Method for identifying trace visible foreign matter in liquid

Abstract

The application provides a method for analyzing and identifying trace visible foreign matters in liquid, and belongs to the technical field of pharmaceutical preparation analysis and detection, and comprises a silicon column method and a suction filtration method, the silicon column method is used in the case of sample scarcity and preciousness, and comprises the following steps: preparation of a silicon column array structure chip, foreign matter separation operation in a to-be-tested solution, analysis and identification operation, the suction filtration method is used for separation and analysis of a small amount of solid insoluble matters in a large amount of liquid preparation, and comprises the following steps: manufacturing of a glass through-hole structure, filter membrane fitting operation, solid insoluble matter separation operation, and microscopic imaging scanning, the application is aimed at insoluble matters in trace liquid and a small amount of micron-level solid insoluble matters in a large amount of liquid in biological pharmaceuticals, the expression structure of the hexagonal packing structure is used to highlight the silicon column structure and the through-hole structure, and finally, the solid insoluble matters in the liquid are obtained on a relatively flat surface, and the microscopic technology is used to facilitate appearance and quantity statistics and chemical component analysis in the mode of fingerprint spectrum.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation analysis and detection technology, and in particular to a method for the analysis and identification of trace visible foreign matter in liquids. Based on solvent separation and combined with fingerprint spectroscopy, it is used for the separation, component detection and traceability of micron-level insoluble foreign matter in liquids during the research and development and production of pharmaceutical preparations. Background Technology

[0002] In the field of pharmaceutical formulations, the detection of visible insoluble matter (especially micron-sized foreign matter) in liquids is of paramount importance, as it is directly related to the safety, efficacy, quality control, and regulatory compliance of drugs. Visible insoluble matter in pharmaceutical formulations, such as biologics (monoclonal antibodies, vaccines, gene therapy drugs, etc.), may include: production process contaminants such as metal particles (from wear and tear of production equipment), glass fragments (from containers / pipes), and microplastics (from filter cartridges or seals); drug degradation products such as protein aggregates, polysaccharide particles, and crystalline impurities; and foreign contaminants such as fibers and microbial cell fragments.

[0003] The presence of these substances can cause a series of harms, including:

[0004] ① Local irritation or inflammation: micron-sized particles may block capillaries or irritate tissues, causing redness, swelling, pain, or even granulomas;

[0005] ② Immune response: Protein aggregates may be recognized as "foreign objects" by the immune system, triggering allergic reactions or the generation of anti-drug antibodies (ADA), leading to reduced drug efficacy or treatment failure;

[0006] ③ Toxicity risk: Some metallic impurities (such as nickel and chromium) may have potential toxicity, and long-term exposure to trace amounts may accumulate into harm.

[0007] Both the Chinese Pharmacopoeia and the US FDA have made clear provisions regarding visible foreign matter in pharmaceutical preparations. The Chinese Pharmacopoeia (ChP) explicitly stipulates that injectable and ophthalmic preparations must undergo "visible foreign matter testing," requiring that no visible foreign matter be detected under specified conditions (ChP 2025 General Chapter 0904). The US FDA and EMA guidelines require that pharmaceutical preparations implement "foreign matter control strategies" throughout the entire research and development and production cycle, requiring the detection of insoluble matter through methods such as light obscuration and microscopy, and setting thresholds for particle size (e.g., ≥10μm) and quantity.

[0008] In existing technologies, the detection of visible foreign matter during the drug formulation development process can be used for:

[0009] ① Tracing the source of impurities and optimizing the process: For example, in the early stages of biopharmaceutical development (such as cell culture and purification processes), detecting trace amounts of foreign matter can help locate the source of contamination;

[0010] ② Testing during the production stage can control the production process and assess drug stability.

[0011] ③ In the field of chemical pharmaceuticals, the analysis of visible foreign matter in drug formulations can optimize production processes and identify problematic raw materials.

[0012] However, in the actual research and development process of biopharmaceuticals, the following technical challenges exist, for example:

[0013] ① It is necessary to design and screen a large number of protein structures to find an effective active ingredient. The design and preparation of each possible protein structure requires a lot of cost. In order to save costs and improve screening efficiency, the output of each experiment is very small, so these very small products become very precious. It is the common pursuit of researchers to obtain the ideal answer with a small sample when analyzing the insoluble substances in the product.

[0014] ② In liquid injection preparations, such as sodium chloride injection and glucose injection, the liquid volume is usually large, and the entire package may reach hundreds or even thousands of milliliters. The solid insoluble matter present in the liquid is difficult to extract, making it more difficult to monitor the overall quality of the drug.

