On-site containment performance evaluation method and lactose detection kit

Abstract

To provide an on-site containment performance evaluation method using drug surrogate samples.SOLUTION: An on-site containment performance evaluation method for evaluating the dispersion state of a powder in a space to be evaluated, comprises: a step of generating and scattering lactose as the powder in the space to be evaluated; a step of collecting the scattered lactose at a predetermined position of at least one of inside or outside the space to be evaluated; a step of preparing a collection liquid containing the collected lactose, water, and an enzyme, contacting the lactose with the enzyme in the collection liquid, performing an enzyme-substrate reaction using the lactose as a substrate, and quantitatively or qualitatively analyzing a product produced by the enzyme-substrate reaction.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an on-site containment performance evaluation method using a drug substitute sample, and a lactose detection kit. [Background technology]

[0002] In pharmaceutical manufacturing, equipment is often shared, and in such cases, cleaning of containers, equipment, and facilities is important to prevent cross-contamination and the inclusion of foreign matter. Containment performance evaluations of powder handling facilities are carried out from the perspective of preventing cross-contamination of pharmacologically active substances and the occupational safety and health of workers. In particular, in recent years, the handling of highly pharmacologically active substances that can affect the body even with very small amounts of exposure has increased, making containment performance evaluations even more important.

[0003] ISPE (International Society of Pharmaceutical Engineers) has published a technical guide called "The Standardized Measurement of Equipment Particulate Airborne Concentration" (SMEPAC) as a method for evaluating the particle containment performance of pharmaceutical manufacturing equipment. The second edition was published in 2012, and it recommends the evaluation of containment performance using alternative samples such as lactose.

[0004] Lactose is a compound that is also used as an excipient in pharmaceuticals, and has the following characteristics: it is harmless to the person performing the measurement, there is no risk of cross-contamination, it is easily soluble in water, and it has good stability.

[0005] As samples to be used for containment evaluation, in addition to substitute samples such as lactose, adenosine 5'-triphosphate (ATP) powder can be used as a simulated powder (e.g., Patent Document 1). In addition, lactose powder impregnated with ATP (e.g., Patent Document 2 and Patent Document 3) and lactose powder impregnated with riboflavin (e.g., Patent Document 4) have been proposed, and containment performance evaluations and powder scattering state evaluations have been carried out. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2010-276468 A [Patent Document 2] JP 2014-221012 A [Patent Document 3] JP 2014-222164 A [Patent Document 4] JP 2015-194382 A Summary of the Invention [Problem to be solved by the invention]

[0007] When a containment performance evaluation is carried out using a simulant powder such as adenosine 5'-triphosphate (ATP) powder described in Patent Documents 1 to 4, the containment performance can be evaluated on-site in a measurement facility.

[0008] In neither case was an on-site containment evaluation conducted using alternative samples such as lactose, as recommended by SMEPAC. Problems that arise when using simulant powders include differences in particle density, particle size distribution, electrostatic charge, and water solubility compared to alternative samples, resulting in differences in residual properties and dispersibility. Other issues include the need for a new risk assessment because the sample is not recommended by SMEPAC, and the need for thorough cleaning after the containment evaluation using the simulant powder.

[0009] In the containment performance evaluation using lactose recommended by SMEPAC, samples taken at a measurement facility must be transported to an analysis laboratory, where the lactose content must be quantitatively analyzed using expensive and large-scale equipment such as a high-performance liquid chromatograph. For this reason, a technology for evaluating containment performance on-site was required.

[0010] An object of the present invention is to provide a method for evaluating containment performance on-site within a measurement facility using lactose, which is a drug surrogate sample. [Means for solving the problem]

