Rapid sterility test method and rapid sterility test platform for verifying safety of biopharmaceuticals

Abstract

The present invention relates to an integrated system and a method for remarkably reducing the time required for a sterility test for safety verification in a process of manufacturing biopharmaceuticals. Particularly, the present invention provides a rapid sterility test method and a rapid sterility test platform for verifying the safety of biopharmaceuticals, comprising magnetic nanobeads coated with a protein capable of binding to microorganisms constituting the system, a biochip specialized in the analysis of nanoparticles, automated imaging equipment and analysis software, and a step and a method in which rapid sterility verification is performed.

Description

Rapid sterility test method and rapid sterility test platform for verifying the safety of biopharmaceuticals.

[0001] The present invention relates to a rapid sterility test method and a rapid sterility test platform for verifying the safety of biopharmaceuticals, and more particularly, to an integrated system and a methodology therefor that drastically shorten the time required for a sterility test for verifying the safety in the manufacturing process of biopharmaceuticals, and more specifically, to a rapid sterility test method and a rapid sterility test platform for verifying the safety of biopharmaceuticals, which includes a magnetic particle coated with a protein capable of binding to a microorganism constituting the system, a biochip specialized in analyzing microorganisms combined with nanoparticles, automated imaging equipment and analysis software, and a process and method for performing rapid sterility verification.

[0002] Sterility testing is a quality control method for pharmaceuticals. It detects bacteria and fungi. It is applied to raw materials and formulations during the manufacturing of sterile pharmaceuticals. It involves taking a specified amount of sample, using a specified medium, and conducting an experiment using a specified method to determine the presence of bacteria and fungi believed to originate from the sample. Currently, there are two types of sterility testing for pharmaceuticals: membrane filtration and direct sterility testing. The membrane filtration method is currently the standard. The membrane filtration method involves passing a biopharmaceutical through a filter to purify and concentrate non-microbial components. The sample is then placed in a microbial growth medium to detect the increased turbidity as the microorganisms divide. This is a labor-intensive process that typically takes two weeks. However, unlike conventional chemically-based synthetic drugs, most biopharmaceuticals have a shelf life much shorter than two weeks. Consequently, sterility test results are typically obtained after immediate administration to patients. This poses a clinical risk of microbial infection in patients. Furthermore, biopharmaceuticals, including cell therapy products, are manufactured on a patient-specific basis, making standardized management difficult. As cutting-edge pharmaceuticals, their management regulations have only recently been fully established. However, rapid bacterial and fungal tests suitable for sterility testing of cell therapy products have not yet been developed, limiting microbiological safety management for both manufacturers and patients. Consequently, with the increasing use of biopharmaceuticals, there is a growing need to develop rapid sterility verification tests that address the long detection times of existing methods.

[0003] To address this, methods for detecting microorganisms without the culture step have been proposed to shorten the time required for sterility verification testing. Representative technologies include solid-phase cytometry and polymerase chain reaction (PCR). Solid-phase cytometry involves passing a biopharmaceutical through a membrane filter, staining the microorganisms caught on the filter, and then detecting them under a microscope. PCR, on the other hand, utilizes the signal amplification of the microorganism's presence through primers specific to the target microorganism. While both technologies offer the advantage of detecting microorganisms within three hours, they are difficult to apply universally to a wide range of microorganisms and biopharmaceuticals. Furthermore, the high incidence of false positives and false negatives makes them difficult to replace existing biopharmaceutical sterility verification tests. Therefore, current technology development is focused on methods that detect carbon dioxide produced by microorganisms during growth, or metabolites such as adenosine triphosphate (ATP). However, these technologies also cannot effectively separate components and microorganisms within biopharmaceuticals due to the limitations of the purification efficiency of the membrane filtration method, resulting in interference from metabolites produced by cells other than microorganisms and false positives, and requiring a long time of about 5 days to measure a significant signal. To solve the above problems, the present invention proposes a methodology and integrated system for separating microorganisms from biopharmaceuticals containing mixed cells and microorganisms with superior purification and concentration efficiency than the membrane filtration method, and for specifically and rapidly detecting microbial metabolites.

[0004] In order to solve the above-mentioned problem, the present invention provides an integrated system and a methodology therefor that drastically shortens the time required for a sterility test for safety verification in the manufacturing process of biopharmaceuticals, specifically, a rapid sterility test method and a rapid sterility test platform for safety verification of biopharmaceuticals, including a magnetic particle coated with a protein capable of binding to a microorganism constituting the system, a biochip specialized in analyzing microorganisms combined with nanoparticles, automated imaging equipment and analysis software, and a process and method for performing rapid sterility verification.

[0005] In order to solve the above-described problem, the present invention provides a rapid sterility test method using a biochip including one or more chambers into which a sample and a control group are each loaded; and one or more magnets installed on one side of each chamber, the method comprising the steps of: mixing magnetic particles into a test target substance to bind the analyte to the magnetic particles; separating the magnetic particles from the test target substance to prepare a sample; and injecting the separated sample and indicator substance into a chamber of the biochip and culturing them to determine the presence of the analyte, wherein the step of determining the presence of the analyte comprises comparing the fluorescence or color of images of the sample and the control group after culturing to determine the presence of the analyte inside the sample.

[0006] In one embodiment, the test subject may be a biopharmaceutical, a cell therapy product, a clinical sample, a chemical pharmaceutical, a cosmetic, a food, or an environmental sample.

[0007] In one embodiment, the analyte may be a bacterium, mycoplasma, or fungus.

[0008] In one embodiment, the step of preparing the sample may include a step of concentrating the analyte bound to the magnetic particles on a growth medium.

[0009] In one embodiment, the growth medium may include Tryptic soy broth, Muller-Hinton broth, Fluid Thioglyollate Medium, Sabouraud broth, or Luria-Bertani broth.

[0010] In one embodiment, the step of determining the presence of the analyte may include the steps of: separating the chamber from the magnet; injecting an indicator substance, a sample, and a control into the chamber and culturing them for a predetermined period of time; approaching the chamber to the magnet after the culturing is completed; and photographing the interior of the chamber to obtain an image, and then comparing the images photographed from the separated sample and the control.

[0011] In one embodiment, the analyte is combined with the magnetic particles to form a complex, and the complex can be moved to one side of the chamber by the magnet when the chamber is brought close to the magnet.

[0012] In one embodiment, the culturing may be performed for 4 to 48 hours.

[0013] In one embodiment, the image acquisition may be performed at intervals of 10 to 120 minutes.

[0014] In one embodiment, the indicator substance may be one whose fluorescence or color changes due to the analyte.

