Method for validating the ability of a test filter unit to produce a sterile effluent while incorporating pupsit conditions

Abstract

The present invention relates to a method for validating the ability of a test filter unit to produce a sterile effluent. The method according to the present disclosure has the advantage that it allows simulating cumulative stress incurred by the filter during use with consideration to time and pressure, as closely as is feasible. A further advantage of the method according to the present disclosure is that it allows evaluating the impact of the stress incurred by the process filter during PUPSIT, while factoring in lab scale equipment / limitations and differing objectives, e.g., performing PUPSIT during manufacturing vs. filter validation.

Description

Method for validating the ability of a test filter unit to produce a sterile effluent while incorporating PUPSIT conditionsTECHNICAL FIELD

[0001] The present invention relates to the field of biotechnological processes, in particular the validation of filters for use in a biotechnological process. Various exemplary embodiments according to the present disclosure relate to a method for validating the ability of a test filter unit to produce a sterile effluent.BACKGROUND

[0002] Filters, especially sterilizing grade filters, are essential components of biotechnological manufacturing processes, particularly in Good Manufacturing Practice (GMP) environments. These filters are designed to remove microorganisms, bioburdens and other contaminants or particulates from a process fluid that is passed through the filter.

[0003] In biotechnological manufacturing, sterilizing grade filters are utilized at various critical stages. They are deployed during the preparation of raw materials, the fermentation process, and the final formulation and filling of products. For example, in the production of parenteral drugs, sterilizing grade filters are used to ensure that solutions are sterile before they are aseptically filled into vials or syringes. In the biopharmaceutical sector, sterilizing grade filters are essential for the filtration of culture media and buffer solutions, thereby maintaining contamination-free cell culture environments. Their application is critical in preserving the integrity and efficacy of biotechnological products, thereby safeguarding both the manufacturing process and the end-user (e.g., patients receiving a biopharmaceutical product).

[0004] Sterile filtration is vital in processes that produce pharmaceutical products, in particular vaccines, and other biologies, because the effective removal of contaminants is a prerequisite of meeting the stringent regulatory standards imposed on GMP processes. As any deviation from purity and sterility can lead to product recalls, financial losses, and potential harm to patients, only filters that produce a sterile effluent under the designated process conditions should be used in the manufacturing process. It is therefore vital that the filters used in the manufacturing process are validated prior to use.

[0005] Filters can be qualified as sterilizing grade and suitable for use in pharmaceutical and biopharmaceutical processes using standard test conditions, in accordance with compendial methods and industry standards such as ASTM F838-20. However, qualifying a filter as sterilizing grade per se is not sufficient to replace the processspecific validation of the filter under the actual intended use conditions.

[0006] The validation process of sterilizing grade filters is a critical aspect of ensuring their efficacy and reliability in biotechnological applications. The filter validation is part of pharmaceutical process validation and is mandatory to ensure quality and safety of the final product. It is also mandatory from a regulatory point of view to allow sales and distribution of the product. The filter validation process may begin with the end-user conducting or commissioning a Bacterial Challenge Test [BCT], which is designed to demonstrate the filter’s ability to retain microorganisms. During this test, fluid containing a predetermined concentration of a test organism is passed through the filter under controlled conditions. The filter must achieve a minimum retention level to be validated as a sterilizing grade filter. This test confirms that the filter can provide a sterile effluent under specified conditions, ensuring its performance in removing microbial contaminants.

[0007] Another important test of the filter validation process is the bacteria retention test which encompasses batch filtration, which is a method used to assess the performance of a filter in processing a specific volume of a process fluid in a singlefiltration run. The bacteria retention test is particularly useful in ensuring that the filter consistently meets the required specifications for removing contaminants, particles, or microorganisms. In general, the bacteria retention test encompasses passing a process fluid through the filter at a temperature, flow rate / pressure and duration representative of the parameters of the intended process, also referred to hereinafter as "process conditions” or "batch filtration conditions”.

[0008] Once the filters are installed in the designated production line, the end-user may perform the Pre-Use Post-Sterilization Integrity Testing (PUPSIT) to verify the filter’s integrity before its actual use in the production process. Conducting the PUPSIT tests is required in certain regions such as the European Union. PUPSIT aims to ensure that the filter has not been compromised during manufacturing or handling and that it will perform as expected during the process. Following the filtration process, a post-use integrity test is conducted to ascertain that the filter maintained its integrity throughout the operation. This involves an integrity test to confirm that no breaches occurred and may also include analysis of the filtrate to ensure that no breakthrough of microorganisms occurred. For example, a PUPSIT test may detect minor flaws on unused filter elements that could theoretically be masked during the filtration process, thus going undetected during the post-use integrity test. Such minor flaws could theoretically, but unlikely, occur during the sterilization process performed by the filter manufacturer, or during the thermal sterilization process by the end-user. The rigorous validation steps are therefore essential to guarantee that the sterilizing grade filters operate correctly and consistently, thereby ensuring the safety and quality of the biotechnological products.

[0009] Known BCT approaches evaluate the filter's microbial retention capabilities under process-specific conditions, ensuring it meets stringent microbial retention standards. However, this test does not account for potential integrity breaches that may occur during prior integrity testing processes of the filter that are carried out by the end-user.OBJECT OF THE INVENTION

[0010] Against this background, it is an object of the present invention to overcome the shortcomings present in the prior art. In particular, it is an object of the present invention to provide an improved filter validation method, in particular an improved Bacterial Challenge Test method. A further object of the present invention is to provide a combined filter validation test that assesses filter performance both under PUPSIT conditions and batch filtration conditions.BRIEF DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0011] These objects are solved in accordance with the present invention which provides a method for validating the ability of a test filter unit to produce a sterile effluent, the method comprising the steps of: a. arranging the test filter unit inside a test system, wherein the test system is a closed system configured to generate a controllable fluid flow, b. determining at least one validation parameter, c.l if the at least one validation parameter is determined to be above a first threshold, i. placing a first recovery filter downstream of the test filter unit into the test system, ii. passing a first process fluid through the test filter unit, wherein the first process fluid is inoculated with a test organism, iii. passing a sterile gas through the test filter unit, wherein steps ii. and iii. are optionally repeated at least once, iv. optionally flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit, v. passing a second process fluid through the test filter unit, wherein the second process fluid is inoculated with the test organism, vi. flushing and plating the first or optionally second recovery filter; ORc.2 if the at least one validation parameter is determined to be at or below the first threshold and above a second threshold, vii. placing a first recovery filter downstream of the test filter unit into the test system, viii. passing a first process fluid through the test filter unit, wherein the first process fluid is inoculated with a test organism, ix. passing a sterile gas through the test filter unit, wherein steps viii. and ix. are optionally repeated at least once, x. flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit, xi. preconditioning the test filter unit using a second process fluid, wherein the second process fluid is not inoculated with the test organism, xii. passing the second process fluid through the test filter unit, wherein the second process fluid is inoculated with the test organism, xiii. flushing and plating the second recovery filter; OR c.3 if the at least one validation parameter is determined to be at or below the second threshold, xiv. passing a first process fluid through the test filter unit, wherein the first process fluid is not inoculated with a test organism, xv. passing a sterile gas through the test filter unit, wherein steps xiv. and xv. are optionally repeated at least once, xvi. preconditioning the test filter unit using a second process fluid, wherein the second process fluid is not inoculated with the test organism, xvii. removing the second process fluid and flushing the test filter unit with a flush fluid, xviii. placing a first recovery filter downstream of the test filter unit, xix. passing a surrogate fluid through the test filter unit, wherein the surrogate fluid is inoculated with the test organism, xx. passing sterile gas through the test filter unit, xxi. optionally flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit,xxii. passing surrogate fluid through the test filter unit, wherein the surrogate fluid is inoculated with the test organism, xxiii. flushing and plating the first or optionally second recovery filter; wherein in each case the step of plating encompasses placing the respective first or second recovery filter and / or its effluent onto a substrate enabling growth of the test organism and incubating for a period of at least 3 hours; d. determining the amount of the test organism present in or on each of the recovery filters, wherein the ability of the test filter unit to produce a sterile effluent is validated if no recovery filter contains more than a predetermined amount of the test organism.