[0011] In order to solve the above problems and achieve the object, the present invention has the following configuration. [1] An on-site containment performance evaluation method for evaluating a powder dispersion state in an evaluation target space, comprising: A step of dusting and scattering lactose as the powder in the evaluation space; A step of collecting scattered lactose at a predetermined position inside or outside the evaluation space; A step of preparing a collection liquid containing the collected lactose, water, and an enzyme, contacting the lactose with the enzyme in the collection liquid, carrying out an enzyme-substrate reaction using the lactose as a substrate, and quantitatively or qualitatively analyzing a product generated by the enzyme-substrate reaction; An on-site containment performance evaluation method having the above-mentioned features. [2] The on-site containment performance evaluation method according to [1], wherein the enzyme comprises lactase (β-galactosidase). [3] The on-site containment performance evaluation method according to [1] or [2], wherein the enzyme includes at least one of glucose dehydrogenase and galactose dehydrogenase. [4] The on-site containment performance evaluation method according to any one of [1] to [3], wherein the enzyme comprises a proluciferin reductase. [5] The method for evaluating on-site containment performance according to any one of [1] to [4], wherein the enzyme comprises luciferase. [6] The on-site containment performance evaluation method according to any one of [1] to [5], wherein the analysis is carried out within a period of 2 hours from the start of the enzyme substrate reaction. [7] A lactose detection kit for use in the method according to any one of [1] to [6], comprising a reagent for measuring lactose contained in the collection liquid, The lactose detection kit includes a reagent comprising one or more selected from lactase (β-galactosidase), glucose dehydrogenase, galactose dehydrogenase, proluciferin, a reductase required for the reduction reaction of proluciferin, and luciferase. Effect of the Invention

[0012] According to the present invention, a method for evaluating containment performance on-site in a measurement facility using lactose, which is a drug surrogate sample, can be provided. [Brief description of the drawings]

[0013] [Figure 1] FIG. 2 is a diagram showing reaction pathways of enzyme reaction steps 1 to 7 performed in the measurement step of this embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, a first aspect of the present invention is an on-site containment performance evaluation method using lactose, which is a drug substitute sample (hereinafter, may be simply referred to as the "containment performance evaluation method of the present invention"). Hereinafter, this embodiment will be described, but the present invention is not limited to this embodiment.

[0015] The drug surrogate sample is used for the purpose of evaluating the dispersion state of the highly pharmacologically active powder by evaluating the dispersion property and containment performance of the surrogate sample when it is dispersed in facilities that handle highly pharmacologically active powder, such as pharmaceutical manufacturing facilities and research and development facilities. Moreover, it is applicable to evaluate the dispersion state of any target powder, not limited to highly pharmacologically active pharmaceutical powder. <Containment performance evaluation method> An example of a method for evaluating the scattering state of lactose, which is an alternative sample, will be described below.

[0016] The evaluation method of this embodiment is a containment performance evaluation method that uses lactose powder (simply referred to as "lactose" in this specification) instead of a target powder such as a pharmaceutical product to evaluate the dispersion state of a target powder, and includes a process (dispersion process) of generating dust and scattering lactose in the space to be evaluated, a process (capture process) of capturing and recovering the scattered lactose at a predetermined position at least either inside or outside the space to be evaluated, and a process (measurement process) of quantitatively or qualitatively analyzing the recovered lactose.

[0017] When lactose is collected within the evaluation space, the scattering state of lactose within the evaluation space can be evaluated. When lactose is collected outside the evaluation space, the scattering state of lactose that has flowed out or leaked from the evaluation space can be evaluated. If there is an intention to confine lactose or similar powder within the evaluation space, it is preferable that the amount of lactose collected outside the evaluation space is small.

[0018] In the scattering step, the method of generating and scattering lactose dust can be, for example, a method of putting lactose into a rotary drum and rotating it, a method of releasing lactose from a hopper, or a method of blowing it with an air blower. The method of generating and scattering lactose dust can be appropriately selected depending on the size and shape of the space to be evaluated.

[0019] Lactose dispersed in the space to be evaluated usually remains suspended in the space for several minutes to several hours. Lactose powder contained inside and outside the space to be evaluated for containment performance (i.e., the space to be collected) is collected sequentially at preset collection times. The collection time can be set arbitrarily.

[0020] Examples of the spaces to be evaluated include powder handling rooms, powder handling booths, clean booths, glove boxes, powder storage rooms, etc. in pharmaceutical manufacturing plants. The OEL (Occupational Exposure Limit) for highly pharmacologically active pharmaceutical ingredients is usually 1 μg / m 3 The powder dispersion state evaluation method of the present embodiment can be applied to the evaluation of facilities (devices, equipment) for such highly pharmacologically active drugs.