[0015] In one embodiment, the indicator substance may be a redox indicator substance, a pH indicator, a dye-based reagent, an enzyme reaction indicator, or an antibody-based reagent.

[0016] The present invention also provides a biochip for a rapid sterility test platform, comprising: a lower plate including a chamber into which a control group and a sample are loaded; and an upper plate fitted to the lower plate and including a magnet, wherein the lower plate includes a bottom surface; a central axis protruding upward from the bottom surface; and one or more chambers installed at a predetermined distance from the central axis, and the upper plate includes a central axis insertion hole into which the central axis of the lower plate is inserted; a chamber insertion hole into which the chamber is inserted; and a magnet installed on one side of the chamber insertion hole.

[0017] In one embodiment, the chamber may include one or more sample chambers and one or more control chambers.

[0018] In one embodiment, the chambers may be installed at equal intervals at a uniform distance around the central axis.

[0019] In one embodiment, the interior of the chamber may additionally include magnetic particles and an indicator material.

[0020] In one embodiment, the magnetic particles may be combined with the analyte to form a complex.

[0021] In one embodiment, the magnetic particle may include a functional group or substance capable of binding to microorganisms on its surface.

[0022] In one embodiment, the complex may be positioned on one side of the chamber using the magnet during fluorescence or optical observation using the indicator substance.

[0023] In one embodiment, the top plate may be rotated at a certain angle around the central axis.

[0024] In one embodiment, the control chamber insert may have an arc shape centered on the central axis.

[0025] In one embodiment, the magnet may have a distance from the chamber that changes according to the rotation of the top plate.

[0026] In one embodiment, a rotation means capable of rotating the top plate may be installed on the outer surface of the top plate.

[0027] The present invention also provides a rapid sterility test platform including a biochip for the rapid sterility test platform.

[0028] In one embodiment, the rapid sterility test platform may include: the biochip; a rotational power unit that rotates the upper plate of the biochip; a lighting unit installed on the upper or lower side of the biochip; and an image acquisition unit that photographs the interior of the chamber of the biochip.

[0029] In one embodiment, the image acquisition unit can acquire a fluorescent image or an optical image of the inside of the chamber.

[0030] In one embodiment, the rapid sterility test platform can determine the presence of microorganisms within the sample by comparing the fluorescence or color of the acquired first image and the current image.

[0031] The rapid sterility verification method proposed in this invention shortens the sterility verification test, which previously took 14 days, to less than a day (24 hours), thereby quickly ensuring the safety of biopharmaceuticals. Considering that biopharmaceuticals, manufactured using human-derived materials such as cells and proteins, have short shelf lives and must be administered to patients promptly, shortening the overall testing time favorably preserves the quality of the biopharmaceutical at the time of patient administration, while simultaneously improving the therapeutic efficacy of the product through rapid administration. Furthermore, from a social and corporate perspective, the safety verification process is shortened by more than 13 days, resulting in significant savings in logistics costs.

[0032]

[0033] Existing sterility tests require continuous observation for contamination at regular intervals, such as 24, 48, and 72 hours, for up to 14 days after microbial culture. In contrast, the rapid sterility verification method of the present invention performs continuous imaging in real time or at intervals of approximately 30 minutes on the system to determine contamination, thereby providing much higher temporal resolution and enabling more accurate analysis based on fluorescence intensity values. In addition, since the automated system eliminates the need for direct observation at regular intervals, it can minimize the labor and human resources required for practical applications compared to existing methods.

[0034]

[0035] From a technical perspective, existing sterility verification testing methods, as well as recently developed technologies, utilize membrane filtration to concentrate contaminating microorganisms in biopharmaceutical samples. Sterility verification requires universal detection of bacteria as small as 1 μm and mold as small as 10 μm. However, filtration methods can result in loss of microbial samples depending on pore size. Using a filter with small pores can also concentrate cells, proteins, and other substances present in the biopharmaceutical, reducing the sensitivity of observing microorganisms or their metabolites in subsequent analyses. The present invention utilizes functionalized nanoparticles that specifically bind to microorganisms within biopharmaceuticals, enabling the isolation and concentration of microorganisms regardless of size. This enables higher sensitivity in post-concentration detection compared to existing filtration methods. Therefore, the concentration of microorganisms in a small volume promotes the resazurin dye reaction, enabling faster metabolite measurement compared to prior art. In particular, viability testing, which detects metabolites (typically ATP, CO2, and NADH) to target all microbial species, is recommended as a standard for sterility verification testing. However, non-microbial cells and other substances contained within biopharmaceuticals also produce metabolites. Therefore, utilizing these methods for isolating and concentrating microorganisms significantly enhances analytical specificity.

[0036]

[0037] Nanoparticle-based isolation and concentration methods for microorganisms can be applied not only to resazurin dye, but also to analyses utilizing molecular diagnostic methods such as polymerase chain reaction (PCR), next-generation sequencing, and advanced technologies such as fluid analysis. These characteristics of the technology offer flexibility for application depending on the intended use. For example, in the case of biopharmaceutical samples confirmed to be contaminated, molecular diagnostic methods can be used to identify microbial species, trace the contamination route, or prevent secondary contamination. Alternatively, the technology can be applied to samples requiring sterility verification, such as food, cosmetics, and environmental monitoring, for specific purposes.

[0038]

[0039] The invention of a chip specialized in nanoparticle-based analysis prevents growth inhibition of microorganisms bound to nanoparticles, thereby enabling the stable observation of microbial signals. If microorganisms bound to nanoparticles are persistently aggregated against the walls of the biochip, normal growth is inhibited. However, the biochip included in the present invention can easily apply or remove magnetism, enabling normal microbial growth. Therefore, compared to a non-chip setup, the production of microbial metabolites increases, enabling faster and more sensitive observation of signals from microorganisms or their metabolites.

[0040]

[0041] Time-lapse fluorescence imaging is performed in a miniaturized imaging system measuring 224 mm x 120 mm x 140 mm proposed in the present invention. The imaging system includes an xyz motorized stage that can adjust the imaging area and focus for a chip sample, a light source for fluorescence imaging, a 4X objective lens, and a CCD camera, and is manufactured in a simplified form specifically for the purpose of a rapid sterility verification method. This allows it to be distributed to the industry in a much cheaper and simpler form than existing commercially available equipment such as plate readers, and it can be easily placed in an incubator, which is advantageous for temperature maintenance and space optimization.

[0042] Figure 1 illustrates the configuration and operating principle of a biochip for a rapid sterility test platform according to one embodiment of the present invention.