[0013] The above-named objects are further solved in accordance with the present invention by a computer program configured to carry out at least steps c. and d. of the method according to the present invention by controlling a test system, which is a closed system configured to generate a controllable fluid flow, wherein the test system comprises a first or optionally second recovery filter downstream of the test filter unit.

[0015] The above-named objects are further solved in accordance with the present invention by a data storage device carrying the information whether the test filter is validated according to a method of the present disclosure and information on process conditions under which the method of the present disclosure was performed.

[0017] The above-named objects are further solved in accordance with the present invention by a method for creating a data set, comprising carrying out multiples of the method according to the present disclosure.

[0019] The object named above is further solved in accordance with the present invention by a use of the data set according to the present disclosure to determine whether a test filter unit produces a sterile effluent under predetermined process conditions. In this context, the term "determining” can be understood as calculating or computing, especially modelling, whether the test filter unit will produce a sterileeffluent. Advantageously, by using a data set to determine whether a test filter unit produces a sterile effluent under predetermined process conditions, a filter can be validated without having to carry out a (physical) validation method of the present disclosure, which may save time and costs.

[0020] The method for validating the ability of a test filter unit to produce a sterile effluent is also referred to herein as "validation method”.

[0021] In the context of the present disclosure, a filter is understood to be a device configured to selectively separate contaminants or particles from a medium, which hereinafter is also referred to as a "process fluid”. Typically, the filter retains the contaminants or particles and blocks them from passing through, whereas the remaining parts of the medium pass through the filter. Preferably, a filter according to the present disclosure is a sterilizing grade filter. This is a filter effective in retaining microorganisms, thus separating them from the remaining medium. Generally, a filter has a first surface (hereinafter also referred to as "upstream surface”) and an opposite second surface (hereinafter also referred to as "downstream surface"), wherein the medium passes through the filter in the direction from the first surface to the second surface. In the context of the present disclosure, particles or contaminants that are retained by the filter are collected at its first surface or between the first and second surface (i.e., within the filter), for example by adsorptive retention. A specifically relevant embodiment of particles or contaminants according to the invention are microorganisms. For example, a filter may comprise one or more filter membranes.

[0022] In the context of the present disclosure, a test filter unit is understood as a device that is representative for the filter to be validated. Preferably, the test filter unit is smaller than the filter to be validated, but otherwise has the same structure and function as the filter to be validated. For example, a test filter unit may comprise one or more filter membranes. For example, a test filter unit can comprise or consist of Nylon, PES, PTFE, Cellulose Acetate or mixtures thereof. For example, a test filter unit may have a filter membrane with a pore size (largest pore diameter) of 0.22 pm or lower, such as0.2 pm or 0.1 pm. For example, a test filter unit may have the form of a disc having a diameter in the range of 10 to 50 mm, such as 47 mm. Alternatively, the test filter unit may have the form of a capsule or cartridge, for example. For example, a test filter unit may have a first surface area in the range of 150 to 2000 cm2.

[0023] In an exemplary embodiment, a test filter unit may comprise more than one filter membranes which may be different from one another or the same. For example, different filter membranes may have a different surface area and / or a different pore size, and / or may be of a different filter type and / or may comprise or consist of a different filter material.

[0024] In the context of the present disclosure, a sterile effluent is understood to be an effluent that does not contain living microorganisms in the sense that plating of the effluent onto a substrate enabling growth of microorganisms and incubating for a period of at least 3 hours does not result in significant growth of microorganisms. If a filter is able to produce a sterile effluent under certain process conditions, this indicates that the filter is a sterilizing grade filter under these process conditions.

[0025] In the context of the present disclosure, a test system is understood to be a closed system configured to generate a controllable flow of fluid. Generally, the direction of fluid flow in the test system is from upstream to downstream. A preferred test system comprises a multitude of elements, in particular at least one liquid pressure source for a process fluid, at least one gas pressure source for compressed gas, means to switch between flow of process fluid and compressed gas (e.g.: a valve or similar mechanism), at least one test filter housing, also referred to as a "capsule”, configured to house a test filter unit, and preferably at least one recovery filter housing which is configured to house a recovery filter and which is arranged downstream of the test filter housing. Therein, the capsule may be a filter housing, for example. The test system may additionally comprise one or more pressure sensors. It is preferred that the elements of the test system are connected to one another by fluid-tight lines.

[0026] In an exemplary embodiment where the test filter unit comprises more than one filter membrane, the individual test filter membranes maybe housed in separate test filter housings which are located within the same flow stream of the test system, i.e. downstream / upstream of one another, optionally with a pressure gauge located inbetween the separate test filter housings. In this exemplary embodiment, it is generally understood that the recovery filter housing is located downstream of the test filter housings. In this exemplary embodiment, the test filter unit may comprise filter membranes of more than one different membrane type, which may in turn result in different minimum acceptable bubble point pressures and / or different intended process durations of the different filter membrane types. When carrying out the method with a test filter unit comprising filter membranes of more than one different membrane type, it is generally preferred that the method is carried out at a pressure corresponding to the highest of the minimum acceptable bubble point pressures and at a duration corresponding to the longest of the different intended process durations of the different filter membrane types of the test filter unit to be validated.

[0027] In the context of the present disclosure, a fluid is understood to be a liquid or a gas. Preferred fluids are sterile gas, a (liquid) process fluid, and a (liquid) surrogate fluid.

[0028] In the context of the present disclosure, a recovery filter is understood to be a filter membrane configured to retain microorganisms. For example, the role of the recovery filter in the validation method according to the present disclosure is to retain any bacterial or fungal cells that may have penetrated through the test filter units during the bacterial challenge phase of testing. The recovery filter thus allows for an evaluation of the ability of the test filter unit to produce a sterile effluent. For example, a recovery filter may comprise or consist of cellulose acetate, cellulose nitrate, PES, or the like, preferably in a single layer (i.e. single membrane). For example, a recovery filter may have a pore size (largest pore diameter) of 0.5 pm or below, such as 0.45 pm or 0.22 pm. Without being limited thereto, a recovery filter may have the shape of a disc, e.g. having a diameter of 47 mm or 136 mm.

[0029] In the context of the present disclosure, a process fluid is understood to be the fluid intended to flow through the filter during the biotechnological process for which the filter is intended. In particular, the process fluid can be a liquid intended to be sterile filtered by the filter to be validated. The process fluid can be toxic to the at least one test organism, can have an intermediate toxicity towards the test organism, or can be nontoxic. The process fluid may encompass aqueous or non-aqueous liquids / buffers. The process fluid may contain complex additives, for example, at least one surfactant, micelle-forming entities such as liposomes, or the like. The process fluid may be a first process fluid or a second process fluid, for example.

[0030] For example, a first process fluid may be selected from the group consisting of water, saline, phosphate buffered saline (PBS), ophthalmic solution, vaccine, culture medium, and antibiotic. For example, the first process fluid may additionally contain at least one added substance, such as a preservative, a surfactant, a defoamer, a stabilizer, a buffering agent, a solubilizer, a chelating agent, an antioxidant, a cryoprotectant, and the like.

