Evaluation of sanitary methods for bioprocessing equipment

Abstract

A method 10 is disclosed for evaluating a sanitization method used to sanitize a bioprocessing apparatus or to evaluate the sanitary design of a bioprocessing apparatus. The method includes the steps of: pre-washing the bioprocessing apparatus 100; preparing a mixture of test microorganisms containing two or more different microbial strains 101; applying the mixture of test microorganisms to the bioprocessing apparatus 102; waiting for at least two hours; sanitizing the bioprocessing apparatus 104; collecting at least one test sample potentially containing the test microorganisms from at least one location of the bioprocessing apparatus after sanitization 105; transferring at least a portion of the collected test sample to a culture medium 106; incubating the culture medium to allow any potentially present test microorganisms to grow and differentiate in the culture medium 107; and identifying and / or counting the amount of test microorganisms in each microbial strain grown in the culture medium 108.

Description

[Technical Field]

[0001] This disclosure relates to the evaluation of sanitization methods used for sanitizing bioprocessing equipment or for evaluating the sanitary design of bioprocessing equipment. [Background technology]

[0002] Microbial contamination, specifically bacteria, yeast, and mold, is common in many laboratory and production environments. Under favorable conditions, these microorganisms can rapidly multiply in enormous quantities, potentially impairing the function and performance of bioprocessing equipment, such as chromatography apparatus and chromatography resins. Furthermore, microorganisms can remain as contaminants in bioproducts throughout the manufacturing process, resulting in batch failures and associated costs. Therefore, adhering to hygiene routines throughout the entire production process is crucial for patient safety, in order to comply with Good Manufacturing Practices (GMP) and meet stringent regulations.

[0003] Sanitization is defined as the use of chemical agents to reduce microbial communities and is typically used to maintain microbial presence in bioprocessing equipment at a level that minimizes the risk of batch failure and product contamination. Sanitization may also be used to evaluate the sanitary design of bioprocessing equipment. Evaluating the sanitization methods used to sanitize bioprocessing equipment, or evaluating the sanitary design of bioprocessing equipment and whether changes to the sanitization methods or design are necessary, are both time-consuming and costly. [Overview of the project]

[0004] The object of this disclosure is to provide a faster and / or improved method for evaluating sanitization methods used to sanitize bioprocessing equipment or to evaluate the sanitary design of bioprocessing equipment.

[0005] The present invention is defined by the appended independent claims. Non-limiting embodiments will become apparent from the dependent claims, the appended drawings, and the following specification.

[0006] According to the first aspect, a method is provided for evaluating the effectiveness of a sanitization method used to sanitize a bioprocessing apparatus or to evaluate the sanitary design of a bioprocessing apparatus, the method comprising the steps of: pre-washing the bioprocessing apparatus; preparing a mixture of test microorganisms containing two or more different microbial strains; applying the mixture of test microorganisms to the bioprocessing apparatus; waiting for at least two hours, sanitizing the bioprocessing apparatus; collecting at least one test sample potentially containing the test microorganisms from at least one location in the bioprocessing apparatus after the sanitization step; transferring at least a portion of the collected test sample to a culture medium; incubating the culture medium to allow any potentially present test microorganisms to grow and differentiate in the culture medium, and identifying and / or counting the amount of test microorganisms in each microbial strain grown in the culture medium. The later steps, which involve identifying the test microorganisms and / or counting the quantity of each microbial strain grown in the culture medium, may preferably include a step to separately quantify the quantity of each microorganism, since each microorganism grows simultaneously with other microorganisms in the same culture medium (e.g., the same agar plate).

[0007] In this specification, a bioprocessing apparatus refers to an apparatus or component used in any stage of bioprocessing, i.e., an upstream, midstream, or downstream process. A bioprocessing apparatus, as used herein, includes components, sets of components (modules), complete systems, and complete process lines having several systems. Examples include chromatography systems, in-line preparation systems, columns, and 3D printed components. The methods described are used to evaluate the effectiveness of sanitization methods used to sanitize a bioprocessing apparatus or to evaluate the sanitary design of a bioprocessing apparatus. The methods provide information on whether changes to the sanitization method or sanitary design are necessary.