[0043] FIG. 2 illustrates the shape of a biochip for a rapid sterility test platform according to one embodiment of the present invention. (a) illustrates the overall shape, (b) illustrates the position of the chamber during reverse rotation, and (c) illustrates the position of the chamber during forward rotation.

[0044] Figure 3 illustrates the configuration of the upper plate of a biochip for a rapid sterility test platform according to one embodiment of the present invention.

[0045] Figure 4 illustrates a rapid sterility test platform according to one embodiment of the present invention.

[0046] Figure 5 illustrates a rapid sterility test method using a rapid sterility test platform according to one embodiment of the present invention.

[0047] Figure 6 illustrates a concentration method using magnetic nanoparticles according to one embodiment of the present invention.

[0048] Figure 7 illustrates the results of microbial growth according to the presence or absence of magnetism according to one embodiment of the present invention.

[0049] Figure 8 compares a rapid sterility test method according to one embodiment of the present invention with a conventional sterility verification method.

[0050] Figure 9 shows the results of a rapid sterility test for various biopharmaceuticals according to one embodiment of the present invention.

[0051] Figure 10 shows the presence or absence of microbial contamination through visual observation according to one embodiment of the present invention.

[0052] Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if a detailed description of related known technology is judged to obscure the gist of the present invention, the detailed description will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as “comprise” or “have” should be understood to indicate the presence of described features, numbers, steps, operations, components, parts, or combinations thereof, but do not preemptively exclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. In addition, when performing a method or a manufacturing method, each step constituting the method may occur in a different order from the stated order, unless the context clearly indicates a specific order. That is, each step may occur in the same order as the stated order, may be performed substantially simultaneously, or may be performed in the opposite order.

[0053] The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the technical spirit of the present technology can be sufficiently conveyed to those skilled in the art. In the drawings, the dimensions of each device component, such as width and thickness, are somewhat enlarged to clearly represent the components. Overall, the drawings are described from the observer's perspective, and when an element is mentioned as being positioned above another element, this includes the meaning that the element is positioned directly above the other element or that additional elements may be interposed between the elements. Furthermore, those skilled in the art will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings indicate substantially the same elements.

[0054] As used herein, the term "and / or" includes a combination of multiple listed items or any one of multiple listed items. As used herein, "A or B" may include "A," "B," or "both A and B."

[0055] The present invention relates to a rapid sterility test method using a biochip including one or more chambers into which a sample and a control group are each loaded; and one or more magnets installed on one side of each chamber, the method comprising: a step of mixing magnetic particles with a test target substance to bind the analyte to the magnetic particles; a step of separating the magnetic particles from the test target substance; and a step of injecting the separated magnetic particles and an indicator substance into a chamber of the biochip and culturing them to determine the presence of the analyte, wherein the step of determining the presence of the analyte comprises comparing the fluorescence or color of images of a sample and a control group after culturing to determine the presence of the analyte inside the sample.

[0056] For biological products such as biopharmaceuticals, the presence of unwanted microorganisms can significantly reduce their safety. The present invention provides a rapid sterility test method that can detect the presence of microorganisms more quickly than conventional methods to ensure the safety of such pharmaceuticals.

[0057] The biochip of the present invention may include one or more chambers into which samples and controls are respectively loaded, and one or more magnets installed on one side of each chamber. The structure of such a biochip will be described later.

[0058] By mixing magnetic particles into the test target substance, the analyte can be bound to the magnetic particles. The magnetic particles refer to particles that bind to the analyte present in the test target substance, and may contain a paramagnetic material or a ferromagnetic material therein. Through this, in the case of the present invention, the analyte can be separated from the test target substance, and also the magnetic particles can be easily moved using magnetism. In addition, the analyte can be bound to the magnetic particles to form a complex. The complex refers to the complex being bound to the surface of the magnetic particle as described above, and as described below, since the surface of the magnetic particle contains a functional group or substance that can bind to a microorganism, it can selectively bind to the analyte to form a complex.

[0059] At this time, the magnetic particles may be used without limitation as long as they are magnetic particles that are conventionally used for microbial separation, and may be manufactured in the form of microparticles or microrods. In addition, the magnetic particles may be magnetic nanoparticles having a nano-sized size.

[0060] The magnetic particles may include a functional group or substance capable of binding to microorganisms on their surface. This allows them to selectively bind to an analyte present in the test target material. In this case, the functional group or substance may be beta-2-glycoprotein 1, a mannose-binding lectin, an antibody, an aptamer, an antibiotic, or a polymer.

[0061] Here, the above-mentioned test subject substance refers to a substance that requires ensuring stability against microorganisms, and specifically, it may be a biopharmaceutical, cell therapy product, clinical sample, chemical medicine, cosmetic, food, or environmental sample. In particular, the above-mentioned biopharmaceutical may be manufactured based on proteins, peptides, antibodies, or vaccines, or may be manufactured based on genes, stem cells, human-derived cells, human cells, or tissues, and the same applies to the above-mentioned clinical samples and cell therapy products. In the case of such test subject substances, since they can have a fatal effect on the human body when contaminated with microorganisms, ensuring the above-mentioned safety is essential.

[0062] The above-mentioned analysis target refers to microorganisms that may compromise the safety of the test substance, particularly the pharmaceutical, and may include bacteria, mycoplasma, or fungi. If such microorganisms are found in the pharmaceutical, it is generally considered to be contaminated. Given the nature of pharmaceuticals, if contamination leads to a decrease in safety, not only the individual product but also the entire lot, production date, or line of products must be discarded.

[0063] As described above, after the analyte is bound to the magnetic particles, the magnetic particles can be separated from the test target material. At this time, since the analyte is bound to the magnetic particles, the separation can be performed simply by washing. In addition, a magnet can be used to remove the washing solution and foreign substances after the washing is complete. As described above, in the case of the present invention, since the analyte is separated using magnetic particles, the use of a magnet allows the magnetic particles and the analyte bound thereto to be easily moved. That is, after the washing is complete, the magnetic particles can be separated by inserting the magnetic rod into the washing container, and separately, the magnet can be brought close to the side or bottom of the washing container, and then the washing solution and foreign substances inside can be discharged, thereby enabling separation without loss of the magnetic particles. Through this, even if the concentration of the analyte is very low, it can be separated together with the magnetic particles, which can significantly reduce the probability of a false negative.

[0064] In addition, it is preferable to concentrate the analyte combined with the magnetic particles on a growth medium. Concentration on the growth medium can be achieved by fixing the magnetic particles to the side of the container using a magnet, removing the test target corresponding to the supernatant, and then resuspending the growth medium. In addition, an appropriate amount of growth medium can be used depending on the size of the container and the amount of magnetic particles, and specifically, it is preferable to resuspend using about 100 microliters of growth medium. At this time, it is preferable to wash with the growth medium three or more times in order to effectively recover the magnetic particles remaining on the side of the container. The growth medium promotes the growth of the analyte and thus reduces the detection time, and concentration with a small amount of growth medium can increase the concentration of the analyte and further shorten the detection time.