[0031] In the context of the present disclosure, a second process fluid is intended to be understood as a fluid which is unsuitable for a PUPSIT test as described e.g. in steps ii., viii., and xiv., respectively, of the method of the present disclosure. For example, the second process fluid may be unsuitable for a PUPSIT test due to incompatibility with the PUPSIT conditions or due to its high value. Consequently, the end-user of the filter might opt to utilize an alternative fluid - specifically, the first process fluid - to conduct the PUPSIT test. In another exemplary embodiment, the method of the present disclosure may achieve in steps ii., viii., and xiv., respectively, a saturation of the filter with a buffer component of a first process fluid, e.g. to reduce adsorption of valuable fluid components to the filter, such that, if the second process fluid is used after the saturation of the filter has been achieved, the method has the advantage that valuable fluid components of the second process fluid are not adsorbed by the filter.

[0032] For example, the second process fluid may be selected from the group consisting of ophthalmic solution, vaccine, antibiotic, and culture medium. For example, a second process fluid may additionally contain at least one added substance, such as a preservative, a surfactant, a defoamer, a stabilizer, a buffering agent, a solubilizer, a chelating agent, an antioxidant, a cryoprotectant, and the like. It is generally appreciated that the first and second process fluids can be identical. In an alternative example, the second process fluid may contain a valuable component such as, for example, an active pharmaceutical ingredient (API), and be otherwise identical to the first process fluid.

[0033] Similarly, in the context of the present disclosure, a surrogate fluid is understood to be a liquid intended to be sterile filtered by the filter to be validated, wherein the surrogate fluid is non-toxic to the at least one test organism. Exemplary surrogate fluids may include water, saline (preferably isotonic), and phosphate buffered saline, without being limited thereto.

[0034] In the context of the present disclosure, a validation parameter can be understood to encompass at least one metric pertinent to the method’s operational suitability. In this context, the determining at least one validation parameter may be understood as obtaining at least one measurement value, for instance by measuring the at least one validation parameter and / or by obtaining at least one predetermined validation parameter, e.g. from a database. Alternatively or additionally, the determining may be understood to mean calculating or computing the at least one validation parameter, e.g. from at least one predetermined value.

[0035] In the context of the present disclosure, a value is considered to be above a threshold if its numerical value is greater than the value of said threshold. Similarly, a value is considered to be at or below a threshold if its numerical value is equal to or lower than the value of said threshold.

[0036] In the context of the present disclosure, a test organism is a microorganism, such as a bacterium or mycoplasma. Moreover, in the context of the present disclosure, theterms "a microorganism”, "a bacterium”, "a mycoplasma" and "a fungus” are meant to encompass multiple microorganisms / bacteria / mycoplasmas / fungal cells of the same strain or species.

[0037] In the context of the present disclosure, the term "inoculated” can be understood to mean that a substance, such as an organism, has been introduced into a liquid, e.g. into a process fluid or surrogate fluid. Typically, in the context of the present disclosure, an inoculated fluid is provided upstream of the test filter unit.

[0038] In the context of the present disclosure, a sterile gas is a gas that has been treated to remove all viable microorganisms, ensuring that it is free from any contaminating bacteria, viruses, or other bioburdens such as spores. The skilled person is aware of methods to sterilize gas, including sterile filtration and irradiation. In the context of the present disclosure, it is generally appreciated that the sterilized gas is provided as a compressed gas. A preferred sterile gas is compressed sterile air.

[0039] In the context of the present disclosure, the term "flushing” comprises passing a flush fluid through the test filter unit with the intent to remove / neutralize any residual bactericidal and / or fungicidal impact of the previous fluid. Preferably, a minimum volume of flush fluid is passed through the test filter unit, preferably using a single pass method.

[0040] In the context of the present disclosure, a substrate enabling growth of the test organism can be understood as a liquid or solid substrate, such as an agar plate or liquid broth. In this context, the term "incubating" may imply that during the incubation period, the growth substrate is kept at conditions optimal for growth of the test organism, such as a temperature in the range of 28 to 32 °C. The duration of the incubation should be sufficient to determine the amount of the test organism by counting colonies, which, depending on the test organism, can be as short as 3 hours, or can, for example, be up to 14 days depending on the test organism, e.g. 3-5 days.

[0041] In the context of the present disclosure, determining the amount of test organism in or on a recovery filter can be understood as manually or automatically counting the number of colonies of the test organism. Moreover, the skilled person is aware of other means to determine the amount of test organism, such as turbidity measurements, fluorescence staining, Polymerase Chain Reaction and others, which are all intended to be encompassed by the present invention.

[0042] In the context of the present disclosure, the term "preconditioning the test filter unit” is to be understood to encompass passing a process fluid through the test filter unit under predetermined conditions designed to simulate the end-user’s process conditions scaled to the size of the test filter unit. For example, the predetermined conditions may encompass a predetermined duration and / or a predetermined temperature and / or a predetermined pressure or flow rate and / or a predetermined batch volume. For example, the passing may be carried out in recirculation or in a single pass.

[0043] In the context of the present disclosure, the term "process conditions" or "process parameters" shall be understood as a set of parameters reflective of the process that the filter is validated for using the method of the present invention. For example, the process conditions may comprise values predetermined by the end-user of the filter to be validated. For example, process conditions may encompass one or more of a duration (also referred to hereinafter as a "process duration”), a temperature (also referred to hereinafter as a "process temperature”), a flow rate or pressure (also referred to hereinafter as a "process flowrate” and "process pressure”), and a batch volume (also referred to hereinafter as a "process batch volume").

[0044] For example, the computer program comprises program instructions which cause a processor to perform and / or control the method according to the present disclosure when the computer program runs on a processor. For example, a processor is intended to be understood as meaning, inter alia, control units, microprocessors, microcontrol units such as microcontrollers, digital signal processors (DSP), application-specific integrated circuits (ASICs) or field programmable gate arrays (FPGAs). In this case,either all steps of the method can be controlled or all steps of the method can be performed or one or more steps can be controlled and one or more steps can be performed. The computer program may be distributable, for example, via a network such as the Internet, a telephone or mobile radio network and / or a local area network. The computer program may be at least partially software and / or firmware of a processor. It may likewise be at least partially implemented as hardware. The computer program may be stored, for example, on a computer-readable storage medium, for example a magnetic, electrical, optical and / or other type of storage medium. The storage medium may be, for example, part of the processor, for example a (non-volatile or volatile] program memory of the processor or a part thereof. The storage medium may be a tangible or physical storage medium, for example.

[0045] In the context of the present disclosure, a data set may comprise multiple entries representing aspects of at least one process condition under which the method of the present disclosure was performed and may additionally comprise entries representing whether a test filter unit produced a sterile effluent under said process conditions.

[0046] In the context of the present disclosure, the term multiples of the method is intended to be understood as carrying out the validation method with more than one different test filter unit and / or after determining more than one different validation parameter and / or under more than one different process conditions. For example, each repeat of the validation method may result in at least one data point or group of data points contributing to the data set.

[0047] For example, a data storage device is intended to be understood as a computer- readable storage device, for example a magnetic, electrical, optical and / or other type of storage device. The data storage device may be a tangible or physical storage device, for example.

[0048] The validation method according to the present disclosure has the advantage that it allows simulating cumulative stress incurred by the filter during use withconsideration to time and pressure, as closely as is feasible. A further advantage of the validation method according to the present disclosure is that it allows evaluating the impact of the stress incurred by the process filter during PUPSIT, while factoring in lab scale equipment / limitations and differing objectives, e.g., performing PUPSIT during manufacturing vs. filter validation.