[0015] Therefore, there is an urgent need for a method for the separation and identification of solid insoluble substances applicable to both of the above application scenarios. Currently, the main technologies for detecting micron-sized insoluble substances include: centrifugation, filtration, microfluidics, light scattering, and machine vision inspection, among which:

[0016] ① Centrifugation: This is a conventional technique in the pharmaceutical field for separating micron-sized particles. It is widely used in laboratories and production processes and is a well-known and publicly available technology. This technique uses high-speed rotation to generate centrifugal force and utilizes the density difference between particles and liquid to achieve separation.

[0017] This method has the advantages of being relatively simple to operate and capable of handling large volume samples, but it also has obvious disadvantages. High centrifugal force may cause fragile particles (such as protein aggregates) to break or denature, and there is particle loss. It is less efficient when processing small amounts of liquid (such as microliters) and it is difficult to accurately separate particles of similar size. The operation is cumbersome, requiring manual control of centrifugation parameters, making it difficult to automate, and it is also time-consuming (such as density gradient centrifugation which takes several hours).

[0018] ② Filtration: This is a common method for separating particles in pharmaceutical preparations, including dead-end filtration and tangential flow filtration (TFF), which are well-known and widely used technologies. It utilizes filter membranes with different pore sizes to retain target particles; for example, bacteria or larger impurities can be removed through a 0.22μm filter membrane.

[0019] This method has low equipment cost and simple operation. However, the filter membrane may adsorb target particles (such as proteins), resulting in a reduced recovery rate. High-concentration samples are prone to clogging the filter membrane, requiring frequent replacement. Furthermore, the separation accuracy is limited, and residual liquid on the filter membrane surface may lead to the loss of trace amounts of sample. Therefore, it is not suitable for precious biological agents.

[0020] ③ Microfluidic technology: Its application in biological separation has been widely studied, such as magnetic field driving, dielectrophoresis, and acoustic sorting methods.

[0021] This technology boasts strong micro-volume processing capabilities, making it suitable for micro-volume samples. It reduces sample consumption and offers high separation accuracy, enabling precise sorting based on particle size, charge, and dielectric properties. However, the manufacturing process of microfluidic chips is complex, requiring specialized equipment (such as photolithography) and relying on high-precision pump control systems. This results in slower processing speeds, making it difficult to meet the demands of large-scale production (unlike traditional centrifugation, which can handle liter-level samples). Furthermore, the technology has a high barrier to entry, requiring specialized knowledge to design chip structures and optimize fluid parameters, making widespread adoption challenging.

[0022] ④ Light scattering method: This is a method for detecting visible foreign matter recommended by pharmacopoeias (such as the Chinese Pharmacopoeia). By irradiating a liquid with a laser and analyzing the energy or image of the scattered light from the particles, the presence and size of the foreign matter can be determined. This method is suitable for detecting particles larger than 50 μm in transparent liquids and can distinguish between foreign matter such as metal shavings and glass shards.

[0023] However, this method can only detect the presence of particles and cannot achieve the physical separation of particles for component analysis. Furthermore, its applicability is limited; samples in suspensions, emulsions, or dark-colored containers cannot be analyzed using light scattering.

[0024] While the methods mentioned above all have the ability to separate visible foreign matter from samples, none of them have the ability to conduct systematic analysis of visible foreign matter. Systematic analysis includes the identification of the material composition of visible foreign matter, quantity statistics, particle size statistics, and source tracing. These are the problems that urgently need to be solved in the field of visible foreign matter detection in pharmaceutical preparations. Summary of the Invention

[0025] To address the aforementioned technical problems, this invention provides a method for analyzing and identifying trace visible foreign matter in liquids. This method separates the liquid from the visible solid insoluble matter it contains, allowing the micron-sized insoluble matter in the formulation to be isolated. Molecular fingerprinting is used to identify the molecular composition of the solid foreign matter, and microscopic optical statistical analysis is employed to statistically analyze the quantity and size of the solid insoluble matter. This enables R&D and manufacturing companies to investigate the source of the solid insoluble matter and formulate corresponding improvement strategies.