[0065] In addition, the growth medium may be used without limitation as long as it is a medium capable of culturing the analyte, but preferably may include Tryptic soy broth, Muller-Hinton broth, Fluid Thioglyollate Medium, Sabouraud broth, and Luria-Bertani broth.

[0066] The separated magnetic particles and indicator substances can be injected into the chambers of a biochip and cultured to determine the presence of the analyte. The biochip used in this case may include one or more chambers into which samples and controls are each loaded; and one or more magnets installed on one side of each chamber, as will be described later.

[0067] The step of determining the presence of the analyte may include the steps of: distancing the chamber from the magnet; injecting an indicator substance, a sample, and a control into the chamber and culturing them for a predetermined period of time; bringing the chamber closer to the magnet after the culturing is completed; and photographing the inside of the chamber to obtain an image, and then comparing the images photographed from the sample and the control.

[0068] The chamber and magnet can be separated. As described above, when the analyte attached to the magnetic particles is cultured, it is desirable for the complex bound to the magnetic particles to float within the chamber. Therefore, by separating the chamber and the magnet, the magnetic attraction can be reduced, and the complex can float freely within the chamber.

[0069] Indicator substances, samples, and controls can be injected into the chamber and cultured for a certain period of time. The sample may be separated from the test subject for which sterility testing is desired, as described above, and may be a complex in which the analyte is bound to the surface of the magnetic particles.

[0070] The control group refers to a substance with proven safety, i.e., a negative control group. Preferably, this refers to a specimen identical to the sample, except for the analyte. Specifically, the control group may include magnetic particles that do not form a complex and the same growth medium as the sample. By culturing the control group and the sample simultaneously and then observing changes within the chamber, the presence of the analyte within the chamber can be confirmed.

[0071] In addition, the sample may be manufactured and used separately from the biochip as described above, but it may also be used after being concentrated inside the biochip. In this case, the biochip contains the magnetic particles inside, and the test target substance is supplied inside the biochip, and then a washing process can be performed using a magnet installed in the biochip. As described above, during the washing process inside the biochip, the complex can be collected to one side of the chamber by the magnet, and thereafter, the washing solution and foreign substances can be removed.

[0072] That is, when the sample is produced outside the biochip as described above, a separate chamber or container can be used, and when the sample is produced using the biochip, the sample can be produced using the internal chamber of the biochip.

[0073] The above indicator substance is used to confirm the presence of an analyte that forms a complex with the sample during culture of the sample within the chamber, and may be a redox indicator substance, a pH indicator, a dye-based reagent, an enzyme reaction indicator, or an antibody-based reagent. Such indicator substances may detect chemical changes within the chamber according to respiration, digestion, or reproduction of microorganisms, thereby changing color or fluorescence, thereby confirming the presence of microorganisms within the sample chamber.

[0074] The indicator substance can be injected before or after the sample and control groups, but it can also be supplied loaded inside the biochip. While this significantly improves user convenience, it can also increase analysis costs, as the biochip containing the indicator substance must be replaced depending on the sample type. Therefore, it is advisable to appropriately adjust the timing of the indicator substance injection based on the user's circumstances and surrounding environment.

[0075] The above redox indicator substance refers to a substance whose color or fluorescence changes due to an oxidation or reduction reaction, and may preferably be resazurin. Generally, all cells including microorganisms perform various oxidation and reduction reactions internally. At this time, if a redox indicator substance such as the above is present, the color or fluorescence may change due to such redox reaction. In particular, since the above resazurin has cell permeability, such fluorescence or color change may be more clearly observed when an oxidation-reduction reaction is performed within the cell.

[0076] The above resazurin is a type of phenoxazine dye that is non-toxic, cell-permeable, and redox-sensitive. When reacting with living cells, it changes color from blue to red when seen with the naked eye, and at the same time exhibits fluorescence, which can be measured through optical observation or fluorescence observation. In other words, if the above color change appears, it can be confirmed that living cells exist inside the sample, and if there is no change in fluorescence or color after culturing, it means that sterility can be proven.

[0077] As described above, the sample and control group can be injected into the chamber respectively and then cultured for a certain period of time. At this time, the culture is preferably performed for 4 to 48 hours. In the case of the existing sterility verification test, since the turbidity caused by the microorganism itself is used, a long-term culture period (2 weeks) is required. However, in the case of the present invention, the presence of microorganisms is detected using the color change of the redox indicator substance, so the presence of microorganisms can be confirmed even with a short culture period as described above. If the culture period is performed for less than 4 hours, it may be difficult to observe changes because the culture period is too short, and if it exceeds 48 hours, the overall test time is increased, which is inefficient.

[0078] After the culture is completed as described above, the chamber can be brought close to the magnet. During this process, the magnetic particles of the control chamber and the complex (magnetic particles + microorganisms) of the sample chamber can be captured by the magnet to one side of the chamber.

[0079] When the above capturing occurs, the magnetic particles and microorganisms inside the chamber are located off-center, so it is possible to obtain a clearer image when acquiring the image described later.

[0080] The interior of the chamber can be photographed to obtain images, and then the images captured from the sample and the control can be compared. As described above, since the control has been proven to be sterile, changes can only occur due to the external environment. Therefore, if the changes in the sample are the same as those in the control, the sample can be determined to be free of microorganisms. If the color or fluorescence of the sample changes, the presence of microorganisms can be determined.

[0081] At this time, it is preferable that the image acquisition is performed at intervals of 10 to 120 minutes. In the case of the existing method, since it has a culture period of 2 weeks, it was common to acquire images at 1-day (24-hour) intervals. However, in the case of the present invention, the culture period is very short (4 to 48 hours) and the presence of microorganisms can be immediately determined if there is a change in color, so images can be acquired at intervals of 10 to 120 minutes, preferably 40 to 80 minutes, and most preferably 60 minutes.

[0082]

[0083] The present invention also provides a biochip for a rapid sterility test platform, comprising: a lower plate including a chamber into which a control group and a sample are loaded; and an upper plate fitted to the lower plate and including a magnet, wherein the lower plate includes a bottom surface; a central axis protruding upward from the bottom surface; and one or more chambers installed at a predetermined distance from the central axis, and the upper plate includes a central axis insertion hole into which the central axis of the lower plate is inserted; a chamber insertion hole into which the chamber is inserted; and a magnet installed on one side of the chamber insertion hole.