[0049] Surprisingly, the inventors of the validation method according to the present invention have found that the validation test according to the present invention results in a better reflection of the intended process conditions for the filter to be validated, and thus in an advantageous increase in the predictive value of the validation result. Currently, it is theorized that the specific positioning and / or timing of the PUPSIT simulation step within the bacterial retention test causes the advantages according to the invention.

[0050] Surprisingly, the inventors of the method according to the present invention have found that performing the PUPSIT simulation after the batch filtration simulation phase of the bacterial retention test may have the disadvantage that it may increase the risk for generating a false negative bacterial challenge test result, due to potential masking effects that may occur, especially when testing process fluids with a high propensity for plugging.

[0051] For example, the validation method according to the present disclosure incorporates test parameters that represent the worst-case process conditions of the process for which the filter is intended to be used, including product composition, contact time, temperature, batch volume, type of filter, and the effective area of filtration. Worst case process parameters like product formulation, contact time, temperature, batch size, filter type and effective filtration area are incorporated and set the test parameters for the design of the method.

[0052] In some exemplary embodiments of the validation method of the present disclosure, the first recovery filter is not exchanged during the method. The advantage ofsuch an approach is minimization of manual manipulation of the test setup and by extension minimization of the risk associated with an inadvertent contamination event. However, if bacterial breakthrough does occur, the limitation of such an approach would be the inability to distinguish the phase in which the breakthrough occurred, during the PUPSIT challenge or the batch filtration challenge phase.

[0053] In alternative exemplary embodiments, the first recovery filter is exchanged for a second recovery filter. In such embodiments, it is preferred that the first (set of) recovery filter(s) is utilized during the PUPSIT challenge phase and that the second (set of) recovery filter(s) is designated for the batch filtration challenge phase of the validation method. In the event of bacterial breakthrough, this approach would allow distinction between the phase of testing in which the breakthrough occurred. However, it is important to keep in mind that with every added step (attachment, detachment, and analytical filter plating) the risk of an inadvertent contamination event increases.

[0055] In the following, further exemplary features and exemplary embodiments according to the present disclosure will be described in more detail. The individual embodiments are in each case individually applicable to the method for validating the ability of a test filter unit to produce a sterile effluent and the computer program and the data storage device and the method for creating a data set and the use of the data set. The individual embodiments may furthermore be combined with each other at will.

[0057] According to an exemplary embodiment, the method of the present disclosure is characterized in that the test organism is one or more bacteria and / or one or more mycoplasm(s). Preferably, the test organism is or comprises Brevundimonas diminuta, more preferably Brevundimonas diminuta as deposited with American Type Culture Collection (ATCC) under deposition No. 19146 or a progeny thereof. Alternatively, the test organism can comprise a microorganism which is present in the surrounding of the location where a filter unit validated through the test filter units is to be used.

[0058] Alternatively, the test organism can be Ralstonia pickettii, for example.

[0060] In an embodiment, the method of the present disclosure is characterized in that the at least one validation parameter comprises an average viability period of the test organism in the second process fluid.

[0061] In this context, an average viability period is meant to be understood as the duration of time after which at least 10% of the initial population of the test organism remains viable in the process fluid. For example, the viability period may be influenced by various factors including the composition of the process fluid, at least one process condition such as temperature, pH and / or the specific test organism.

[0062] In certain exemplary embodiments, multiple process fluids may be used, e.g., a first process fluid and a second process fluid which are not identical. In these embodiments, more than a single viability study may be performed.

[0063] The skilled person is aware of methods to determine the average viability period of the test organism in the process fluid. The average viability may be calculated or computed, or may be determined from a data base, or may be directly measured. For example, such a measurement may encompass inoculating a process fluid with a predetermined amount of test organism, incubating in the presence of a filter membrane, flushing the filter membrane, plating the filter membrane onto an appropriate growth substrate such as an agar plate, incubating, and counting colonies of the test organism.

[0065] In an embodiment, the method of the present disclosure is characterized in that the first threshold is defined in that the test organism has a viability of at least approx. 10 % after being present for a first duration at a predetermined temperature in the second process fluid, and the second threshold is defined in that the test organism has a viability of at least approx. 10 % after being present for a second duration at the predetermined temperature in the second process fluid, wherein the first duration is longer than the second duration.

[0066] In the context of the first threshold, the first duration may preferably be defined as the duration of the entire method as described in step c.l (i.e., steps i. through v.J. The first duration may be about 20 days, about 15 days, about 10 days, about 5 days, about 48 hours, about 24 hours, or about 8 hours, for example.

[0067] In the context of the second threshold, the second duration may be approx. 120 minutes, approx. 60 minutes, or approx. 30 minutes.

[0068] In the context of the first and second threshold, the predetermined temperature may preferably be the process temperature.

[0070] In an exemplary embodiment, the method of the present disclosure is characterized in that the first and second process fluids are identical.

[0071] For example, without being limited thereto, the first and second process fluids may be buffer solution, ophthalmic solution, preferably void of preservative, cell culture medium, or any other fluid described as first or second process fluid throughout this disclosure.

[0073] In a first alternative embodiment, the method of the present disclosure is characterized in that in steps iii., ix., xv., and / or xx., the sterile gas is applied to the test filter unit at an upstream pressure that is increased, e.g. incrementally increased, until a target pressure of the filter is reached, and is subsequently maintained at said target pressure for at least 60 seconds. In this embodiment, it is generally appreciated that the target pressure is equivalent to or greater than a minimum accepted bubble point pressure of the test filter unit.

[0074] In the context of the present disclosure, the term minimum accepted bubble point pressure is intended to be understood as a pressure value which is predetermined under standardized conditions for a given combination of a filter type and a wettingmedia such as a process fluid. The minimum accepted bubble point is intended to represent a minimum pressure at which the bubble point should be reached. In contrast, if a tested filter or test filter unit reaches its bubble point at a pressure below the minimum accepted bubble point, this might be indicative of structural damage, for example. The person skilled in the art is aware of methods to determine the minimum accepted bubble point.

[0075] In the context of the present disclosure, the bubble point pressure of a test filter unit is to be understood as the minimum gas pressure required to force the sterile gas through the one or more wetted test filter unit’s membranes, thereby expelling the wetting agent (e.g.: a process fluid) from the largest pores of the one or more filter membranes. For example, the bubble point pressure can be empirically determined for each test filter unit, or can e.g. be determined by determining a value from a database containing predetermined values for the bubble point pressure of the test filter unit, or can e.g. be calculated or computed. For example, the predetermined value for bubble point pressures may be a value of the minimum accepted bubble point.

[0076] According to this alternative embodiment, the passing of sterile gas through the test filter unit according to steps iii., ix., xv., and / or xx. mimics a bubble point test which is part of the Pre-Use Post-Sterilization Integrity Testing (PUPSIT) procedure usually carried out at the end user’s facility. By including a simulation of such an integrity test into the method of the present disclosure, the method has the advantage that it better reflects the worst-case conditions to which the filter is exposed at the end user’s site, so that the method has a higher predictive value that a validated filter will generate a sterile effluent under the process conditions present at the site of the end user.

[0078] In a second alternative of the above embodiment, the method of the present disclosure is characterized in that in steps iii., ix., xv., and / or xx., the sterile gas is applied to the test filter unit at an upstream pressure of 65-95 % of the bubble point pressure of the test filter unit for a predetermined period, preferably for at least 5 minutes.