[0026] A method for analyzing and identifying trace visible foreign matter in a liquid includes: a silica column method and a suction filtration method, wherein:

[0027] The silicon column method is used when samples are scarce and valuable during the research and development and production stages of biopharmaceuticals, including:

[0028] Step 1: Fabrication of silicon pillar array structure chips;

[0029] Multiple sets of silicon pillars are arranged on the sorting chip in a hexagonal stacked structure with equal spacing, so that the spacing between each set of silicon pillars is equal; at the same time, the diameter and height of each set of silicon pillars are the same, forming a silicon pillar array structure; the spacing between each set of silicon pillars and the diameter of the silicon pillars are dynamically adjusted according to the size of the solid insoluble matter to be analyzed.

[0030] The silicon pillar array structure and sorting chip are subjected to surface hydroxylation or amination treatment to make them superhydrophilic, and thus a silicon pillar array structure chip is fabricated.

[0031] As an example, the spacing between two adjacent sets of silicon pillars is set between 0.5 and 10 μm.

[0032] As an example, the diameter of each set of silicon pillars is set between 0.5 and 10 μm.

[0033] As an example, the height of each set of silicon pillars is set between 500nm and 10μm.

[0034] As a preferred example, when dealing with trace samples of less than 10 microliters containing 1 μm of insoluble matter, a silicon pillar array chip with a spacing of 0.5 μm (the spacing should be smaller than the diameter of the insoluble matter), a diameter of 0.5-1 μm, and a height of 500 nm is used.

[0035] Step 2: Separation of foreign matter from the solution to be tested;

[0036] Take the solution to be tested and drop it onto the surface of the silicon pillar array structure chip;

[0037] Because the silicon pillar array structure chip has undergone hydrophilic treatment, the silicon pillars themselves have superhydrophilic properties. The liquid quickly bonds with the surface of the silicon pillars and flows into the bottom of the silicon pillars along the sidewalls and diffuses in all directions. Meanwhile, the solid insoluble substances contained in the liquid are supported on the surface of the silicon pillars due to the influence of steric hindrance. They cannot flow with the liquid and will not sink into the gaps between the silicon pillars. This completes the solid-liquid separation process.

[0038] Step 3: Analysis and identification procedures;

[0039] The solid insoluble matter in the test solution eventually deposits on the surface of the silicon pillar, forming a circular region through the internal stress of the liquid itself. The fixed insoluble matter (the target to be tested) is distributed within this circular region, and the fixed insoluble matter is scanned by microscopic imaging using a micro Raman spectroscopy system.

[0040] As an example, the microscopic imaging scan includes:

[0041] ① Count the number and size of particles and generate a particle count report;

[0042] ② Raman spectroscopy is performed on each particle. Raman spectroscopy has fingerprint recognition capabilities and can identify the chemical composition of each substance. Pollution can be traced based on all the links involved in the process.

[0043] By performing solid-liquid separation, quantity statistics, and source tracing of solid insoluble substances, it provides strong support for drug development, industrial upgrading, and quality control.

[0044] As an example, when the test solution is 10 μL of liquid, the area that ultimately contacts the substrate surface is approximately a circular region with a diameter of 5 mm. The test targets are all distributed within this 5 mm circular region, which facilitates microscopic observation and spectral acquisition.

[0045] As an example, in practical applications, there are some applications where the size of the fixed insoluble matter is greater than 1 μm. Depending on the size of the solid insoluble matter, silicon pillar array structure chips of different specifications can be selected to separate different solid insoluble matter for subsequent microscopic or spectroscopic analysis.

[0046] The aforementioned suction filtration method is used for the separation and analysis of small amounts of solid insoluble matter in large quantities of liquid formulations, including:

[0047] Step 1: Fabrication of the glass through-hole structure;

[0048] Using a standard cover glass, multiple identical through holes are machined at equal intervals on its surface in a hexagonal stacking structure to form a glass through-hole structure;

[0049] As an example, the diameter of each group of through holes is set to be between 5 and 50 μm; the spacing between each group of through holes is set to be between 10 and 50 μm.

[0050] As an example, the hexagonal stacked structure can also be replaced by a square stacked structure, with multiple identical through holes machined at equal intervals.

[0051] Step 2: Filter membrane bonding operation;

[0052] The filter membrane is flatly attached to the glass through-hole structure; the filter membrane is selected based on the amount of liquid to be tested and the size range of the insoluble solid to be tested.

[0053] As an example, the selection criteria for the filter membrane are as follows: when the size of the solid insoluble matter is 5μm, a filter membrane with a pore size of 2μm is selected to ensure that the target matter is separated, while the liquid flow rate is fast enough to improve the detection efficiency.

[0054] As an example, the filter membrane is a filter membrane with nanoscale pores.

