measured by the filter’s efficiency in retaining particles of specific sizes. Virus filters typically have very high retention with a log reduction value of greater than 4, indicating removal of 99.99% of viruses present in the fluid stream.

[0066] Forward flow is a measure of the amount of gas that diffuses through a wetted filter membrane under constant pressure. The gas typically flows across the wetted filter membrane by diffusion due to the difference in pressure across the membrane. This performance parameter is useful to check the integrity of the filter membrane by indicating defects if the forward flow exceeds a validated maximum.

[0067] The contact angle of a filter is a measure the wettability of the surface of the filter membrane by a liquid. The contact angle is defined as the angle formed between the tangent to the liquid surface and the solid surface at the point of contact. The contact angle indicates the wettability of the filter, and indicates whether the surface is hydrophilic or hydrophobic. A lower contact angle means the liquid spreads out more, indicating a hydrophilic surface. A higher contact angle means the liquid forms droplets, indicating a hydrophobic surface. This performance measurement is useful in designing filtration processes because it affects how fluids pass through the filter and how efficiently the filter can retain particles or contaminants.

[0068] The fluid used in measuring the performance of the filter may be a test fluid containing filtrate to help in comparing performance of different filters or assessing the filter’s condition over time. A suitable test fluid would be for example human or bovine immunoglobulin G (IgG), optionally diluted in a buffer, for example an acetate buffer.

[0069] The inventors have discovered that there is a predictable change in filter performance as measured by at least one of the above defined performance characteristics when the filter is sterilized by submerged autoclaving.

[0070] Sterilizing a small-scale filter by submerged autoclaving was also discovered to emulate the change in filter performance of a large-scale filter that was sterilized by another sterilization method. The other sterilization method may be an in-place sterilization method, using at least one of steam, dry heat, radiation, or gas to perform the sterilization. Steam in-place sterilization may involve using steam at highP2023-4233 temperatures, typically 120 °C to 135 °C, to sterilize static equipment such as a filter in a bioprocess. Dry heat in-place sterilization may involve using hot air to sterilize equipment for items that might be damaged by moisture. Gas in-place sterilization may involve using gases like ethylene oxide to sterilize equipment for items that cannot withstand high temperatures. Radiation in-place sterilization may use ionizing radiation to sterilize equipment, for example gamma rays, x-rays or electron beams.

[0071] The large-scale filter may be a virus-retentive filter typically used in a bioprocess. The large-scale filter may have an effective filter area of greater than about 200 cm2.

[0072] As shown in Figure 2, there is a predictable relationship between maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles on the mean filterability at 75% flux decay (V75) of a small-scale example filter after sterilization by submerged autoclaving. A predictable relationship means that it may be possible to extrapolate the effect on mean filterability of the filter after submerged autoclaving for any combination and value of maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles, within reason. Figure 2 shows that increased maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles increase the mean filterability at 75% flux decay (V75).

[0073] As shown in Figure 3, there is a predictable relationship between maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles on the mean increase in water flux of a small-scale example filter after sterilization by submerged autoclaving. A predictable relationship means that it may be possible to extrapolate the effect on water flux of the filter after submerged autoclaving for any combination and value of maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles, within reason. Figure 3 shows that increased maximum chamber temperature and number of autoclave cycles increase the mean increase in water flux. Figure 3 additionally shows that increased time at maximum chamber temperature reduces the mean increase in water flux.

[0074] As shown in Figure 4, the permeability of a small-scale example filter before and after sterilization by submerged autoclaving compares favourably to the change in permeability of a large-scale filter before and after being sterilized by a steam in-place method. The water permeability of the filter pre-sterilization for submerged autoclaving, gamma radiation, and steam in-place are all approximately the same. The water permeability of the filter post-sterilization for submerged autoclaving and steam in-place increases by approximately 10%. However, the water permeability of the filter post-sterilization for gamma radiation increases by approximately 35%. This shows that, comparatively, the small-scale filter sterilized by gamma radiation does not emulate the permeability performance change of the steam in-place method as well as the submerged autoclaving method.

[0075] As shown in Figure 5, the capacity (filterability) of a small-scale example filter after sterilization by submerged autoclaving compares favourably to the capacity (filterability) of a large-scale filter after being sterilized by a steam in-place method. This shows that the small-scale example filter subjected to sterilization by the submerged autoclaving method exhibits scalability within 80-90% of a large-scale filter subjected to sterilization by a steam-in-place method.P2023-4233

[0076] As shown in Figure 6. the contact angle, indicative of surface tension, of a small-scale example filter after sterilization by submerged autoclaving compares favourably to the contact angle of a large-scale filter after being sterilized by a steam in-place method. The contact angle (surface tension) of the filter post-sterilization for submerged autoclaving was about 76.2° and steam-in-place was about 73.1°. The contact angle of the filter pre-sterilization by autoclaving or steaming was about 65.9°.