[0008] Effective sanitization of bioprocessing equipment is crucial for patient safety and compliance with Good Manufacturing Practices (GMP) and regulations. Failure to comply results in batch losses and costly investigations for pharmaceutical companies. Therefore, it is essential to understand whether the chosen sanitization method meets the requirements for the product to comply with GMP and regulations.

[0009] Traditionally, sanitization methods have been evaluated by loading a system with one type of test microorganism at a time and then assessing the results. If additional test microorganisms are used, they are tested sequentially. The more test microorganisms used, the longer the evaluation time becomes. Such sanitization research is therefore time-consuming and costly.

[0010] The methods described above, like conventional methods, provide information on how the sanitization methods used function and whether changes to the sanitization design and / or methods are necessary. The presence and number of one or more test microorganisms in the culture medium indicates that changes to the sanitization design and / or methods are necessary.

[0011] Before the microorganisms are applied to the system, all components of the system are pre-cleaned, for example, using detergent and a soft brush, followed by treatment with a sanitizing agent or autoclaving (taking into account the material properties and / or size, where appropriate). The pre-cleaning / pre-sanitization process is carried out to reduce the risk of microorganisms already being present in the system when the test microorganism mixture is applied, otherwise it may affect the results / conclusions drawn.

[0012] The amount of each microorganism in the mixture of test microorganisms, which includes two or more different microbial strains applied to the system, is 10 3 More than CFU / ml, or 10 4 More than CFU / ml, or 10 5 CFU / ml or more than 10 6 It's acceptable if the CFU / ml level exceeds this.

[0013] The test microorganisms in the mixture should be applied to the bioprocessing apparatus at the specified concentration for each microorganism. The test microorganisms are mixed in an isotonic solution, such as 0.9% NaCl, before being applied to the system. Preferably, the test microorganisms are present in the mixture at approximately the same concentration.

[0014] The microbial mixture may be applied at one or more locations in the bioprocessing apparatus (depending on the apparatus and its size).

[0015] After the mixture of test microorganisms is applied to the bioprocessing apparatus, there is a waiting step of at least 2 hours. The waiting period may be 2 to 48 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, 4 to 20 hours, or 10 to 18 hours.

[0016] The chosen waiting time can depend on the mixture of test microorganisms used. Various microbial deposits begin to form boundaries on the surface of the bioprocessing apparatus and eventually form a biofilm after varying amounts of time.

[0017] After a waiting period, the bioprocessing equipment is sanitized according to the prescribed sanitization method.

[0018] After sanitization, at least one test sample potentially containing the test microorganism is collected from at least one location, or 1 to 70 different locations, or 10 to 20 different locations in the bioprocessing apparatus. From each location in the processing apparatus, 1 to 70, or 10 to 20, or 20 to 70 separate samples may be taken. The samples may then be analyzed in parallel. The samples may be, for example, liquid volumes, or samples taken using contact plates, swabs, or tops. Test samples may be collected at several locations in the apparatus and analyzed in parallel.

[0019] Next, at least a portion of the collected test sample is transferred to a culture medium, which promotes the growth and differentiation of all test microbial strains in the mixture of test microorganisms applied to the bioprocessing device, thereby causing any potentially present test microorganisms to grow and differentiate in the same culture medium sample. Furthermore, any unintended organisms in the collected sample that are not part of the test mixture but are present in the bioprocessing system are also allowed to grow and differentiate in the culture medium.

[0020] After culturing, the test microorganisms and any unintended organisms present are identified, and / or the number of test microorganisms or any unintended organisms in each microbial strain is counted.

[0021] Depending on the acceptance criteria for a specific bioprocessing device, or for various microorganisms, results derived from the identification and / or counting of various microbial strains may indicate inadequate sanitization methods and the need for alternative / improved sanitization methods. The results may also indicate whether the sanitary design of the bioprocessing device is adequate or requires redesign.