[0055] As an example, when the pore size of the filter membrane is greater than 3μm, there is tension in the through-pores, causing size fluctuations; when the pore size of the filter membrane is less than 0.5μm, the filtration speed is slow. The pore size of the filter membrane is set to be between 0.5μm and 3μm.

[0056] Step 3: Separation of solid insoluble substances;

[0057] Place the glass perforated structure with the filter membrane attached inside the Buchner funnel of the suction flask; control the dropping speed of the liquid to be tested according to the viscosity of the liquid to be tested and the suction pump speed; after the dropping is completed, the separation of solid insoluble matter is completed, and at this time the solid insoluble matter is placed on the filter membrane.

[0058] As an example, this method can be used not only for the separation of solid insoluble substances in liquid pharmaceutical preparations, but also for solid-liquid separation of environmental water samples and drinking water quality monitoring.

[0059] As an example, the dropping rate is 0.1 mL / s to 10 mL / s, which can achieve a liquid processing rate of 0.6 L / min.

[0060] Step 4: Place the glass porous filter membrane with one side facing up under the micro Raman spectroscopy system, and use the microscopic imaging function to scan the entire area enriched with solid insoluble matter.

[0061] As an example, the microscopic imaging scan includes:

[0062] ① Count the number and size of particles and generate a particle count report;

[0063] ② Raman spectroscopy is performed on each particle. Raman spectroscopy has fingerprint recognition capabilities and can identify the chemical composition of each substance. Pollution can be traced based on all the links involved in the process.

[0064] By performing solid-liquid separation, quantity statistics, and source tracing of solid insoluble substances, it provides strong support for drug development, industrial upgrading, and quality control.

[0065] As an example, when using a filter membrane alone to separate solid insoluble substances, the separated solid insoluble substances need to be observed under a microscope and have their spectra collected. Therefore, the flatness of the filter membrane is required to be very high. The flatness of the filter membrane needs to fluctuate less than 5μm within a millimeter range to meet the requirements of subsequent operations. However, the filter membranes with micron-sized pores in the existing technology are mainly made of thin polymer materials, which are prone to wrinkles and deformation under suction filtration conditions. They cannot meet the required flatness, resulting in large errors in subsequent observations and causing misjudgments by pharmaceutical companies and other production units.

[0066] The beneficial effects of this invention are:

[0067] This invention addresses the issue of insoluble matter in trace amounts of liquids and trace amounts of micron-sized solid insoluble matter in large amounts of liquids in the biopharmaceutical field. By using a hexagonal stacked structure to express the structure, the invention achieves a prominent silicon pillar structure and an internally permeable through-hole structure. Ultimately, the solid insoluble matter in the liquid is obtained on a relatively flat surface. Microscopic techniques are used to perform appearance and quantity statistics, and fingerprinting is used for chemical composition analysis.

[0068] For the detection of trace samples, especially the analysis of precious micro-level samples, an original filtration architecture design is adopted, which is well used for the separation of micron-sized insoluble matter from the solution in micro-level samples. This method overcomes the shortcomings of existing designs, such as centrifugation and filtration methods being unable to handle trace samples, the complexity of microfluidic technology, and the inability of light scattering methods to analyze and detect small-sized targets.

[0069] Suction filtration can rapidly extract liquids, and if the rate of liquid addition is properly controlled, the spatial distribution of solid insoluble matter can be limited to within 10 mm, thereby improving the efficiency of subsequent microscopic observation and spectral acquisition.

[0070] The suction filtration method involves laying a filter membrane on a glass substrate to achieve adhesion and support of the filter membrane. At this time, the filter membrane is completely adhered to the glass substrate, and the surface maintains the same flatness as the glass substrate. Furthermore, no deformation occurs during the suction filtration process, which meets the conditions for subsequent microscopic observation. Attached Figure Description

[0071] Figure 1 This is a flowchart illustrating a method for analyzing and identifying trace visible foreign matter in a liquid according to the present invention.

[0072] Figure 2 This is a schematic diagram of a silicon pillar array structure for a method of analyzing and identifying trace visible foreign matter in liquids according to the present invention.

[0073] Figure 3 The images show a comparison of two silicon pillar array structures with different spacings for a method for analyzing and identifying trace visible foreign matter in liquids according to the present invention (top view of the product, where the spacing between each group of silicon pillars in image P1 is smaller than the spacing between each group of silicon pillars in image P2).

[0074] Figure 4 This is a comparison diagram (product side view) of two silicon pillar array structure chips with different spacings for the analysis and identification method of trace visible foreign matter in liquid according to the present invention.