[0077] Having both a predictable change in performance and a change in performance that emulates that of the steam in-place sterilization method makes the submerged autoclaving sterilization method desirable when performing filterability studies on small-scale filters to predict large-scale filter performance when designing a bioprocess. Filterability studies evaluate how well a filter can handle the specific biological fluids, which can vary in viscosity and particulate or viral load. By testing different filters, it is possible to determine the performance in terms of throughput, flow rate, and retention of contaminants. Small-scale filterability studies help predict how the filter will perform at larger scales, ensuring that the process can be scaled up without significant issues.

[0078] In a filterability study, a representative sample of the biological fluid is prepared mimicking the actual conditions of the bioprocess, including concentration and viscosity. The data from the filterability study may be used to predict the performance at a large-scale. When scaling from small-scale to large-scale, parameters may be adjusted. For example, surface area may be increased proportionally to handle larger volumes, flow rates may be adjusted to maintain similar linear velocities, and pressure across the filter may be managed to avoid damage. Performing predictable filterability studies may be a requirement to meet regulatory standards. The scaling may or may not be linear.

[0079] The predictability of the change in performance allows autoclave parameters to be selected such that the small-scale filter performance after sterilization by submerged autoclaving is known. The small-scale filter performance after sterilizing by submerged autoclaving using the selected autoclave parameters can then be chosen to emulate the performance of a large-scale filter sterilized by a steam-in-place method using selected steam-in-place parameters.

[0080] It is possible to provide a set of parameters so selected to achieve a desired change in filter performance after sterilization by submerged autoclaving. The parameters may be part of a kit of parts including the filter to be sterilized by submerged autoclaving using the provided parameters. Providing a set of parameters with a filter in this manner makes it straightforward for a user to emulate the performance of a large-scale filter that will be used in a bioprocess. In particular, the user may select the parameters for submerged autoclaving of a small-scale filter to match the change in performance of a large-scale filter when sterilized using a different set of parameters, thus giving a known performance to be used when carrying out filterability studies.

[0081] The different set of parameters for the large-scale filter may be a set of parameters for steam-in-place sterilization, for example a temperature of 125 °C for 15 minutes or 135 °C for 10 minutes, or any reasonable temperature or length of sterilization time.P2023-4233

[0082] It is thus desirable to provide a set of alternative parameters for an alternative sterilization method, such as the submerged autoclaving method which better emulates a steam in-place sterilization process performed with specific steam in-place parameters.

[0083] An exemplar design of experiments (DoE) was undertaken to determine a possible suitable window for autoclaving Pegasus™ Prime microdiscs (filters) using the above-described submerged autoclaving technique. The DoE used:• 3M sodium Acetate Buffer Solution pH 5.2+ / - 0.1 (0.2um filtered; the acetate buffer is supplied filtered so, additional filtration was not required)• Acrodisc™ Syringe filter 0.1 pm Supor™ Membrane• Tubing size: Size 16• Pendotech filter screening / control systems 1, 2 and 3 with balances communicating • IL pressure pots• Human IgG (hlgG feed stock) - approx 50g / L (Monomer particle size lOnM).• Pegasus™ Prime microdiscs (filters) comprising:i. Run 1: Nominal membrane microdiscs (N)ii. Run 2: Thin membrane microdiscs (W)• Priorclave 230L Rectangular Section Autoclave (Non-Vacuum)

[0084] A buffer consisting of 2 litres of 10 mM Acetate by diluting 6.67ml of 3M Sodium Acetate in 1993.33ml of 0.1 pm filtered ultrapure water.

[0085] A 50g / L stock solution of hlgG was defrosted and filtered through a 0.1 pm syringe filter, before being added to 1494 ml of the 10 mM acetate buffer to make 1.5 litres of 0.2 g / L of final feed solution.

[0086] All filters were tested for pre-autoclave water flow at a constant pressure of 30 psi with ultrapure water for 10 minutes. The water flow test was followed by submerged autoclaving with temperature, time and cycle parameters as stipulated by the DoE design. After the submerged autoclaving, and allowing the filters to cool to room temperature, a 10 minute post-autoclave water flow was performed. An IgG feed challenge was then performed using the 0.2g / L hlgG in acetate buffer. The water flow and IgG feed challenge tests were performed at a constant pressure of 30psi.