[0022] The amount of each test microorganism in the mixture of microorganisms applied to the bioprocessing device may be, for example, 10 6 CFU / ml. In that case, the criteria for a successful sanitation method can be that, after sanitation and after culturing, the number of each test microorganism in the collected sample is less than 100 CFU / ml or less than 10 CFU / ml. If the amount is larger, the sanitation method does not meet the criteria and another sanitation method is required.

[0023] Compared to the conventional evaluation of sanitation methods, the method of the present invention uses two or more test microorganisms that are mixed together before application. The collected sample that may contain test microorganisms is then analyzed in the same culture medium. Thereby, there is a significant reduction in time compared to the case where two test microorganisms are sequentially evaluated. Therefore, the described method can reduce the time required for sanitation evaluation from three to four months to possibly only one month in some cases. More data is generated in a shorter time, thereby reducing the total project time and optimizing the use of funds in a better manner.

[0024] The mixture of test microorganisms can include two or more different microbial strains selected from Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Bacillus subtilis, Aspergillus niger, Pichia pastoris, and Saccharomyces cerevisiae.

[0025] Such microorganisms are preferably USP (US Pharmacopeia) recommended organisms for use in testing non-sterile products. The selected microbial strains (Escherichia coli (Gram-negative bacterium), Pseudomonas aeruginosa (Gram-negative bacterium), Staphylococcus aureus (Gram-positive bacterium), Candida albicans (yeast), Bacillus subtilis (Gram-positive bacterium), Aspergillus niger (fungus), Pichia pastoris (yeast, not USP recommended) and Saccharomyces cerevisiae (yeast, not USP recommended)) cover a wide range of microorganisms commonly encountered in the bioprocessing industry. Since these microorganisms are different from each other in terms of morphology, size and attraction to different substances, a more stringent and realistic test scenario regarding hygienic design is achieved, thereby reducing the risk of time delay (redesign of the product or test method).

[0026] The mixture of selected microorganisms may contain 2 to 4 microbial strains.

[0027] When using a mixture of 2 or 3 test microorganisms, the test microorganisms may include, for example, Escherichia coli, Pseudomonas aeruginosa, and / or Staphylococcus aureus.

[0028] When using 4 test microorganisms in the mixture, the microorganisms used may be, for example, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.

[0029] All the listed test microorganisms can be co-cultured in the same culture medium.

[0030] The step of waiting for at least 2 hours may be carried out at a temperature of 15 to 30 °C.

[0031] Alternatively, the temperature used may be less than 15 °C, for example 4 to 14 °C.

[0032] Sanitization of bioprocessing equipment may be carried out using any one or more of the following: sodium hydroxide, isopropanol, ethanol, peracetic acid, hydrogen peroxide, acetic acid, citric acid, sodium hypochlorite (chlorine), quaternary ammonium compounds (e.g., benzalkonium chloride), iodine-based disinfectants (e.g., povidone-iodine), phenols (e.g., chlorophenol), aldehydes (e.g., formaldehyde and glutaraldehyde), ammonia, nitric acid, phosphoric acid, sulfuric acid, peroxyacetic acid, potassium permanganate, potassium hydroxide, ammonium chloride, sodium bicarbonate, chlorhexidine, and perethylene.

[0033] The listed sanitizing agents may be in various concentrations and may be used in various combinations. For example, there may be a sanitizing step with one or more acids first, followed by a sanitizing step with one or more bases, or vice versa. Sanitization is defined as the use of chemical agents to reduce microbial communities and is typically used in chromatography systems to maintain the presence of microorganisms at a level that minimizes the risk of contamination of bioproducts.

[0034] Depending on the bioprocessing equipment and materials used, sanitization may also include gamma irradiation.

[0035] Test samples may be further collected from at least one location in the bioprocessing apparatus after application and / or immediately before the sanitization step, each test sample being transferred to a culture medium and incubated to allow any potentially present microorganisms to grow and differentiate, and then each microbial strain grown in the culture medium being identified and / or the number of microorganisms being counted.