[0079] According to this alternative embodiment, the passing of sterile gas through the test filter unit according to steps iii., ix., xv., and / or xx. mimics a diffusion test which is part of the Pre-Use Post-Sterilization Integrity Testing (PUPSIT) procedure usually carried out at the end user’s facility. By including a simulation of such an integrity test into the method of the present disclosure, the method has the advantage that it better reflects the worst-case conditions to which the filter is exposed at the end user’s site, so that the method has a higher predictive value that a validated filter will generate a sterile effluent under the process conditions present at the site of the end user.

[0080] Including a diffusion test according to the aforementioned embodiment is particularly advantageous in a case where the maximum allowable pressure tolerance for the test filter unit included in a single-use system is lower than the bubble point pressure reached during manufacturing using a production process filter. Under these conditions, use of a lower test pressure is advantageous because the lower test pressure can be sustained for a longer duration, thus accounting for the overall stress incurred by the process filter, which in turn results in an improved predictive value of the method according to the present disclosure. This embodiment may be understood as a comprehensive approach combining aspects of a bubble point and a diffusion integrity test.

[0082] In an exemplary embodiment, the method of the present disclosure is characterized in that in steps ii., viii., and xix., the inoculated process fluids and surrogate fluids each comprise the test organism in an amount determined on the basis of a maximum allowable wetting volume, a maximum number of trial repeats, a maximum permitted bioburden level, and a safety factor, wherein preferably the amount is at least 102CFU / cm2of a first surface area of the test filter unit.

[0083] For example, the amount of test organism can be determined by the following formula:C = l / x nPUPSITx CPx S,wherein C represents the amount of test organism, which is also referred to as "PUPSIT challenge level". The numerical value of C represents the amount of test organism in CFU / cm2of the first surface area of the test filter unit;Vw represents the Maximum Allowable Wetting Volume for the respective step of the method; npupsiT may represent the maximum number of repeats of steps ii., viii., and xix., respectively; for example, npupsiT may correspond to the number of PUPSIT repeats intended to be carried out at the end-user’s facility;CB represents the Maximum Permitted process Bioburden Level for the specific test filter unit, andS represents a safety factor, which can usually be in the range of 1 to 100.

[0084] In this exemplary embodiment, an advantage of the validation method is that it provides a customizable and process specific PUPSIT simulation phase challenge level option based on process specific needs. Incorporating a process specific PUPSIT simulation in the presence of the test bacteria in the system as part of the bacterial retention study has the particular advantage that it provides an extra measure to simulate and substantiate the use of worst-case PUPSIT conditions, which in turn results in an improved predictive value of the method according to the present disclosure.

[0085] Incorporation of the safety factor when determining the PUPSIT challenge level has the advantage of providing means to accommodate unexpected variations during filter manufacture and validation testing, such as the necessity for an increased PUPSIT wetting volume.

[0087] In an exemplary embodiment, the method of the present disclosure is characterized in that in step v., xii., and xxii., the inoculated process fluids and surrogate fluids each comprise the test organism in an amount of at least 107CFU / cm2of the first surface area of the test filter unit.

[0088] In the above-mentioned process steps, the method of the present disclosure mimics a bacterial challenge test [BCT] usually carried out at the filter manufacturer’s site. By having an amount of at least 107CFU / cm2of the first surface area of the test filter unit, it is ensured that the method mimics a worst-case scenario that the filter to be validated might be exposed to at the end user’s site, which in turn results in an improved predictive value of the method according to the present disclosure.

[0090] In an exemplary embodiment, the method of the present disclosure is characterized in that in step d., the predetermined amount of the test organism is 0 CFU of the test organism in or on each recovery filter.

[0091] By having a strict requirement of 0 CFU, the method has an improved predictive value that a validated filter will produce a sterile effluent under the intended process conditions.

[0093] In an exemplary embodiment, the method according to the present disclosure is characterized in that prior to step ii. and v., viii. and xii., or xix. and xxii., respectively, the method comprises the step of acquiring a monitoring sample upstream of the test filter unit. In this embodiment, the method is further characterized in that the method additionally comprises the steps of (1) plating each monitoring sample onto a substrate enabling growth of the test organism and incubating for a period of at least 3 hours, and (2) determining the amount of the test organism present in each of the monitoring samples. In this embodiment, the method is still further characterized in that in step d. the ability of the test filter unit to produce a sterile effluent is validated if no recovery filter contains more than 0 CFU of the test organism and if each of the monitoring samples contains at least a predetermined amount of the test organism.

[0094] In this exemplary embodiment, the predetermined amount of the test organism may be approximately equal to the amount of test organism that is present in the process fluid, for example at least 102CFU / cm of the first surface area or at least 107CFU / cm2of the first surface area.

[0095] In this exemplary embodiment, the method allows for controlling whether the intended amount of test organism has been reached, whereby the predictive value of the filter validation method according to the present disclosure is improved.

[0097] In a further exemplary embodiment, the method according to the present disclosure is characterized in that in step d., the amount of the test organism present in each of the recovery filters is determined by determining the number of colonies of the test organism present on the respective recovery filter.

[0098] In this context, the determining can be understood as manually or automatically counting the number of colonies, or may encompass real-time and in-line detection technology.

[0100] In an exemplary embodiment, the method according to the present disclosure is characterized in that step a. further comprises determining at least one process parameter, and in that at least steps v., xi., xii., xvi., and xxii. are carried out according to the at least one process parameter.

[0101] In the context of the present disclosure, a process parameter is intended to represent the process of intended use of the filter to be validated by the method of the present disclosure, i.e., the process to be carried out with the validated filter at the enduser site. For example, the at least one process parameter may be selected from the group consisting of process temperature, process duration, maximum process differential pressure, and process flow rate.

[0102] In the context of the present disclosure, carrying out the respective method step according to the at least one process parameter can be understood as carrying out the respective method step for a duration equal to the process duration and / or at a temperature equal to the process temperature, and so on.

[0103] In this exemplary embodiment, the method of the present disclosure has the advantage that it better mimics the process conditions present at the end-user site, whereby the predictive value of the method is improved.

[0105] In the context of the above embodiment, the method may further comprise the step of adapting at least one process parameter and / or repeating the method with a different test filter unit if, in step d., it is determined that any recovery filter contains more than the predetermined amount of the test organism.

[0106] In this context, the adapting may be understood as changing a value in one or more process parameter, e.g., decreasing or increasing a process duration, and / or decreasing or increasing a process temperature, and / or decreasing or increasing a process pressure / flowrate, and / or decreasing or increasing a process batch size.

[0107] In the context of repeating the method with a different test filter unit, it is preferred that the different test filter unit is a different type of filter, i.e., a filter having a different structure and / or consisting of a different combination of materials.

[0108] Further features and advantages of the method for validating the ability of a test filter unit to produce a sterile effluent, the computer program, the data set, the method for creating the data set, and the use of the data set emerge from the following description of exemplary embodiments where reference is made to the attached drawings.

[0110] In the drawings,

[0111] Fig. 1 is a schematic drawing of a test system configured to carry out the invention, and

[0112] Fig. 2 is a schematic drawing of the method for validating the ability of a test filter unit to produce a sterile effluent according to the present disclosure.

[0115] Fig. 1 shows an exemplary embodiment of a test system 100 configured to carry out the invention. According to the exemplary embodiment shown, the test system 100 encompasses a gas pressure source 110 for compressed gas, and a liquid pressure source 120. The liquid pressure source 120 is supplied with a fluid, which may be a first process fluid or a second process fluid or a flush fluid or a surrogate fluid, by a reservoir 121.