[0084] The above lower plate is installed with a chamber, and the control group and sample can be loaded into the chamber.

[0085] The bottom surface (110) of the above lower plate is generally in the form of a flat plate, but it is preferable to manufacture it in various shapes depending on the location of the chamber and the shape of the rapid sterility test platform. In the case of the present invention, it is described based on manufacturing it in a circular shape, but it can also be manufactured in the shape of a square, oval, triangle, or polygon, and it can also be manufactured in the shape of a plate having a curve or a certain shape at the top or bottom rather than a general plate shape. In addition to the plate shape, it can also be manufactured in the shape of a column or with an incline in one or various directions.

[0086] In the present invention, the bottom surface (110) is preferably manufactured in a rectangular plate shape as described above. As described below, when installed on a rapid sterile test platform, such a rectangular plate shape can be prevented from rotating along with the rotation of the top plate, which will be described below, by simply inserting it into a groove corresponding to the shape of the bottom surface (110) without installing a separate fixing device.

[0087] A central axis (120) and a chamber can be installed on the above lower plate.

[0088] The central axis (120) described above may be installed to protrude upward from the lower plate as a center of rotation around which the upper plate, which will be described later, is fitted. At this time, the central axis (120) may be manufactured to have a circular cross-section to facilitate smooth rotation of the upper plate.

[0089] In addition, it is preferable that the central axis (120) be installed at the center of the bottom surface (110), but it is also possible to install it at a location spaced a certain distance from the center of the lower plate for fixing the lower plate or for installing the chamber.

[0090] In addition, the central axis (120) may be simply manufactured in a cylindrical shape, but a separation prevention means may be installed at the end inserted into the upper plate. The separation prevention means is a part that prevents the upper plate from coming off after the upper plate is fitted and is manufactured to have a diameter larger than the insertion hole of the central axis (120) installed in the upper plate, thereby preventing the upper plate from coming off after being coupled with the upper plate. In addition, in order to facilitate the coupling of the central axis (120) and the central axis insertion hole (210), the central axis (120) may be manufactured in a pipe shape with an empty center, but may have a structure in which a slit is installed in a portion of the upper portion so that the diameter is reduced when passing through the central axis insertion hole (210).

[0091] The chamber may be manufactured in a form in which the lower part is closed and the upper part is open, where the sample and control are loaded. In addition, it is preferable to manufacture it in the shape of a pipe with an empty interior for the loading as described above, and it is preferable that the lower part is manufactured optically transparent to facilitate image acquisition as described later. In addition, the chamber is preferably manufactured in a cross-section with a circular, oval, square, or polygonal shape, but may be manufactured in a circular shape for mixing efficiency and uniformity of culture. In other words, the chamber is most preferably manufactured in the shape of a circular pipe with a closed lower part and an open upper part.

[0092] In addition, the upper part of the chamber may be manufactured in a simple open form, but preferably, a polymer membrane (film) may be installed. The polymer membrane is a part that plays a role in preventing contact with external contamination after the sample and control group are loaded, and a “-” or “+” shaped incision may be installed at the upper part for the convenience of loading the sample and control group. The sample and control group are generally loaded using a dropper, pipette, or syringe. Therefore, when loading the sample and control group, they can be loaded through the incision, and after loading is completed, the incision is closed by elasticity, so that contact with external contaminants, especially microorganisms, can be blocked.

[0093] The chamber may include one or more sample chambers (131) and one or more control chambers (132). The sample chamber (131) is a chamber into which a sample for which sterility testing is desired is loaded, and the control chamber (132) is a chamber into which a solution proven to be sterile, i.e., a control, is loaded (see (a) of Fig. 1). By loading the sample and control simultaneously as described above, the sample and control can be compared under identical conditions, and thus, if the sample is contaminated, it can be easily identified.

[0094] In addition, the sample chamber (131) and the control chamber (132) may be installed only one at a time, but it is also possible to install multiple ones. If multiple sample chambers (131) are installed, sterility tests can be performed on multiple samples simultaneously, and if multiple control chambers (132) are installed, sterility tests can be performed under various conditions.

[0095] The chambers may be installed at equal intervals at a uniform distance around the central axis (120). In the case of the chambers, as described later, after being inserted into the chamber insertion port, the upper plate rotates. Therefore, in order to facilitate the rotation of the upper plate, the chambers are preferably arranged at a constant interval from the central axis (120). In addition, it is preferable that each chamber is installed at equal intervals so as to maintain the same interval between the magnet (230) described later and each chamber. If the interval between each chamber is not uniform, the distance from the magnet (230) described later will be different, so that the magnetic particles inside each chamber will receive different magnetization, which means that the experimental results of each chamber may be different.

[0096] The interior of the above chamber may additionally contain magnetic particles and redox indicator substances.

[0097] The magnetic particles may be combined with microorganisms to form a complex (see (a) of FIG. 1). These magnetic particles may be moved to one side of the chamber wall by magnetism as the magnet (230) approaches one side of the chamber during the forward rotation process of the upper plate described later ((b) of FIG. 1). Through this, the microorganisms (complex) combined with the magnetic particles may be removed from the central portion of the chamber, and a clearer image may be obtained. That is, the complex may be positioned on one side of the chamber by using the magnet (230) during fluorescence or optical observation using the indicator substance described later.

[0098] The above microorganisms can be combined with the magnetic particles using a known method, but preferably, a substance that selectively binds to a protein present on the surface of a microorganism can be combined with the surface of the magnetic particles and then used. At this time, the substance that selectively binds to a protein present on the surface of a microorganism can be appropriately selected and used depending on the type of each microorganism, but preferably, it can be apolipoprotein H, beta-2-glycoprotein 1, mannose binding lectin, antibody, aptamer, antibiotic, or polymer.

[0099] In addition, the magnetic particles may be directly mixed into the sample as described above, but a concentration process using the magnetic particles may be performed (see Fig. 6). This concentration process may be performed inside the chamber, or may be performed in a separate chamber from the chamber. That is, if the magnetic particles are mixed with the test target material that requires testing and the magnetic particles are separated, the microorganisms (analyte) attached to the magnetic particles can also be separated. Using this, microorganisms can be separated at a high concentration from the test target material, and they can be cultured to verify sterility.

[0100] Since the above indicator material is the same as described above, its description will be omitted.