[0075] Figure 5 This is a schematic diagram of the microscopic imaging scanning principle of the suction filtration method for the analysis and identification of trace visible foreign matter in liquid according to the present invention.

[0076] Figure 6 This is a schematic diagram of the glass through-hole structure of the suction filtration method for the analysis and identification of trace visible foreign matter in liquid according to the present invention.

[0077] Figure 7 This is a product rendering of a glass porous structure filter membrane after lamination, which is part of the suction filtration method for the analysis and identification of trace visible foreign matter in liquids according to the present invention.

[0078] Figure 8 This is a microscopic image of 169 solid insoluble substances separated by the suction filtration method of the present invention for the analysis and identification of trace visible foreign matter in liquid.

[0079] Figure 9 This is a Raman spectrum obtained by microscopic imaging scanning of a solid insoluble substance separated by the absorption filtration method in a method for analyzing and identifying trace visible foreign matter in liquids according to the present invention (the solid insoluble substance is PET). Detailed Implementation

[0080] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Figures 1 to 9 As shown.

[0081] A method for analyzing and identifying trace visible foreign matter in a liquid includes: a silica column method and a suction filtration method, wherein:

[0082] The silicon column method is used when samples are scarce and valuable during the research and development and production stages of biopharmaceuticals, including:

[0083] Step 1: Fabrication of silicon pillar array structure chips;

[0084] Multiple sets of silicon pillars are arranged on the sorting chip in a hexagonal stacked structure with equal spacing, so that the spacing between each set of silicon pillars is equal; at the same time, the diameter and height of each set of silicon pillars are the same, forming a silicon pillar array structure; the spacing between each set of silicon pillars and the diameter of the silicon pillars are dynamically adjusted according to the size of the solid insoluble matter to be analyzed.

[0085] The silicon pillar array structure and sorting chip are subjected to surface hydroxylation or amination treatment to make them superhydrophilic, and thus a silicon pillar array structure chip is fabricated.

[0086] As an example, the spacing between two adjacent sets of silicon pillars is set between 0.5 and 10 μm.

[0087] As an example, the diameter of each set of silicon pillars is set between 0.5 and 10 μm.

[0088] As an example, the height of each set of silicon pillars is set between 500nm and 10μm.

[0089] As a preferred example, when dealing with trace samples of less than 10 microliters containing 1 μm of insoluble matter, a silicon pillar array chip with a spacing of 0.5 μm (the spacing should be smaller than the diameter of the insoluble matter), a diameter of 0.5-1 μm, and a height of 500 nm is used.

[0090] Step 2: Separation of foreign matter from the solution to be tested;

[0091] Take the solution to be tested and drop it onto the surface of the silicon pillar array structure chip;

[0092] Because the silicon pillar array structure chip has undergone hydrophilic treatment, the silicon pillars themselves have superhydrophilic properties. The liquid quickly bonds with the surface of the silicon pillars and flows into the bottom of the silicon pillars along the sidewalls and diffuses in all directions. Meanwhile, the solid insoluble substances contained in the liquid are supported on the surface of the silicon pillars due to the influence of steric hindrance. They cannot flow with the liquid and will not sink into the gaps between the silicon pillars. This completes the solid-liquid separation process.

[0093] Step 3: Analysis and identification procedures;

[0094] The solid insoluble matter in the test solution eventually deposits on the surface of the silicon pillar, forming a circular region through the internal stress of the liquid itself. The fixed insoluble matter (the target to be tested) is distributed within this circular region, and the fixed insoluble matter is scanned by microscopic imaging using a micro Raman spectroscopy system.

[0095] As an example, the microscopic imaging scan includes:

[0096] ① Count the number and size of particles and generate a particle count report;

[0097] ② Raman spectroscopy is performed on each particle. Raman spectroscopy has fingerprint recognition capabilities and can identify the chemical composition of each substance. Pollution can be traced based on all the links involved in the process.

[0098] By performing solid-liquid separation, quantity statistics, and source tracing of solid insoluble substances, it provides strong support for drug development, industrial upgrading, and quality control.

[0099] As an example, when the test solution is 10 μL of liquid, the area that ultimately contacts the substrate surface is approximately a circular region with a diameter of 5 mm. The test targets are all distributed within this 5 mm circular region, which facilitates microscopic observation and spectral acquisition.

[0100] As an example, in practical applications, there are some applications where the size of the fixed insoluble matter is greater than 1 μm. Depending on the size of the solid insoluble matter, silicon pillar array structure chips of different specifications can be selected to separate different solid insoluble matter for subsequent microscopic or spectroscopic analysis.