[0116] According to the exemplary embodiment shown in Fig. 1, the gas pressure source 110 and the liquid pressure source 120 are both connected to a valve 130 which is set up to direct either the compressed gas from the gas pressure source 110 or the liquid from the liquid pressure source 120 further downstream towards an optional pressure sensor 135 and the test filter housings 140. According to Fig. 1, the direction of flow is indicated by arrows.

[0117] Each of the test filter housings 140 is configured to house a test filter unit. Downstream of each test filter housing 140, an optional pressure sensor 145 and a recovery filter housing 150 is arranged. Each recovery filter housing 150 is configured to house a recovery filter, which can be a first or second or further recovery filter.

[0118] The elements of the test system are connected to one another in a fluid-tight manner by means of lines 160. According to the exemplary embodiment shown in Fig. 1, the lines 160 connect the recovery filter housings 150 back to the reservoir 121, such that the first process fluid or second process fluid or flush fluid or surrogate fluid or sterile gas are circulated through the test system. In an alternate embodiment (not shown here), the recovery filter housings 150 can be connected to a waste reservoir, for example.

[0119] In the direction of flow, a first process fluid or a second process fluid or a flush fluid or a surrogate fluid originate from the respective reservoir 121, and are pumpedthrough the test system 100, successively passing through the liquid pressure source 120, the valve 130, the optional pressure sensor 135, a test filter housing 140, an optional pressure sensor 145, and the recovery filter housing 150, before returning to the reservoir 121.

[0120] The skilled person appreciates that the number of parallel test filter housings 140 and pressure sensors 145 and recovery filter housings 150, which are 3 according to the exemplary embodiment shown in Fig. 1, can vary, e.g., between 1 and 10, especially between 1 and 5.

[0122] Fig. 2 shows a schematic overview of the method for validating the ability of a test filter unit to produce a sterile effluent according to the present disclosure.

[0123] The method for validating the ability of a test filter unit to produce a sterile effluent according to the present disclosure comprises a step 310 / a. of arranging the test filter unit inside a test system, wherein the test system is a closed system configured to generate a controllable fluid flow. The test system may be a test system 100 as shown in Fig. 1.

[0124] The method further comprises a step 320 / b. of determining at least one validation parameter. As described above, the determining may, for example, be achieved by direct measurement, and / or by obtaining at least one predetermined validation parameter, and / or by calculating or computing the at least one validation parameter.

[0125] According to the at least one validation parameter determined in step 320 / b., a decision is made: If (330 / c.l) the at least one validation parameter is determined to be above a first threshold, the method steps 331 / i., 332 / ii., 333 / iii., optionally 334 / iv., 335 / v., and 336 / vi. are carried out in succession, wherein optionally the steps 332 / ii. and 333 / iii. can be repeated at least once. If (340 / c.2) the at least one validation parameter is determined to be at or below the first threshold and above a secondthreshold, the method steps 341 / vii., 342 / viii., 343 / ix., 344 / x., 345 / xi., 346 / xii., and 347 / xiii. are carried out in succession, wherein optionally the steps 342 / viii. and 343 / ix. can be repeated at least once. If (350 / c.3J the at least one validation parameter is determined to be at or below the second threshold, the method steps 351 / xiv., 352 / xv., 353 / xvi., 354 / xvii., 355 / xviii., 356 / xix., 357 / xx., optionally 358 / xxi., 359 / xxii., and 360 / xxiii. are carried out in succession, wherein optionally the steps 351 / xiv. and 352 / xv. can be repeated at least once.

[0126] It is generally preferred that the at least one validation parameter pertains to the average survival time of the test organism inside the first or second process fluid.Therefore, if the process fluid is non-toxic to the test organism, method steps 331 / i. to 336 / vi. maybe carried out, whereas if the process fluid shows intermediate toxicity to the test organism, method steps 341 / vii. to 347 / xiii. are carried out, and whereas if the process fluid shows high toxicity to the test organism, method steps 351 / xiv. to 360 / xxiii. are carried out. In each case, the method ensures that the validation comprises both a robust bacterial challenge and a significant structural challenge of the test filter unit.

[0127] Method steps 331 / i. to 336 / vi. encompass a structural challenge testofthe test filter unit by passing first liquid (step 332 / ii.J and then gas (step 333 / iii.) through the test filter unit. Afterwards, the first recovery filter can optionally be analyzed and exchanged for the second recovery filter, before a bacterial challenge test is carried out in steps 335 / v. and 336 / vi.. Because the first process fluid is non-toxic towards the test organism, no additional steps apart from step 370 / d. are necessary to validate the test filter unit.

[0128] Method steps 341 / vii. to 347 / xiii. encompass a structural challenge test of the test filter unit by passing first liquid (step 342 / viii.) and then gas (step 343 / ix . through the test filter unit. Since the first process fluid is partially toxic towards the test organism, analysis and exchange of the recovery filter according to step 344 / x. is - contrary to the method according to steps 331 / i. to 336 / vi. - mandatory in order toavoid false negative results. Afterwards, in step 345 / xi. the test filter unit is preconditioned with the second process fluid not inoculated with the test organism, wherein the preconditioning preferably is carried out to mimic the intended process conditions present at the end user. Afterwards, in steps 346 / xii. and 347 / xiii., a bacterial challenge test is carried out with the second process fluid.

[0129] Method steps 351 / xiv. to 360 / xxiii. encompass a structural challenge test of the test filter unit by passing first liquid (step 351 / xiv.) and then gas (step 352 / xv.) through the test filter unit. Given that the first process fluid is toxic for the test organism, the first process fluid is not inoculated, and no recovery filter is present at this point. In the subsequent step 353 / xvi., the test filter unit is preconditioned with a second process fluid, which may be identical to the first process fluid, to mimic the intended process conditions present at the end user. After the preconditioning, in step 354 / xvii. the second process fluid is removed and the test filter unit is flushed with a flush fluid, wherein preferably the flush fluid is non-toxic for the test organism. Subsequently, in step 355 / xviii. a recovery filter is placed downstream of the test filter unit. In steps 356 / xix., and 357 / xx., a structural test is carried out by passing first a non-toxic surrogate fluid and then a sterile gas through the test filter unit, with optional analysis and exchange of the recovery filter in step 358 / xxi.. Finally, in steps 359 / xxii. and 360 / xxiii., a bacterial challenge test is carried out with the non-toxic surrogate fluid.

[0130] Method steps 331 / i., 341 / vii., and 355 / xviii. each encompass placing a first recovery filter downstream of the test filter unit. When using a test system 100 as shown in Fig. 1, the first recovery filter may be placed inside the recovery filter housing 150. In this case, the test filter unit is present in the test filter housing 140, such that the recovery filter is arranged downstream of the test filter unit in the test system 100.

[0131] Method steps 332 / ii., and 342 / viii. each encompass passing a first process fluid through the test filter unit, wherein the first process fluid is inoculated with a test organism. According to one exemplary embodiment, the amount of test organism contained in the first process fluid may be at least 102CFU / cm2of a first surface area ofthe test filter unit, preferably greater than or equal to 107CFU / cm2of the first surface area of the test filter unit. In these embodiments, the method has the particular advantage of allowing customization of the target challenge level based on the actual process conditions of the end-user, whereas known validation tests could lead to unnecessary false failures due to unrealistic test conditions.

[0132] In contrast, method step 351 / xiv. encompasses passing a first process fluid through the test filter unit, wherein the first process fluid is not inoculated with a test organism. In this case, because the first process fluid is toxic for the test organism, an accumulation of test organism fragments in the filter is avoided by not inoculating the first process fluid with the test organism. In this case, even if the first process fluid was inoculated with the test organism, it would be impossible to determine the bacterial retention ability of the test filter unit because units of the test organism that pass though the test filter unit are killed due to toxicity and thus, bacterial growth on a recovery filter cannot be detected.