[0036] This method is performed to determine the viability and concentration of each test microorganism in a mixture of microorganisms at various time points. The amount of each test microorganism in the mixture of microorganisms at the start is, for example, about 10 6It may also be CFU / ml. After application and / or immediately before sanitization, the viability and concentration of each test microorganism in the collected test sample are analyzed. If the concentration of the test microorganism decreases below a predetermined minimum amount (e.g., 10) after application or immediately before sanitization, 6 If the CFU / ml level is less than this, it indicates that the experimental method or equipment is not functioning, and that the method was interrupted before sanitization, as it is impossible to evaluate the sanitization process used.

[0037] The effectiveness of a sanitary design or selected sanitization method can be evaluated if the quantity / types of microorganisms present before / during sanitization are known. First, the number of organisms in the system is established, and then the absence of organisms is checked after sanitization.

[0038] The method may include a step of filtering the collected test sample, after which at least a portion of the filtered sample or at least a portion of the filter is transferred to a culture medium.

[0039] Filtration may be performed with a pore size (e.g., 0.45 μm) suitable for capturing the presence of microorganisms and then transferring the filter to a suitable culture medium. Filtration may also be performed to remove non-microbial components from the liquid test sample.

[0040] The step of incubating the culture medium to grow and differentiate any potentially present microorganisms may be carried out at a temperature of 20-42°C for a period of 1-5 days, with a pH of 6.0-8.0 and an osmotic pressure of 0.05-2.5 M.

[0041] The culture medium may be solid or semi-solid.

[0042] The solid culture medium may contain agar at a concentration of 1.5–2.0%. The semi-solid culture medium may contain agar at a concentration of 0.2–0.5%.

[0043] Alternatively, the culture medium may be broth.

[0044] The culture medium may contain a carbon source selected from any one or more of glucose, lactose, sucrose, glycerol, fructose, mannitol, succinate, maltose, xylose, and galactose.

[0045] The culture medium may contain a nitrogen source selected from any one or more of peptone, tryptone, yeast extract, ammonium sulfate, and ammonium chloride.

[0046] The culture medium may contain any one or more of the following: iron, manganese, copper, cobalt, molybdenum, nickel, selenium, biotin, folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, vitamin K, inositol, and para-aminobenzoic acid. [Brief explanation of the drawing]

[0047] [Figure 1] This diagram schematically shows the general steps of a sanitization method 10 used to sanitize bioprocessing equipment or to evaluate the sanitary design of bioprocessing equipment. [Figure 2] This image shows an agar plate 20 containing three co-cultured microorganisms: 1) Staphylococcus aureus, 2) Escherichia coli, and 3) Pseudomonas aeruginosa. [Modes for carrying out the invention]

[0048] Figure 1 schematically illustrates a sanitization method 10 used to sanitize bioprocessing equipment or to evaluate the sanitary design of bioprocessing equipment. Effective sanitization is crucial for compliance with Good Manufacturing Practices (GMP) and regulations. Failure to comply can lead to high costs. The method of the present invention provides information on how the sanitization process used functions and whether changes to the sanitization process are necessary. Compared with conventional sanitization evaluation processes, the method described herein is faster and less expensive. The method can also be used to evaluate the sanitary design of bioprocessing equipment and whether changes to the sanitary design are necessary.

[0049] The method includes a step 100 of pre-cleaning the bioprocessing equipment using, for example, detergent, autoclave treatment, or antimicrobial agents. A mixture of two or more, for example, two to four, different strains of test microorganisms is prepared 101. The strains may be selected from Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Bacillus subtilis, Aspergillus niger, Pichia pastris, and Saccharomyces cerevisiae. The selected test microorganisms cover a broad range of microorganisms commonly encountered in the bioprocessing industry, as these microorganisms differ from one another in terms of morphology, size, and attraction to different substances.

[0050] Next, the mixture of test microorganisms is applied to at least one location in the bioprocessing apparatus 102. The test microorganisms are allowed to accumulate and adhere to the surface of the apparatus for a waiting period of at least 2 hours 103, forming a biofilm. The bioprocessing apparatus is then sanitized using, for example, sodium hydroxide, isopropanol, ethanol, peracetic acid, and / or hydrogen peroxide 104. The agents used for sanitization may remain in the bioprocessing apparatus for about 1 hour, i.e., the waiting time may be about 1 hour.