[0101] The aforementioned suction filtration method is used for the separation and analysis of small amounts of solid insoluble matter in large quantities of liquid formulations, including:

[0102] Step 1: Fabrication of the glass through-hole structure;

[0103] Using a standard cover glass, multiple identical through holes are machined at equal intervals on its surface in a hexagonal stacking structure to form a glass through-hole structure;

[0104] As an example, the diameter of each group of through holes is set to be between 5 and 50 μm; the spacing between each group of through holes is set to be between 10 and 50 μm.

[0105] As an example, the hexagonal stacked structure can also be replaced by a square stacked structure, with multiple identical through holes machined at equal intervals.

[0106] Step 2: Filter membrane bonding operation;

[0107] The filter membrane is flatly attached to the glass through-hole structure; the filter membrane is selected based on the amount of liquid to be tested and the size range of the insoluble solid to be tested.

[0108] As an example, the selection criteria for the filter membrane are as follows: when the size of the solid insoluble matter is 5μm, a filter membrane with a pore size of 2μm is selected to ensure that the target matter is separated, while the liquid flow rate is fast enough to improve the detection efficiency.

[0109] As an example, the filter membrane is a filter membrane with nanoscale pores.

[0110] As an example, when the pore size of the filter membrane is greater than 3μm, there is tension in the through-pores, causing size fluctuations; when the pore size of the filter membrane is less than 0.5μm, the filtration speed is slow. The pore size of the filter membrane is set to be between 0.5μm and 3μm.

[0111] Step 3: Separation of solid insoluble substances;

[0112] Place the glass perforated structure with the filter membrane attached inside the Buchner funnel of the suction flask; control the dropping speed of the liquid to be tested according to the viscosity of the liquid to be tested and the suction pump speed; after the dropping is completed, the separation of solid insoluble matter is completed, and at this time the solid insoluble matter is placed on the filter membrane.

[0113] As an example, this method can be used not only for the separation of solid insoluble substances in liquid pharmaceutical preparations, but also for solid-liquid separation of environmental water samples and drinking water quality monitoring.

[0114] As an example, the dropping rate is 0.1 mL / s to 10 mL / s, which can achieve a liquid processing rate of 0.6 L / min.

[0115] Step 4: Place the glass porous filter membrane with one side facing up under the micro Raman spectroscopy system, and use the microscopic imaging function to scan the entire area enriched with solid insoluble matter.

[0116] As an example, the microscopic imaging scan includes:

[0117] ① Count the number and size of particles and generate a particle count report;

[0118] ② Raman spectroscopy is performed on each particle. Raman spectroscopy has fingerprint recognition capabilities and can identify the chemical composition of each substance. Pollution can be traced based on all the links involved in the process.

[0119] By performing solid-liquid separation, quantity statistics, and source tracing of solid insoluble substances, it provides strong support for drug development, industrial upgrading, and quality control.

[0120] As an example, when using a filter membrane alone to separate solid insoluble substances, the separated solid insoluble substances need to be observed under a microscope and have their spectra collected. Therefore, the flatness of the filter membrane is required to be very high. The flatness of the filter membrane needs to fluctuate less than 5μm within a millimeter range to meet the requirements of subsequent operations. However, the filter membranes with micron-sized pores in the existing technology are mainly made of thin polymer materials, which are prone to wrinkles and deformation under suction filtration conditions. They cannot meet the required flatness, resulting in large errors in subsequent observations and causing misjudgments by pharmaceutical companies and other production units.

[0121] To better illustrate the design principles of this invention, specific embodiments 1 and 2 are provided below:

[0122] Example 1, Silicon Pillar Method;

[0123] First, this invention designs a fabrication scheme for a hexagonal close-packed silicon pillar array structure, such as... Figure 2 As shown, and using this as a structural model, a silicon pillar array structure chip for filtering solid insoluble matter from trace amounts of liquid was designed, such as... Figure 3 , Figure 4 As shown.

[0124] In this scheme, the diameter of the silicon pillars is an adjustable structural parameter with a size ≥500nm, the minimum gap between the silicon pillars is 500nm, and the entire silicon pillars undergo hydrophilic treatment, giving the entire chip surface strong hydrophilicity.

[0125] When the test solution is dropped onto the chip surface, the water-based biological solution quickly wets the entire chip. The solution flows through the silicon pillars to the bottom of the pillars, while solid insoluble substances larger than 1 μm are supported on the chip surface by the silicon pillars, allowing researchers to analyze their composition and origin, thus providing a basis for research and development.