[0101] The upper plate is a part that is fitted into the lower plate, and as described above, a central axis (120) formed on the lower plate can be inserted into a central axis insertion hole (210) installed on the upper plate to be fitted thereto. That is, the upper plate can be fitted so as to rotate around the central axis (120) (Fig. 2 (b) and (c)). Since the upper plate has a magnet (230) installed thereon, which will be described later, the gap between the magnet (230) and the chamber of the lower plate can change due to the rotation as described above. At this time, when the magnet (230) moves away, the magnetic nanoparticles inside the chamber can float freely inside the chamber, and when the magnet (230) moves closer, the magnetic nanoparticles can be captured to one side of the chamber by the magnet (230) (see Fig. 1 (b)). This will be described later.

[0102] The upper plate may include a central axis insertion hole (210) into which the central axis (120) of the lower plate is inserted; a chamber insertion hole into which the chamber is inserted; and a magnet (230) installed on one side of the chamber insertion hole (see FIG. 3).

[0103] The above-mentioned central axis insertion hole (210) is a portion into which the central axis (120) installed in the lower plate is inserted, and may be manufactured in a circular shape corresponding to the central axis (120) so that the central axis (120) can be easily rotated. At this time, the central axis insertion hole (210) is preferably located in the center of the upper plate, but in cases where multiple chambers are installed and eccentric rotation is required, it may be located on one side of the upper plate rather than the center.

[0104]

[0105] In addition, in the case of the above-mentioned top plate, it is preferable to manufacture it in a circular or oval shape as shown in Fig. 3, but it is also possible to manufacture it in a square or polygonal shape for the arrangement of internal magnets and chambers or for coupling with an external system.

[0106] In addition, if a separation prevention means is installed on the central axis (120), separation of the upper and lower plates can be prevented by manufacturing it in a corresponding form.

[0107] The chamber insertion portion may have an arc shape centered on the central axis (120). As described above, the nanoparticle-based biochip for the rapid sterility test platform of the present invention can change the position of the magnet (230) by rotating the upper plate. In this case, if the chamber insertion portion has the same shape as the chamber, the upper plate cannot rotate, and even if it is simply formed in a straight line, the upper plate may not rotate. Therefore, if it has an arc shape as described above, the chamber can move along the arc shape, and as a result, the upper plate can rotate smoothly.

[0108] At this time, it is preferable that one chamber insertion part is installed for each chamber. That is, in the case of the present invention, since the sample chamber (131) and the control chamber (132) are installed in the lower plate, the sample chamber insertion port (221) and the control chamber insertion port (222) can be formed at positions corresponding to each chamber (see (a) of FIG. 2).

[0109] In addition, in the case of the present invention, as will be described later, the magnetic nanoparticles inside the chamber are moved using a magnet (230), so it is preferable that each chamber be provided with its own magnet (230) and chamber insertion port. If multiple chambers share the same chamber insertion port, the positions of the magnets (230) corresponding to each chamber are not uniform, so they may be differently affected by magnetism, which may result in differences in observation between each chamber.

[0110] A magnet (230) may be installed on one side of the chamber insertion port. The magnet (230) is used to capture magnetic nanoparticles inside the chamber to one side of the chamber, and is preferably installed on one side of the chamber insertion port.

[0111] That is, the distance between the chamber and the magnet changes due to the rotation of the upper plate, and when the chamber is located at a position away from the magnet (230), the influence of magnetism is reduced, allowing the magnetic nanoparticles to float within the chamber. In addition, when the chamber is located at a position close to the magnet (230), the magnetic nanoparticles can be captured on one side of the chamber due to magnetism (see Fig. 1).

[0112] In addition, in the case of the magnet, the distance from the chamber may change due to the rotation of the upper plate, but in addition, the distance from the chamber may be spaced apart due to the movement of the upper plate. That is, when the chamber insertion port is formed in a straight line, the upper plate moves in a straight line rather than a rotational movement, and at this time, the magnet may also change the distance from the chamber by the straight line movement on the upper plate as described above. In addition, in this case, it is preferable that the upper plate be manufactured in a polyhedral shape rather than a circle to facilitate the straight line movement as described above.

[0113] The capture of these magnetic particles can have two effects. First, it can increase the ease and accuracy of observation. The magnetic particles can be combined with microorganisms to form a complex as described above, and at this time, the complex can be captured on one side of the chamber by the magnet (230). Consequently, after the capture is completed as described above, the concentration of microorganisms in the center of the chamber can be drastically reduced. Generally, when magnetic particles are used, a large amount of magnetic particles float within the chamber, so light cannot penetrate and it is difficult to observe changes in color or fluorescence due to the indicator substance. Therefore, in existing methods, after long-term cultivation, the magnetic particles (complex) are removed and changes in turbidity or color are observed. However, in the case of the present invention, by capturing the complex on one side of the chamber using the magnet as described above, it is possible to obtain a clear image even if only a small change in color or fluorescence such as the above appears.

[0114] The second effect can promote microbial growth. Generally, when microorganisms reach a certain concentration, their population no longer increases. As discussed above, this is due to substrate competition, nutrient deficiency, and external contamination, and for the continuous growth of microorganisms, the concentration needs to be controlled below a certain level. In the present invention, by applying this, when the upper plate is rotated forward to bring the distance between the chamber and the magnet (230) closer, the concentration of the complex in the center of the chamber is lowered, facilitating image acquisition. In addition, when the upper plate is rotated backward to increase the distance from the magnet (230), the local increase in microbial concentration is resolved, thereby increasing the overall culture speed. Therefore, after image acquisition is complete or when rapid culture is required, the upper plate can be rotated backward to increase the distance between the magnet (230) and the vacuum chamber. When image acquisition is complete or when culture is complete and no additional culture is required, the upper plate can be rotated forward to bring the distance between the chamber and the vacuum chamber closer.

[0115] Therefore, even when the concentration of microorganisms is very low, a high concentration of microorganisms can be obtained by repeating the image acquisition and culture as described above multiple times, and through this, higher sterility can be proven in a shorter period of time than with existing methods.

[0116] The magnet (230) may be installed on one side of the chamber insertion port. In particular, it is preferable that the magnet (230) be installed in the same direction as the direction in which the chamber rotates (see FIG. 2). When the chamber is spaced apart from the magnet (230), it is preferable that the magnetic nanoparticles float within the chamber by minimizing the influence of magnetism. At this time, when the chambers are installed at equal intervals, if the rotation angle of the chamber is half the installation angle of the magnet (230), each chamber is arranged at equal intervals from each magnet (230). In this case, since the chamber is located between two magnets (230), the magnetic nanoparticles inside the chamber are minimized by the influence of the magnetic force, and can float within the chamber (see FIG. 3).

[0117] When the magnet (230) is installed on the outside or inside of the direction in which the chamber rotates, the magnet (230) is positioned on the inside or outside despite the rotation, so the magnetic particles can be captured on the inside or outside of the chamber due to the influence of the magnet (230).