[0051] Next, the test sample, liquid volume, contact plate or swab or top test are collected from at least one location in the bioprocessing apparatus after the sanitization process105, and optionally also after application and / or immediately before the sanitization process105. (In some cases, the bioprocessing apparatus may have to be disassembled in order to collect the test sample at a specific location in the apparatus, e.g., in a flow path).

[0052] If the test sample is in liquid volume, or is to be transferred to a liquid volume, the liquid volume may be filtered through a filter having a suitable pore size (e.g., 0.45 μm) to capture the presence of organisms in the filter material or to filter out microorganisms into the filtrate.110

[0053] The collected single or multiple test samples are each transferred to the same culture medium that promotes the growth and colonization of all microbial strains in the mixture of microorganisms applied to the bioprocessing apparatus, so that the strains can be individually distinguished from one another.106 The test samples may be serially diluted, for example, with sodium chloride, before being transferred to a suitable culture medium. The culture medium is incubated until the individual CFUs become countable, from which the concentration of the loaded microbial strains in the original test sample is estimated.

[0054] The culture medium may be a broth, or a solid or semi-solid culture medium (i.e., containing agar at a concentration of 1.5-2.0% or 0.2-0.5%, respectively). The broth / agar used may be, for example, Luria-Bertani (LB) broth, tripty soy broth (TSB), tripty soy agar (TSA), nutrient broth, Sabouraud dextrose broth, Brain Heart Infusion (BHI) broth, Mueller-Hinton broth, MacConkey broth, cetrimido broth, chocolate agar, Colombian broth, mannitol salt broth, and blood broth.

[0055] The step of incubating the culture medium to grow and differentiate any potentially present microorganisms may be carried out at a temperature of 20-42°C for a period of 1-5 days, with a pH of 6.0-8.0 and an osmotic pressure of 0.05-2.5 M.

[0056] The osmotic pressure range may have an optimal range of 0.05 to 0.5 M (i.e., NaCl). The optimal pH range may be 6.5 to 7.5. The optimal temperature range may be 30 to 37°C. Incubation may be carried out at atmospheric pressure (101.3 kPa).

[0057] The culture medium may contain a carbon source selected from any one or more of the following: glucose (1-2%), lactose (0.5-2%), sucrose (0.5-1%), glycerol (0.1-1%), fructose (1-2%), mannitol (0.5-2%), succinate (0.1-1%), maltose (0.5-1%), xylose (0.5-1%), galactose (1-2%), and other simple sugars and organic acids.

[0058] The culture medium may contain a nitrogen source selected from any one or more of the following: peptone: 5-20 g / L, tryptone: 5-20 g / L, yeast extract: 1-10 g / L, ammonium sulfate: 0.5-2 g / L, and ammonium chloride: 0.5-2 g / L.

[0059] The culture medium contains trace elements and cofactors: iron: 1-10 mg / L, manganese: 0.1-1 mg / L, zinc: 0.1-1 mg / L, copper: 0.1-1 mg / L, cobalt: 0.1-1 mg / L, molybdenum: 0.1-1 mg / L, nickel: 0.1-1 mg / L, selenium: 0.1-1 mg / L, biotin: 0.1-1 μg / L, folic acid: 0.1-1 μg / L, nicotinic acid: 1-10 μg / L. It may contain any one or more of the following: pantothenic acid: 0.1-1 μg / L, pyridoxine: 0.1-1 μg / L, riboflavin: 0.1-1 μg / L, thiamine: 0.1-1 μg / L, vitamin B12: 0.01-0.1 μg / L, vitamin K: 0.1-10 μg / L, inositol: 1-100 mg / L, and PABA (para-aminobenzoic acid): 1-10 mg / L.

[0060] The microbial mixture may grow in buffers such as the following: phosphate buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, and Tris (tris(hydroxymethyl)aminomethane) buffer.

[0061] All of the organisms mentioned above may be grown in a shaken or stirred liquid culture medium or broth with an aeration rate of approximately 150-250 rpm and an upper space to medium ratio of 1:2-1:10.