[0087] The above test design was undertaken and submerged autoclaving was performed on filters across a range of maximum chamber temperature, time at maximum chamber temperature, and number of cycles. For example, a set of parameters of 135 °C for 60 minutes for 1 cycle was referred to as 135-60-1. Other parameter sets were 121-30-1, 135-60-4, 121-60-4, 135-30-1, 121-60-1, 128-45-2, 121-30-4, and 135-30-4. The filter was also varied between a thin membrane type and a nominal membrane type. The thin membrane type comprised a membrane having a thickness of approximately the lower set limit of manufacturing. The nominal membrane type comprised a membrane having a thickness of approximately the mean of the upper set limit and lower set limit of manufacturing. Measurements were taken for preautoclave water flow, post-autoclave water flow, and IgG feed challenge across all parameter sets.P2023-4233

[0088] Figure 7 shows flux / water flux against throughput using the 0.2g / L hlgG in acetate buffer solution for a number of the parameter sets.

[0089] To determine the relationship between the parameters and the resulting filterability, a statistical model was fit to the data obtained by the measurements and partially shown in Figure 7. The overall model f-value in regression of 11.62 and an overall model p-value in regression of 0.000 for filterability at V75% throughput indicate that the model is statistically significant. This means that there is a very low probability that this model’s results for filterability at 75% flux decay (V75) are due to random chance.

[0090] Linear terms (maximum chamber temperature, time at maximum chamber temperature, number of cycles, and membrane type) are all significant, with p-values of 0.000 for maximum chamber temperature, time at maximum chamber temperature, number of cycles. This indicates a strong relationship with the response variable. Type of membrane also shows significance with a p-value of 0.011. The 2-way interaction between maximum chamber temperature and number of cycles is particularly significant (p-value of 0.000), suggesting that the combined effect of these two factors on the response is strong. The 3 -way interactions are also significant between maximum chamber temperature, time at maximum chamber temperature, and number of cycles with a p-value of 0.000, which indicates that the interaction between these three factors is important to the response.

[0091] The r-squared value of 90.72% suggests that 90.72% of the variability in the response can be explained by the model, which is quite high. The adjusted r-squared value of 82.91% is also high, indicating that the model fits well even after adjusting for the number of predictors. The lack-of-fit p-value of 0.982 is high, indicating that the lack of fit is not significant, and the model fits the data well.

[0092] Figure 8 shows interaction plots of fitted means for filterability at 75% flux decay (V75) showing that the maximum chamber temperature with number of cycles, followed by maximum chamber temperature with time at maximum chamber temperature, interactions have the most significant effects of the 2-way interactions.

[0093] Figure 9A shows contour plots for filterability at 75% flux decay (V75) having linear responses for maximum chamber temperature, time at maximum chamber temperature, and number of cycles for thin membrane type. Figure 9B shows contour plots for filterability at 75% flux decay (V75) having linear responses for maximum chamber temperature, time at maximum chamber temperature, and number of cycles for nominal membrane type, having a higher filterability than the thin membrane type.

[0094] To determine the relationship between the parameters and the resulting percentage increase in water flow, a statistical model was fit to the data obtained by the measurements and partially shown in Figure 7. The overall model f-value in regression of 3.45 and an overall model p-value in regression of 0.006 for percentage increase in water flow indicate that the model is statistically significant. This means that there is a strong relationship between the factors and the response.

[0095] The time at maximum chamber temperature is highly significant with an f-value in regression of 17.04 and a p-value in regression of 0.001. The maximum chamber temperature, number of cycles, and type of membrane may not be individually significant because their p-values in regression areP2023-4233 greater than 0.05. The 2-way interaction between maximum chamber temperature and number of cycles is significant (p-value of 0.024), suggesting that the combined effect of these two factors on the response is strong. The 3 -way interactions between maximum chamber temperature, time at maximum chamber temperature, and number of cycles has a p-value of 0.001, and maximum chamber temperature, number of cycles, and type of membrane has a p-value of 0.024, which indicates that the interaction between these sets of three factors is important to the response.

[0096] The r-squared value of 74.39% indicates that 74.39% of the variability in the response can be explained by the model. The predicted r-squared value of 2.41% is very low, indicating that the model may not predict new observations accurately. The lack-of-fit p-value of 0.574 suggests that the lack of fit is not significant, meaning the model adequately fits the data.