[0126] This approach creatively reduces the gaps between silicon pillars, allowing for optimal filtration control of solid insolubles in the triangular areas between each pillar.

[0127] Secondly, by controlling the diameter of the silicon pillars, the spacing between the silicon pillars can be effectively adjusted, from a minimum of 500 nm to tens of micrometers, to meet different particulate matter analysis needs.

[0128] When the target to be tested is an insoluble substance of 1 μm or larger, a structure size with a silicon pillar gap of 1 μm can be selected. When the target to be tested is an insoluble substance of 5 μm or larger, a structure size with a silicon pillar gap of 5 μm can be selected to meet the optimal use case.

[0129] Furthermore, the structural dimensions of the silicon pillars can be adjusted by the size of the processing template. When a small gap structure is required, the diameter of the silicon pillars can be designed to be 500 nm or 1 μm. By adjusting the etching process, silicon pillars with a depth of hundreds of nanometers to several micrometers can be prepared, while the gap between the silicon pillars is always kept to be no more than 500 nm. This can satisfy the separation of particles with a diameter of 1 μm.

[0130] When the target particle size is 5 μm or larger, the diameter of the silicon pillars can be designed to be 5 μm or larger. By adjusting the etching process, the gap between the silicon pillars can be kept ≤5 μm, thus ensuring that target particles larger than 5 μm can be separated. At the same time, as the size of the silicon pillar structure increases, the processing technology can also ensure that the silicon pillars have a higher aspect ratio, thereby meeting the requirements for larger volume solution analysis.

[0131] Finally, this separation method can separate solid insolubles from bioactive substance solutions with a minimum solution volume of 1 μL and a maximum volume of 1 mL. The separated solid insolubles are supported on the surface of silicon pillars, and each solid insoluble is larger than 1 μm in size, which can be clearly observed under a microscope. Therefore, further microscopic observation can be performed. At the same time, it can be combined with micro Raman spectroscopy to conduct detailed analysis of the type, structure, and chemical composition of the particles. Alternatively, the chip can be placed in a scanning electron microscope for fine characterization of the fine surface morphology and elemental composition of the particles.

[0132] Example 2, suction filtration method;

[0133] First, adopt such as Figure 5 The suction filtration scheme places a glass substrate with a microporous array inside the Buchner funnel of the suction flask, and drips the test liquid onto the surface of the filter membrane at a certain speed. Under the suction action of the vacuum pump, the liquid is rapidly drawn into the suction flask. The dripped liquid continuously passes through the filter membrane at the same position, collecting solid insoluble matter in the reagent within a minimal area. By minimizing the area of ​​action, the scanning area of ​​the microscopic image can be reduced, thus improving the efficiency of microscopic statistics.

[0134] Then, as Figure 6 , Figure 7 The diagram shows a design of a through-hole glass substrate. The diameter of the glass holes is designed to be 5μm and the spacing is 5μm. This size of glass hole can maximize the flatness of the filter membrane and the liquid flow efficiency, while ensuring that the processing cost is acceptable.

[0135] As the density and diameter of the glass pores increase, the processing cost rises exponentially, which is detrimental to the universal testing of large numbers of samples. While increasing the pore size offers advantages in terms of processing cost and liquid permeability, it may cause deformation of the filter membrane, adversely affecting microscopic observation. Therefore, it is necessary to select appropriate pore array parameters based on the specific circumstances.

[0136] Figure 7 This is a microscopic scanning image of solid particles in a pharmaceutical preparation under a microscope. The image is composed of 13×13 stitched microscopic images and represents a microscopic scan of a specific area. Based on such microscopic images, the number and size parameters of all solid particles contained in the test sample can be statistically determined. This method is a direct observation method, which more intuitively reflects the specific information of solid insoluble substances and is more accurate than methods such as dynamic light scattering.

[0137] Furthermore, combined with Raman spectroscopy, the Raman spectrum of each particle can be collected, such as... Figure 8The image shown is a Raman spectrum of PET. Based on Raman spectroscopy, the specific chemical composition of each particle can be identified, such as whether the particle is a protein aggregate, inorganic salt particle, or debris from a pipe or container, facilitating process traceability.

[0138] It should be noted that, for the sake of simplicity, the method embodiments are all described as a series of actions. However, those skilled in the art should understand that this application is not limited to the described order of actions, because according to this application, some steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also understand that the embodiments described in the specification are all optional embodiments, and the actions involved are not necessarily required by this application.