[0062] Examples of isotonic solutions that can be used when culturing microorganisms in culture media include: phosphate-buffered saline (PBS) (10 mM to 100 mM, pH 7.2 to 7.4), sodium chloride solution (50 mM to 1 M), Tris-EDTA (TE) buffer (10 mM to 100 mM, EDTA 0.1 mM to 10 mM, pH 8.0), and MOPS (3-(N-morpholino)propanesulfonic acid) buffer (10 mM to 5 These are 0 mM, pH 7.0-7.4), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (10 mM-50 mM, pH 7.0-8.0), potassium phosphate buffer (10 mM-100 mM, pH 7.0-7.4), sodium acetate buffer (10 mM-100 mM, pH 4.5-5.5), and Tris-HCl buffer (10 mM-100 mM, pH 7.4).

[0063] During incubation of the culture medium, 107 any potentially present microorganisms are grown and differentiated. The microorganisms are identified and / or the amount of each microbial strain grown in the culture medium is counted.108 In the broth, motile bacteria create a uniform turbidity. Non-motile bacteria with waxy cell walls tend to float on the surface of the broth. Other types of non-motile bacteria may form sediment at the bottom of the broth.

[0064] Figure 2 shows an agar plate 20 containing three co-cultured microorganisms: 1) Staphylococcus aureus (ATCC6538), 2) Escherichia coli (ATCC8739), and 3) Pseudomonas aeruginosa (ATCC9027).

[0065] Traditionally, sanitization methods have been evaluated by loading a system with one type of test microorganism at a time and then assessing the results. If additional microorganisms are used, they are tested sequentially. The more test microorganisms used, the longer the evaluation time becomes. Such sanitization research is therefore time-consuming and expensive.

[0066] The methods described above, like conventional methods, provide information on how the sanitary design and / or the sanitization methods used function, and whether changes to the sanitary design and / or sanitization process are necessary. The presence of one or more test microorganisms, and optionally other organisms present in the system, and their amounts in the culture medium, indicate that changes to the sanitary design and / or sanitization methods may be necessary.

[0067] Compared to conventional methods, the present invention uses two or more test microorganisms that are mixed together before application. This results in a significant reduction in time, sometimes from 3 / 4 months to 1 month, compared to when microorganisms are evaluated sequentially.

[0068] The mixture of test microbial strains used may be prepared by preparing a separate culture plate containing a suitable culture medium and one loaded microbial strain for each plate. The plate is incubated until distinct colonies are formed. Then, single colonies from each microbial strain are transferred to a separate suitable culture medium and incubated with shaking at 32-37°C until a suitable optical density (OD) is obtained. The incubated organisms are then fed to a predetermined concentration, for example, 10 6 The microbial strain is diluted and mixed in a solution that provides a steady-state environment for microbial strains exceeding CFU / ml. An example of a steady-state solution is 0.05–1 M sodium chloride.

[0069] The prepared solution containing the mixed test microorganisms is applied to one or more locations in a bioprocessing apparatus using preferred and realistic parameters. If the bioprocessing apparatus is a chromatography system, such realistic parameters may include flow rate, back pressure, and time.

[0070] After sanitization, the sanitizing agent may be replaced with a suitable neutralizing solution until no traceable amount of the sanitizing agent is found. An example of a neutralizing solution is: 0.2 M sodium acetate, 20% ethanol, pH 5.5.

Example

[0071] Experimental Example Parts of a bioprocessing device containing hydrophobic / inert substances were used. The parts included several different test sample collection sites and were evaluated based on the sanitary design and sanitation methods used. Using 1M NaOH as the sanitizing agent, the study was carried out with a contact time of 1 hour or more. The concentration of each organism was 10 6 CFU / mL or more of Escherichia coli (Ec), Pseudomonas aeruginosa (Pa), and Staphylococcus aureus (Sa) mixed in suspension was used to load the parts.

[0072] Pre - cleaning The bioprocessing device parts were pre - cleaned with a detergent and a small brush. Ethanol (70%) was sprayed on the cleaned surface and left to air - dry. In Study 3, ethanol was replaced with a sporicidal agent called Prochlor. This agent was applied using the same method.