[0133] Method steps 333 / iii., 343 / ix., 352 / xv., and 357 / xx. each encompass passing sterile gas through the test filter unit. In exemplary embodiments of the present disclosure described above, the gas pressure can be the bubble point pressure of the respective test filter unit, or can be below the bubble point pressure.

[0134] In the optional method steps 334 / iv. and 358 / xxi., and in method steps 336 / vi., 344 / x., 347 / xiii., and 360 / xxiii., the first recovery filter is exchanged and analyzed. This is generally done, for example, by minimally flushing and then aseptically disconnecting the recovery filter housing from the downstream side of the test filter unit, extracting the recovery filter and placing it onto a growth substrate such as a TSA (tryptic soy agar) plate, and incubating for 3 to 5 days at 30±2 °C. A second recovery filter is placed into the test system, for example into the recovery filter housing 150, and the method is continued. In method steps 336 / vi., 347 / xiii., and 360 / xxiii., the recovery filter is removed and analyzed in a similar fashion.

[0135] In method steps 335 / v., 346 / xii., and 359 / xxii., a second process fluid or surrogate fluid inoculated with the test organism is passed through the test filter unit as part of a bacterial challenge test. In an exemplary embodiment of the present disclosure described above, the amount of test organism is at least 107CFU per cm2of the first surface area of the test filter unit. This test is generally aimed to mimic the worst-case conditions of a bacterial contamination under the intended use case of the filter to be validated, optionally in combination with a process condition test as described, for example, in method steps 345 / xi. and 353 / xvi. throughout this disclosure. For example, step 359 / xxii. may be carried out using standard conditions, such as a pressure of 2 bar for a duration of 30 minutes. For example, step 346 / xii. may be carried out for a short duration of 15 minutes or less, for example, at a pressure representing the process pressure.

[0136] In method steps 345 / xi. and 353 / xvi., the test filter unit is preconditioned using a second process fluid. As generally appreciated by a skilled person, the second process fluid and the first process fluid may be identical or may be different from each other.

[0137] For example, the preconditioning accordingto step 345 / xi. and 353 / xvi., as well as the method steps 335 / v., 346 / xii., and 359 / xxii. may be carried out according to at least one process parameter, which may be an intended process duration and / or an intended process temperature.

[0138] Example 1The ability of a test filter unit to produce a sterile effluent was tested by a method according to the present disclosure.

[0139] A cellulose acetate membrane type, size 4 capsule, 0.2 gm rated, with first surface area of 150 cm2, was used as a test filter unit. The test filter unit was placed inside a closed test system configured to generate a controllable fluid flow.

[0140] To determine a validation parameter, a predetermined amount of test organism was suspended in a first process fluid. Brevundimonas diminuta was used as test organism. The first process fluid was DMEM cell culture medium, which is commonly known in the field. After 30 minutes of incubating the test organism in the first process fluid (second threshold), it was found that the viability of the test organism was 100 %. After 60 minutes of said incubation (first threshold), the viability of the test organism was still 100 %. Therefore, the validation parameter was determined to be above the first threshold, such that the method steps i. to vi. were carried out.

[0141] A single-layer cellulose acetate filter disc having a pore size of 0.45 pm and a diameter of 136 mm was placed as recovery filter into a recovery filter housing in the test system downstream of the capsule housing the test filter unit.

[0142] A total volume of 2 L DMEM inoculated with 2 x 102CFU I cm2of the first surface area was passed through the test filter unit at a temperature of 20-25 °C for a duration of 2 min. Subsequently, sterile air was passed through the test filter unit at an upstream pressure of 4 bar, corresponding to 125 % of the predetermined minimum bubble point pressure of the test filter type, for a duration of 10 min. These steps were repeated a total of 3 times.

[0143] The first recovery filter was flushed with 1 L of sterile saline at a pressure / flowrate below the target gas pressure applied (<4 bar), removed from the recovery filter housing, and placed onto an agar plate. In its stead, a second recovery filter was placed into the recovery filter housing.

[0144] As a second process fluid, DMEM culture medium identical to the first process fluid was used. DMEM inoculated with > 107CFU I cm2of the first surface area was passed through the test filter unit at a flow rate of 0.08 L / min and at a temperature of 20-25 °C for a duration of 10 hours. Subsequently, the second recovery filter was flushed, removed and plated as described above with respect to the first recovery filter.

[0145] The first and second recovery filters were incubated on the agar plates for a period of at least 3 days. The number of colonies of the test organism was determined by manual counting. No colonies were found on any agar plate. Therefore, the ability of the test filter unit to produce a sterile effluent was validated.

[0146] Example 2:In a variant of the method of Example 1 above, the same test filter unit was tested with the method according to the present disclosure using a different process fluid.

[0147] In this experiment, an aqueous buffer having a pH of 5, which has an intermediate toxicity towards the test organism B. diminuta, was used as first process fluid. By obtaining predetermined viability values from a database, it was determined that the test organism had a viability of >10 % after 30 minutes of incubation in the first process fluid (second threshold), and had a viability of <10 % after 60 minutes of incubation in the first process fluid (first threshold). Accordingly, the validation parameter was determined to be below the first threshold and above the second threshold, and the method comprising steps vii. to xiii. was carried out.

[0148] As described above for Example 1, a first recovery filter was placed into the test system. The first process fluid subsequently passed through the test filter unit was inoculated with B. diminuta in an amount equal to 105CFU / cm2of the first surface area, which was controlled and confirmed by acquiring a sample of the first process fluid upstream of the test filter unit, diluting the sample 1:100 in 0.9% saline and plating the sample onto agar plates.

[0149] As described for example 1 above, sterile air was passed through the test filter unit and the first recovery filter was exchanged for a second recovery filter.

[0150] The test filter unit was preconditioned for a duration of 5.5 hours at a pressure of3 bar with a second process fluid. The second process fluid was Ophthalmic solutioncontaining a preservative. The second process fluid was not inoculated with the test organism.

[0151] Subsequently, second process fluid inoculated with B. diminuta in an amount equal to >107CFU / cm2of the first surface area, which was controlled and confirmed as described above, was passed through the test filter unit for a duration of 30 minutes at a pressure of 3 bar.

[0152] The second recovery filter was flushed and plated as described above. The amount of test organism was determined as described above in example 1. No bacterial colonies were found on any agar plate. Therefore, the ability of the test filter unit to produce a sterile effluent under the used process parameters was validated.

[0153] Example 3:In a third test, the ability of the test filter unit described above in example 1 to produce a sterile effluent was again tested in a different environment.

[0154] As a first process fluid, 60 % isopropyl alcohol was used. The test described above for Example 1 was used to determine the viability of the test organism in the process fluid. No viable bacteria were found after 5 minutes of incubation in the first process fluid [below second threshold]. Accordingly, the validation parameter was determined to be at or below the second threshold and the method comprising steps xiv. to xxiii. was carried out.

[0155] The first process fluid was passed through the test filter unit at a flow rate of 0.25 L / min and a temperature of 20-25 °C for a duration of 4 min. Subsequently, sterile nitrogen gas was passed through the test filter unit at a pressure of 3.2 bar, which corresponds to the bubble point pressure of the test filter unit, for 10 min. These steps were repeated for 5 times in total.