[0118] A rotation means (240) capable of rotating the upper plate may be installed on the outer surface of the upper plate. As described above, the distance between the chamber and the magnet (230) may be adjusted by the rotation of the upper plate in the present invention. For this rotation, it is preferable that a rotation means (240) be installed on the outer surface of the upper plate, and the rotation means (240) may rotate the upper plate forward or backward by a force transmitted from a rotation power unit (300) to be described later.

[0119] At this time, the rotating means (240) may be installed without limitation as long as it can rotate the upper plate, but preferably, it may be a gear-shaped device installed on the outer surface of the upper plate. That is, the upper plate of the present invention may have a cylindrical gear shape with gear teeth formed on the outer surface as a whole.

[0120] In addition, it is preferable that the shape of the above gear teeth be manufactured as a spur gear, but it may be manufactured as a bevel gear or screw gear depending on the installation position of the rotation power unit (300) described later, and it is also possible to manufacture it as a helical gear for noise reduction, etc.

[0121] The present invention also relates to a rapid sterility test platform comprising a biochip for the rapid sterility test platform.

[0122] The rapid sterility test platform may include: the biochip; a rotational power unit (300) that rotates the upper plate of the biochip; a lighting unit (400) installed on the upper or lower side of the biochip; and an image acquisition unit (500) that photographs the interior of the chamber of the biochip (see FIG. 4).

[0123] The above biochip is the same as described above, so its description will be omitted.

[0124] The above-mentioned rotational power unit (300) may be a gear connected to an electric motor that supplies power for the rotation of the upper plate. At this time, the gear may be directly coupled to the rotation means (240) of the upper plate to supply power, but may also be connected via a chain, belt, or the like. In addition, the gear installed in the above-mentioned rotational power unit (300) preferably has a shape corresponding to the rotation means (240) of the upper plate, and specifically may have a shape of a spur gear, a helical gear, a rack gear, a bevel gear, a screw gear, or a worm gear.

[0125] A lighting unit (400) may be installed on the top or bottom of the biochip. The lighting unit (400) supplies lighting for observing the inside of the chamber. When observing color changes, white lighting may be used, and when observing fluorescence, ultraviolet or visible light may be supplied. In addition, the top or bottom surface (110) of the chamber is preferably made of a transparent material for the above-described lighting and observation.

[0126] The above lighting can be used without limitation as long as it supplies light of a desired color, but an LED light source is preferably used. In this case, the LED light source may be a light source that emits visible light or ultraviolet light, and a laser LED may also be used for fluorescence observation.

[0127] An image acquisition unit (500) may be installed to photograph the inside of the chamber of the biochip. The image acquisition unit (500) is used to observe changes in color or fluorescence inside the chamber, and an image acquisition device using a CCD may be used. In the case of the present invention, since color or fluorescence may change due to the redox indicator substance as described above during a sterility test, the presence or absence of microorganisms can be easily confirmed by acquiring images at regular time intervals using the image acquisition unit (500) as described above. In addition, in order to increase accuracy during the image acquisition, the upper plate is rotated to bring the chamber and the magnet (230) closer, as described above.

[0128] Looking at the image acquisition unit (500) in detail, the present invention can use a miniaturized imaging system of 224 mm x 120 mm x 140 mm for time-series fluorescence imaging. The imaging system includes an xyz motorized stage capable of adjusting the imaging area and focus for a chip sample, a 4X objective lens, a CCD camera, and may further include the lighting (400). In addition, it is preferable that the imaging system of the present invention be manufactured in a simplified form specifically for the purpose of a rapid sterility verification method. This allows it to be distributed to the industry in a much cheaper and simplified form than existing commercialized equipment such as plate readers, and can be easily incorporated into incubators and the sterility test platform of the present invention, which is advantageous for temperature maintenance and space optimization.

[0129]

[0130] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Furthermore, when describing the present invention, detailed descriptions of related, known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, certain features presented in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0131]

[0132] Example 1

[0133] The separation and concentration of microorganisms using nanoparticles is performed according to the process presented in Figure 3. Nanoparticles coated with a substance that binds to microorganisms are added in proportion to the sample volume of the biopharmaceutical being tested, and then mixed at 35°C for approximately 15 minutes to induce binding of the nanoparticles to the microorganisms.

[0134] At this time, the nanoparticles were coated with Apolipoprotein H, a type of protein. Afterwards, the nanoparticles were isolated toward the wall by attaching them to a magnetic bar for 5 minutes, and the supernatant excluding the nanoparticles was removed. Finally, a small amount of culture medium was added to the sample in which only the nanoparticles bound to microorganisms remained and resuspension was performed. Specifically, Tryptic soy broth was used as the culture medium for detecting aerobic microorganisms, and Fluid Thioglycollate Medium was used as the culture medium for detecting anaerobic microorganisms.

[0135] The resuspended sample was injected into the sample chamber of the biochip, and resazurin, a redox indicator, was added at a concentration of 10 wt%. The control chamber was injected with magnetic nanoparticles, culture medium, and resazurin at the same concentrations as the resuspended sample.

[0136] Afterwards, the biochip was inserted into the imaging system shown in Fig. 4, and fluorescence imaging was performed at regular intervals. The fluorescence signals extracted from each image taken from the sample and the control group without microorganisms were measured using analysis software, and the intensities were compared. If the fluorescence signal measured from the sample was significantly higher than the fluorescence signal measured from the control group, it was judged as positive for contamination.

[0137]

[0138] Experimental results

[0139] Figure 7 illustrates the results of an experiment confirming the performance of the biochip included in the present invention. When the number of microorganisms was compared after 12 hours under the condition of culturing in a growth medium without nanoparticles as a control (ctrl.), the number of microorganisms was measured to be about 5 times less under the condition of culturing in an environment where nanoparticles were aggregated by applying magnetism (w / mag.). This means that the growth of microorganisms was inhibited due to the aggregated environment. On the other hand, when the environment where magnetism was removed (w / o mag.) was simulated using a biochip, the number of microorganisms similar to the control group was observed after the same amount of time of culturing, and this was statistically verified through 5 repeated experiments. In other words, through the above results, it was confirmed that in the case of the present invention, the culture speed can be controlled by a simple operation such as rotating the top plate.