[0097] Figure 10 shows interaction plots of fitted means for percentage increase in water flow showing that the maximum chamber temperature with number of cycles, followed by time at maximum chamber temperature with number of cycles, interactions have the most significant effects of the 2-way interactions.

[0098] Figure 11 A shows contour plots for percentage increase in water flow having linear responses for maximum chamber temperature, time at maximum chamber temperature, and number of cycles for thin membrane type. Figure 1 IB shows contour plots for percentage increase in water flow having linear responses for maximum chamber temperature with time at maximum chamber temperature, and number of cycles with time at maximum chamber temperature for nominal membrane type.However, time at maximum chamber temperature with number of cycles for nominal membrane type shows significant curvature, with the best increases in water flow being towards the highest temperatures and longest cycle times.

[0099] The determined model is linear and significant for analysing both the filterability at 75% flux decay (V75) and percentage increase in water flow with a good lack-of-fit. This exemplar DoE demonstrates a predictable change in filter performance as measured by defined performance characteristics when the filter is sterilized by submerged autoclaving.

[0100] As used herein, terms such as “first”, “second” and “third” are used to provide distinct labels for different features. These terms are not intended to carry their own meaning, or themselves impart structural or functional limitations to the components / features with which they are used.

[0101] The foregoing description is exemplary in nature only. Those skilled in the art will understand that changes and variations on the disclosed embodiments are possible within the scope of the claims. It will also be understood that features described with reference to specific embodiments herein may be combined with those described with reference to other embodiments.

Claims

P2023-4233CLAIMS1. A method for sterilizing a filter, the method comprising:submerging the filter in a liquid; andplacing the submerged filter in a chamber of an autoclave for at least one autoclave cycle.

2. The method of claim 1, wherein the filter comprises at least one microporous polymeric membrane.

3. The method of claim 2, wherein the filter is a small-scale device having a surface area of the at least one microporous polymeric membrane of less than about 20 cm2.

4. The method of claim 2 or 3, wherein the filter comprises a capsule containing the at least one microporous polymeric membrane.

5. The method of any one of the preceding claims, wherein the liquid is water.

6. The method of claim 5, wherein the water is any one or combination of: distilled water, deionized water, ultrapure water, or clean water.

7. The method of any one of the preceding claims, wherein the at least one autoclave cycle is a non- vacuum autoclave cycle.

8. The method of any one of the preceding claims, wherein parameters for the at least one autoclave cycle comprises at least one of: maximum chamber temperature, time at maximum chamber temperature, and number of autoclave cycles.

9. The method of claim 8, wherein the maximum chamber temperature is in the range of about 115 °C to about 145 °C.

10. The method of any claim 8 or 9, wherein the time at maximum chamber temperature is greater than or equal to about 15 minutes per cycle.

11. The method of any one of claims 8 to 10, wherein the number of cycles is in the range of about 1 to 8.P2023-4233 12. The method of claim 8 or 9, wherein a cumulative sterilization time for the at least one autoclave cycle is greater than about 15 minutes, andwherein the cumulative sterilization time is the number of autoclave cycles multiplied by the time at maximum chamber temperature.

13. The method of any one of claims 8 to 12, wherein the performance of the filter is changed by the at least one autoclave cycle, such that the performance of the filter after the at least one autoclave cycle is different to the performance of the filter before the at least one autoclave cycle.

14. The method of claim 13, wherein the performance of the filter is measured by at least one of: flux, water flux, water permeability, throughput, capacity, retention, forward flow, and contact angle.

15. The method of claim 13 or 14, wherein the change in performance of the filter is controlled by selecting the autoclave parameters to result in a desired change in performance after the at least one autoclave cycle.

16. The method of claim 15, wherein the desired change in performance after the at least one autoclave cycle is approximately the change in performance of another filter after a sterilization process.

17. The method of claim 16, wherein the change in performance of the other filter is due to being sterilized in-place.

18. The method of claim 16 or 17, wherein the sterilization process comprises at least one of: steam, heat, radiation, or gas.

19. The method of any one of claims 16 to 18, wherein the other filter is a large-scale device having an effective filter area of greater than about 200 cm2.

20. A filter that has been sterilized by the method of any one of the preceding claims.

21. A filter that has been sterilized by a sterilization process so that the change in performance of the filter due to the sterilization process simulates a change in performance due to a different sterilization process.

22. A kit of parts comprising:a filter; andP2023-4233 a list of parameters for sterilizing the filter by a sterilization process,wherein a change in performance of the filter due to the sterilization process using the list of parameters results in a desired change in performance of the filter.

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