[0156] Afterwards, the test filter unit was preconditioned, using the first process fluid as a second process fluid, for a duration of 3 days at a pressure of 2.5 bar. The second process fluid was removed, and the test filter unit was flushed with sterile water as a flush fluid at a pressure of < 2.5 bar for a duration of 15 minutes.

[0157] A first recovery filter was placed as described above with respect to example 1.

[0158] Subsequently, surrogate fluid and then sterile gas was passed through the test filter unit as described above in Example 3. Sterile Phosphate-Buffered Saline (PBS) was used as a surrogate fluid. The PBS was inoculated with the test organism B. diminuta in an amount of 106CFU / cm2of the first surface area of the test filter unit.

[0159] Without exchanging the recovery filter, PBS inoculated with B. diminuta in an amount of 107CFU / cm2of the first surface area of the test filter unit was passed through the test filter unit. The recovery filter was flushed and plated as described above with respect to Example 1.

[0160] The amount of test organism was determined as described above. No bacterial colonies were found on the agar plate, such that the test filter unit’s ability to produce a sterile effluent was validated.

Claims

C l a i m s1. A method for validating the ability of a test filter unit to produce a sterile effluent, the method comprising the steps of: a. arranging the test filter unit inside a test system, wherein the test system is a closed system configured to generate a controllable fluid flow, b. determining at least one validation parameter, c.l if the at least one validation parameter is determined to be above a first threshold, i. placing a first recovery filter downstream of the test filter unit into the test system, ii. passing a first process fluid through the test filter unit, wherein the first process fluid is inoculated with a test organism, iii. passing a sterile gas through the test filter unit, wherein steps ii. and iii. are optionally repeated at least once, iv. optionally flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit, v. passing a second process fluid through the test filter unit, wherein the second process fluid is inoculated with the test organism, vi. flushing and plating the first or optionally second recovery filter; OR c.2 if the at least one validation parameter is determined to be at or below the first threshold and above a second threshold, vii. placing a first recovery filter downstream of the test filter unit into the test system, viii. passing a first process fluid through the test filter unit, wherein the first process fluid is inoculated with a test organism, ix. passing a sterile gas through the test filter unit, wherein steps viii. and ix. are optionally repeated at least once, x. flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit,xi. preconditioning the test filter unit using a second process fluid, wherein the second process fluid is not inoculated with the test organism, xii. passing the second process fluid through the test filter unit, wherein the second process fluid is inoculated with the test organism, xiii. flushing and plating the second recovery filter; OR c.3 if the at least one validation parameter is determined to be at or below the second threshold, xiv. passing a first process fluid through the test filter unit, wherein the first process fluid is not inoculated with a test organism, xv. passing a sterile gas through the test filter unit, wherein steps xiv. and xv. are optionally repeated at least once, xvi. preconditioning the test filter unit using a second process fluid, wherein the second process fluid is not inoculated with the test organism, xvii. removing the second process fluid and flushing the test filter unit with a flush fluid, xviii. placing a first recovery filter downstream of the test filter unit, xix. passing a surrogate fluid through the test filter unit, wherein the surrogate fluid is inoculated with the test organism, xx. passing sterile gas through the test filter unit, xxi. optionally flushing and plating the first recovery filter and placing a second recovery filter downstream of the test filter unit, xxii. passing surrogate fluid through the test filter unit, wherein the surrogate fluid is inoculated with the test organism, xxiii. flushing and plating the first or optionally second recovery filter; wherein in each case the step of plating encompasses placing the respective first or second recovery filter and / or its effluent onto a substrate enabling growth of the test organism and incubating for a period of at least 3 hours;d. determining the amount of the test organism present in or on each of the recovery filters, wherein the ability of the test filter unit to produce a sterile effluent is validated if no recovery filter contains more than a predetermined amount of the test organism.

2. The method according to claim 1, characterized in that the test organism is one or more bacteria and / or mycoplasma(s), preferably the test organism is or comprises Brevundimonas diminuta, more preferably Brevundimonas diminuta as deposited with American Type Culture Collection (ATCC) under deposition No. 19146, or a microorganism which is present in the surrounding of the location where a filter unit validated through the test filter units is to be used.

3. The method according to claim 1 or claim 2, characterized in that the at least one validation parameter comprises an average viability period of the test organism in the second process fluid.

4. The method according to claim 3, characterized in that the first threshold is defined in that the test organism has a viability of at least approx. 10 % after being present for a first duration at a predetermined temperature in the second process fluid, and the second threshold is defined in that the test organism has a viability of at least approx. 10 % after being present for a second duration at the predetermined temperature in the second process fluid, wherein the first duration is longer than the second duration.

5. The method according to any of the preceding claims, characterized in that the first and second process fluids are identical.

6. The method according to any of the preceding claims, characterized in that in steps iii., ix., xv., and / or xx., the sterile gas is applied to the test filter unit at an upstream pressure that is increased until a target pressure of the filter is reached, and is subsequently maintained at said target pressure for at least 60 seconds.

7. The method according to any of claims 1 to 5, characterized in that in steps iii., ix., xv., and / or xx., the sterile gas is applied to the test filter unit at an upstream pressure of 65-95 % of the bubble point pressure of the test filter unit for a predetermined period, preferably for at least 5 minutes.

8. The method according to any of the preceding claims, characterized in that in steps ii., viii., and xix., the inoculated process fluids and surrogate fluids each comprise the test organism in an amount determined on the basis of a maximum allowable wetting volume, a maximum number of trial repeats, a maximum permitted bioburden level, and a safety factor, wherein preferably the amount is at least 102CFU / cm2of a first surface area of the test filter unit.

9. The method according to any of the preceding claims, characterized in that in step v., xii., and xxii., the inoculated process fluids and surrogate fluids each comprise the test organism in an amount of at least 107CFU / cm2of the first surface area of the test filter unit.

10. The method according to any one of the preceding claims, characterized in that in step d., the predetermined amount of the test organism is 0 CFU of the test organism in each recovery filter.

11. The method according to any of the preceding claims, characterized in that prior to step ii. and v., viii. and xii., or xix. and xxii., respectively, the method further comprises the step of acquiring a monitoring sample upstream of the test filter unit, further characterized in that the method additionally comprises the steps of: plating each monitoring sample onto a substrate enabling growth of the test organism and incubating for a period of at least 3 hours, determining the amount of the test organism present in each of the monitoring samples,and still further characterized in that in step d. the ability of the test filter unit to produce a sterile effluent is validated if no recovery filter contains more than 0 CFU of the test organism and if each of the monitoring samples contains at least a predetermined amount of the test organism.

12. The method according to any one of the preceding claims, characterized in that in step d., the amount of the test organism present in each of the recovery filters is determined by determining the number of colonies of the test organism present on the respective recovery filter.

13. The method according to any of the preceding claims, characterized in that step a. further comprises determining at least one process parameter, and in that at least steps v., xi., xii., xvi., and xxii. are carried out according to the at least one process parameter.

14. The method according to claim 13, further comprising the step of adapting at least one process parameter and / or repeating the method with a different test filter unit if, in step d., it is determined that any recovery filter contains more than the predetermined amount of the test organism.

15. Computer program configured to carry out at least steps c. to d. of the method according to any of the preceding claims by controlling a test system, which is a closed system configured to generate a controllable fluid flow, wherein the test system comprises a first recovery filter downstream of the test filter unit.

16. Data storage device carrying the information whether the test filter is validated according to the method of claims 1 to 14 and under what process conditions of method of claim 1 to 14.

17. A method for creating a data set, comprising carrying out multiples of the method according to any one of claims 1 to 14.

18. Use of the data set according to claim 17 to determine whether a test filter unit produces a sterile effluent under predetermined process conditions.

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