[0139] The above description is only a preferred embodiment of the present invention. It should be understood that the above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention, and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the idea and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for analyzing and identifying trace visible foreign matter in a liquid, characterized in that, include: Silicon column method, wherein: The silicon column method is used when samples are scarce and valuable during the research and development and production stages of biopharmaceuticals, including: Step 1: Fabrication of silicon pillar array structure chips; Multiple sets of silicon pillars are arranged on the sorting chip in a hexagonal stacked structure with equal spacing, so that the spacing between each set of silicon pillars is equal; at the same time, the diameter and height of each set of silicon pillars are the same, forming a silicon pillar array structure; the spacing between each set of silicon pillars and the diameter of the silicon pillars are dynamically adjusted according to the size of the solid insoluble matter to be analyzed. The silicon pillar array structure and sorting chip are subjected to surface hydroxylation or amination treatment to make them superhydrophilic, and thus a silicon pillar array structure chip is fabricated. Step 2: Separation of foreign matter from the solution to be tested; Take the solution to be tested and drop it onto the surface of the silicon pillar array structure chip; Step 3: Analysis and identification procedures; The solid insoluble matter in the test solution eventually deposits on the surface of the silicon pillar, forming a circular region through the internal stress of the liquid itself. The fixed insoluble matter is distributed within this circular region, and the fixed insoluble matter is scanned by a microscopic Raman spectroscopy system.

2. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 1, characterized in that, It also includes suction filtration, which is used for the separation and analysis of small amounts of solid insoluble matter in large quantities of liquid formulations, including: Step 1: Fabrication of the glass through-hole structure; Using a standard cover glass, multiple identical through holes are machined at equal intervals on its surface in a hexagonal stacking structure to form a glass through-hole structure; Step 2: Filter membrane bonding operation; The filter membrane is flatly attached to the glass through-hole structure; the filter membrane is selected based on the amount of liquid to be tested and the size range of the insoluble solid to be tested. Step 3: Separation of solid insoluble substances; Place the glass perforated structure with the filter membrane attached inside the Buchner funnel of the suction flask; control the dropping speed of the liquid to be tested according to the viscosity of the liquid to be tested and the suction pump speed; after the dropping is completed, the separation of solid insoluble matter is completed, and at this time the solid insoluble matter is placed on the filter membrane. Step 4: Place the glass porous filter membrane with one side facing up under the micro Raman spectroscopy system, and use the microscopic imaging function to scan the entire area enriched with solid insoluble matter.

3. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 1, characterized in that, In the silicon pillar method, the spacing between two adjacent groups of silicon pillars is set between 0.5 and 10 μm; the diameter of each group of silicon pillars is set between 0.5 and 10 μm; and the height of each group of silicon pillars is set between 500 nm and 10 μm. When dealing with trace samples of less than 10 μL containing 1 μm of insoluble matter, a silicon pillar array structure chip with a spacing of 0.5 μm, a diameter of 0.5-1 μm, and a height of 500 nm is used.

4. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, The microscopic imaging scan includes: ① Count the number and size of particles and generate a particle count report; ② Raman spectroscopy is performed on each particle. Raman spectroscopy has fingerprint recognition capabilities and can identify the chemical composition of each substance. Pollution can be traced based on all the links involved in the process.

5. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, In the aforementioned suction filtration method, the diameter of each group of through holes is set to be between 5 and 50 μm; the spacing between each group of through holes is set to be between 10 and 50 μm.

6. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, The hexagonal stacked structure can be replaced by a square stacked structure, with multiple identical through holes machined at equal intervals.

7. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, The selection criteria for the filter membrane are as follows: when the size of the solid insoluble matter is 5μm, a filter membrane with a pore size of 2μm is selected.

8. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, When the pore size of the filter membrane is greater than 3μm, there is tension in the through-pores, causing size fluctuations; when the pore size of the filter membrane is less than 0.5μm, the filtration speed is slow. The pore size of the filter membrane is set to be between 0.5μm and 3μm.

9. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, In the aforementioned suction filtration method, the dropping rate is 0.1 mL / s to 10 mL / s.

10. The method for analyzing and identifying trace visible foreign matter in a liquid according to claim 2, characterized in that, When using a filter membrane alone to separate solid insoluble substances, the flatness of the filter membrane needs to fluctuate less than 5 μm within a millimeter range because the separated solid insoluble substances need to be observed under a microscope and have their spectra collected.

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