[0073] Preparation and application of the load - test microbial suspension Tryptic soy agar (TSA) plates streaked with load - test microorganisms were incubated overnight (O.N.) at 37°C. Fresh colonies from those plates were transferred to 200 mL of autoclaved Bacto tryptic soy broth (TSB) medium and left shaking O.N. at 37°C. Based on the premise that the measured OD of the pre - culture and 1 OD corresponded to about 10 9 CFU / mL for Ec, about 2x10 9 CFU / mL for Pa, and about 1.5x10 8 CFU / mL for Sa, calculations were made for the required volume of the pre - culture to be added to 25 - 55 L of filtered 0.9% NaCl solution to obtain a final concentration of about 10 7 CFU / mL for each load - test microorganism in the suspension.

[0074] The suspension was applied to multiple sites on the parts of the bioprocessing device. The infected system was left standing at room temperature (RT) for 16 - 20 hours before the start of the sanitation method.

[0075] Sanitization method The liquid was filtered through a 0.2 μm filter, after removing the 1 M NaOH sanitizing agent. The method was the same as that used for pre-washing.

[0076] Before collecting microbial samples, the liquid was removed from the components of the bioprocessing device.

[0077] Microbial sample collection Microbial samples were collected from predetermined locations on components of the bioprocessing device.

[0078] Test Method 1, Microbial Air Sampling: Air samples for airborne microorganisms were collected using a Microbial Air Sampler (MAS). The MAS, equipped with an agar plate, was placed at a suitable measurement location. When measurement began, a predetermined volume of ambient air passed through the machine. Microorganisms were collected on the agar surface by impact.

[0079] Test method 2, direct filtration: Sample solution (minimum 10 mL) was collected in a sterile tube and then filtered through a 0.45 μm nitrocellulose membrane filter. The filter was incubated on an agar plate at 30-35°C for 5 days, and the plate was then tested for CFU.

[0080] Test Method 3, Swab: Surface samples were collected using a swab. The swab was inserted into a tube containing isotonic swab solution and vortexed for at least 20 seconds. The solution containing the swab was poured into a Petri dish and mixed with 30 mL of temperature-controlled molten agar. The maximum temperature of the molten agar should be 45°C. After solidification, the plate was incubated at 30-35°C for 5 days, and then the plate was tested for CFU.

[0081] Samples of the load test organism suspension were serially diluted with 0.9% NaCl. The diluted suspension-derived samples were seeded onto agar plates and incubated at 30-35°C for 1-2 days. The plates were then examined for CFU (concentrated force units). The concentration of the load test organism was determined in the sampled suspension.

[0082] result First, a sanitization method using 1M NaOH and a 1-hour contact time was used. Two of the three load organisms (Pseudomonas aeruginosa and Escherichia coli) were eradicated, but the third load organism (Staphylococcus aureus) was found in several sample locations on the bioprocessing equipment components. Other contaminants besides the load organisms: Gram-positive cocci, Gram-positive bacilli (spore-forming bacteria), and Bacillus cereus / Bacillus thuringiensis / Bacillus mycoides (spore-forming bacteria) were also found in several horizontal test locations. Spores are generally more difficult to eradicate than vegetative microorganisms. The design and placement of some components can trap liquid, which can be a potential risk of contamination and biofilm formation.

[0083] This sanitization method did not eradicate the spores found in the horizontal areas. Biofilms may also be a potential factor that increases resistance to sanitizing agents.

[0084] Staphylococcus aureus is a Gram-positive cocci with a size of approximately 1 μm, which is smaller than the other two rod-shaped organisms (1–5 μm in length and 0.5–1 μm in diameter). These cocci can likely reach and hide in crevices that the other two load microorganisms cannot access. Therefore, Staphylococcus aureus is more difficult to eradicate than the other two load microorganisms.

[0085] The results indicated that contact time should be increased and the sanitization process should be modified to improve hygiene. The issues with the horizontal sections mentioned suggest that the sanitary design used is not optimal and that problems with biofilms may occur.