[0140] Figure 8 illustrates the results of a comparison between the rapid sterility verification test (NEST) proposed in the present invention and the existing 14-day culture-based sterility verification test for performance evaluation. Six representative microorganisms recommended for sterility verification performance evaluation were injected into biopharmaceuticals at a concentration of 1 CFU / mL, and the two tests were performed. As a result, it was confirmed that the rapid sterility verification test of the present invention provided superior positive determination accuracy along with an average measurement time of approximately 18 times shorter than the existing test method in 60 tests each. The rapid sterility verification test detected 57 out of 60 positive results (95% accuracy), while the existing sterility verification test detected 50 positive results (83.3% accuracy).

[0141] Figure 9 shows the results of rapid sterility tests for various types of biopharmaceuticals. The rapid sterility test, which shows superior performance compared to existing test methods, can be universally applied regardless of the substances contained in the biopharmaceutical by specifically isolating only the microorganism. To demonstrate this, a type of microorganism was administered to various biopharmaceuticals, including clinical-grade CAR-T therapy, cell therapy, and RNA vaccines, and the rapid sterility test was applied. As a result, all samples showed similar signal changes over time, confirming the specific detection of only the signal for the microorganism and the expandability of the industrial application of the invented rapid sterility test.

[0142] Furthermore, the rapid sterility test of the present invention can easily confirm the presence of microorganisms with the naked eye in addition to the method using fluorescence described above. As shown in Fig. 10, this method is not applicable to anaerobic microorganisms, and although it takes a relatively long time compared to analysis using fluorescence imaging for a clear comparison, it can serve as a means to easily perform sterility verification tests even in environments without imaging equipment. For bacteria with a fast division rate, contamination of biopharmaceuticals can be confirmed within 16 hours, and for fungal strains with a relatively slow division rate, contamination can be confirmed within 24 hours.

[0143]

[0144] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid sterility test method using a biochip comprising one or more chambers into which samples and controls are each loaded; and one or more magnets installed on one side of each of the chambers, A step of mixing magnetic particles into a substance to be examined and binding the analysis target to the magnetic particles; A step of preparing a sample by separating magnetic particles from the above-mentioned test target material; and A step of injecting the separated sample and indicator substance into a chamber of a biochip and culturing them to determine the presence of the analyte; Includes, A rapid sterility test method in which the step of determining the presence of the analyte is to determine the presence of the analyte within the sample by comparing the fluorescence or color of the sample and control images after culture.

2. In paragraph 1, A rapid sterility test method characterized in that the above test target material is a biopharmaceutical, cell therapy product, clinical sample, chemical pharmaceutical, cosmetic, food or environmental sample.

3. In paragraph 1, A rapid sterility test method characterized in that the above analysis target is bacteria, mycoplasma or fungi.

4. In paragraph 1, A rapid sterility test method, characterized in that the step of preparing the sample includes a step of concentrating the analyte combined with the magnetic particles on a growth medium.

5. In paragraph 4, The above growth medium is a rapid sterility test method including Tryptic soy broth, Muller-Hinton broth, Fluid Thioglyollate Medium, Sabouraud broth, and Luria-Bertani broth.

6. In paragraph 1, The step of determining the existence of the above analysis target is: A step of separating the chamber from the magnet; A step of injecting indicator substances, samples, and controls into the chamber and culturing them for a certain period of time; A step of bringing the chamber closer to the magnet after the above culturing is completed; and A step of photographing the interior of the chamber to obtain an image, and then comparing the images photographed from the separated sample and the control group; A rapid sterility test method including:

7. In paragraph 6, The above analysis object is combined with the magnetic particles to form a complex, A rapid sterility test method wherein the complex is moved to one side of the chamber by the magnet when the chamber is brought close to the magnet.

8. In paragraph 1, A rapid sterility test method wherein the above culture is performed for 4 to 48 hours.

9. In paragraph 1, A rapid sterility test method in which the above image acquisition is performed at intervals of 10 to 120 minutes.

10. In paragraph 1, The above indicator substance is a rapid sterility test method in which fluorescence or color changes by the analyte.

11. In paragraph 10, A rapid sterility test method characterized in that the above indicator substance is a redox indicator substance, a pH indicator, a dye-based reagent, an enzyme reaction indicator, or an antibody-based reagent.

12. A lower plate including a chamber into which the control and sample are loaded; and An upper plate fitted into the lower plate and including a magnet; In a biochip including, The above lower plate, floor surface; A central axis protruding upward from the above floor surface; and One or more chambers installed at a certain distance from the central axis; Includes, The upper plate has a central axis insertion hole into which the central axis of the lower plate is inserted; a chamber insertion port into which the chamber is inserted; and A magnet installed on one side of the chamber insertion port; Biochip for rapid sterility testing platform including.

13. In paragraph 12, A biochip for a rapid sterility test platform, wherein the chamber comprises one or more sample chambers and one or more control chambers.

14. In paragraph 13, A biochip for a rapid sterility test platform, wherein the chambers are installed at equal intervals at a uniform distance around the central axis.

15. In paragraph 12, A biochip for a rapid sterility test platform further comprising magnetic particles and indicator substances inside the chamber.

16. In paragraph 15, A biochip for a rapid sterility test platform, wherein the magnetic particles are combined with an analyte to form a complex.

17. In paragraph 16, A biochip for a rapid sterility test platform, wherein the magnetic particles include a functional group or substance capable of binding to microorganisms on the surface.

18. In paragraph 16, A biochip for a rapid sterility test platform, wherein the complex is positioned on one side of the chamber using the magnet when observing fluorescence or optically using the indicator substance.

19. In paragraph 12, A biochip for a rapid sterility test platform, wherein the above-mentioned upper plate is rotated at a certain angle around the above-mentioned central axis.

20. In paragraph 19, A biochip for a rapid sterility test platform, wherein the control chamber insert has an arc shape centered on the central axis.

21. In paragraph 19, A biochip for a rapid sterility test platform, wherein the distance between the magnet and the chamber changes according to the rotation of the upper plate.

22. In paragraph 19, A biochip for a rapid sterile test platform having a rotation means for rotating the upper plate installed on the outer surface of the upper plate.

23. A rapid sterility test platform comprising a biochip for a rapid sterility test platform according to any one of claims 12 to 22.

24. In paragraph 23, The above rapid sterility test platform, The above biochip; A rotational power unit that rotates the upper plate of the above biochip; A lighting unit installed on the upper or lower side of the biochip; and An image acquisition unit for photographing the interior of the chamber of the biochip; A rapid sterility testing platform including.

25. In paragraph 24, The above image acquisition unit is a rapid sterility test platform that acquires a fluorescent image or an optical image inside the chamber.

26. In paragraph 24, The above rapid sterility test platform is a rapid sterility test platform that determines the presence of microorganisms within the sample by comparing the fluorescence or color of the first image acquired and the current image.

Images