[0086] Thus, various aspects and embodiments of the present invention provide improved methods for evaluating sanitization methods. The methods involve adding multiple different organisms, keeping them alive, and keeping them measurable until the time of subsequent sanitization (therefore, the method itself is not merely a sanitization method whose purpose is to remove the organisms themselves). Such methods, therefore, allow the use of several different organisms simultaneously, and nevertheless, each of them can be individually identified / quantified because they grow together in the same growth medium (e.g., shared agar plates) throughout the evaluation method.

Claims

1. A method (10) for evaluating a sanitization method used to sanitize a bioprocessing apparatus or to evaluate the sanitary design of a bioprocessing apparatus, - A step (100) to pre-clean the bioprocessing equipment, - A step (101) to prepare a mixture of test microorganisms containing two or more different microbial strains, - Step (102) of applying a mixture of test microorganisms to a bioprocessing apparatus, - A process that requires waiting at least two hours. - Steps to sanitize the bioprocessing equipment (104), - A step (105) of collecting at least one test sample that may contain test microorganisms from at least one location in the bioprocessing apparatus after sanitization. - A step (106) to transfer at least a portion of the collected test samples to the culture medium. - A step (107) of incubating the culture medium to allow any test microorganisms that may be present to grow and differentiate in the culture medium. - A step (108) to identify the test microorganism of each microbial strain grown in the culture medium and / or count the amount of the test microorganism. Methods that include...

2. The method according to claim 1 (10), wherein the mixture of test microorganisms comprises two or more different test microbial strains selected from Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Bacillus subtilis, Aspergillus niger, Pichia pastrius, and Saccharomyces cerevisiae.

3. The method according to claim 2 (10), wherein the mixture of test microorganisms comprises 2 to 4 different strains of microorganisms.

4. The method according to any one of claims 1 to 3 (10), wherein the step of waiting for at least two hours is carried out at a temperature of 15 to 30°C.

5. The method according to any one of claims 1 to 4 (10), wherein the sanitization (104) of the bioprocessing apparatus is carried out using any one or more of sodium hydroxide, isopropanol, ethanol, peracetic acid, hydrogen peroxide, acetic acid, citric acid, sodium hypochlorite, quaternary ammonium compounds, iodine-based disinfectants, phenols, aldehydes, ammonia, nitric acid, phosphoric acid, sulfuric acid, peroxyacetic acid, potassium permanganate, potassium hydroxide, ammonium chloride, sodium bicarbonate, chlorhexidine, and perethylene.

6. The method according to any one of claims 1 to 5 (10), wherein the test sample is further collected from at least one location in the bioprocessing apparatus after application (102) and / or immediately before sanitization (105), each test sample is transferred to a culture medium (106), incubated to allow any test microorganisms present to grow and differentiate (107), the microbial strains grown in the culture medium are identified and / or the number of microorganisms is counted (108).

7. The method according to any one of claims 1 to 6 (10), comprising the step of filtering (110) the collected (105) test sample, and then transferring at least a portion of the filtered sample or at least a portion of the filter to a culture medium (106).

8. The method according to any one of claims 1 to 7 (10), wherein the step of incubating (107) a culture medium to grow and differentiate any potentially present microorganisms is carried out at a temperature of 20 to 42°C for a period of 1 to 5 days, with a pH of 6.0 to 8.0 and an osmotic pressure of 0.05 to 2.5 M.

9. The method according to any one of claims 1 to 8 (10), wherein the culture medium is a solid or semi-solid culture medium.

10. The method according to any one of claims 1 to 8 (10), wherein the culture medium is broth.

11. The method according to any one of claims 1 to 10 (10), wherein the culture medium comprises a carbon source selected from any one or more of glucose, lactose, sucrose, glycerol, fructose, mannitol, succinate, maltose, xylose, and galactose.

12. The method according to any one of claims 1 to 11 (10), wherein the culture medium comprises a nitrogen source selected from any one or more of peptone, tryptone, yeast extract, ammonium sulfate, and ammonium chloride.

13. The method according to any one of claims 1 to 12 (10), wherein the culture medium comprises any one or more of iron, manganese, copper, cobalt, molybdenum, nickel, selenium, biotin, folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, vitamin K, inositol, and para-aminobenzoic acid.

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