[0021] The method of collecting lactose particles in the collection step can be an active sampling method or a swab method. In the active sampling method, the air in the collection target space is collected by a collection material (e.g., SKC's IOM inhalable dust sampler), and the collected particles are extracted into a collection liquid. In the swab method, a predetermined area of ​​any surface, such as a floor or wall surface, in the collection target space is wiped with a swab, and then the attached particles are extracted into a collection liquid. As a swab material, for example, Smart Check, manufactured by AS ONE, can be used. As a collection liquid, for example, pure water or a buffer solution such as phosphate buffered saline can be used.

[0022] The method of collecting lactose dispersed in the space is not limited to the above. Air in the space to be collected may be introduced, and the air (gas) in the space containing lactose powder suspended in the introduced air may be led to a collector equipped with a filter to collect the lactose suspended in the space with the filter. Alternatively, a sampling sheet may be installed on a wall or floor in advance to collect the lactose that falls, and then the collected lactose may be dissolved in pure water or a buffer solution such as phosphate buffered saline, and the lactose may be collected in the aqueous solution. Instead of the collector equipped with the above filter, an impinger containing a solvent capable of dissolving lactose may be used as the collector.

[0023] The containment performance evaluation method of this embodiment can be used to evaluate, for example, whether lactose particles have diffused (leaked) into a space (second space) adjacent to the space to be evaluated (first space). The amount or presence of lactose particles in the first and second spaces can be confirmed by the above-mentioned collection method, and it can be evaluated whether the amount of lactose detected in the second space is within an expected range. If no lactose is detected in the second space or the detected amount is lower than a predetermined value, it can be determined that the containment performance of lactose in the first space is good.

[0024] By quantitatively or qualitatively analyzing the collected lactose, the scattering state of lactose powder in the space of interest can be evaluated. Since lactose is a substance recommended by SMEPAC, it can be considered that the scattering and residual properties of lactose powder are similar to those of the actual target powder (e.g., highly pharmacologically active pharmaceutical ingredients) handled in the space of interest. Therefore, the scattering state of the actual target powder can be known from the scattering state of lactose powder, and its containment performance can be evaluated. That is, in the containment performance evaluation of this embodiment, lactose powder is collected and analyzed at the location of interest, so that the scattering state of the pharmaceutical powder can be directly evaluated.

[0025] In the measurement step, first, a collection liquid containing the lactose obtained in the collection step, water, and an enzyme is prepared. Using this collection liquid, it is preferable to carry out one or more of the following enzyme reaction steps 1 to 7 in this order, or all at once. The enzymatic reaction step 1 is a step in which lactose contained in the collection liquid is hydrolyzed by an enzymatic reaction with lactase (β-galactosidase) to produce galactose and glucose. In the enzymatic reaction step 2, D-glucose produced in the enzymatic reaction step 1 is reacted with glucose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + ) produces D-gluconolactone and reduced nicotinamide adenine dinucleotide (NADH). In the enzymatic reaction step 3, D-galactose produced in the enzymatic reaction step 1 is reacted with galactose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + ) to produce D-galactolactone and reduced nicotinamide adenine dinucleotide (NADH). Enzymatic reaction step 4 is a step in which luciferin is produced by an enzymatic reaction between the reduced nicotinamide adenine dinucleotide (NADH) produced in enzymatic reaction step 2 and proluciferin and a reductase required for the reduction reaction of proluciferin. Enzymatic reaction step 5 is a step in which luciferin is produced by an enzymatic reaction between the reduced nicotinamide adenine dinucleotide (NADH) produced in enzymatic reaction step 3 and proluciferin and a reductase required for the reduction reaction of proluciferin. In the enzymatic reaction step 6, the luciferin produced in the enzymatic reaction step 4, luciferase, and adenosine 5'-triphosphate (ATP) react with magnesium ions (Mg 2+ ) in the presence of oxygen, resulting in an enzymatic reaction that produces bioluminescence. In the enzymatic reaction step 7, the luciferin produced in the enzymatic reaction step 5, luciferase, and adenosine 5'-triphosphate (ATP) react with magnesium ions (Mg 2+ ) in the presence of oxygen, resulting in an enzymatic reaction that produces bioluminescence.

[0026] In the enzyme reaction steps 1 to 7, lactose collection solution, lactase (β-galactosidase), glucose dehydrogenase, galactose dehydrogenase, oxidized nicotinamide adenine dinucleotide (NAD + ), proluciferin, a reductase required for the reduction of proluciferin, adenosine 5'-triphosphate (ATP), magnesium ions (Mg 2+ ) and luciferase are only required to be contacted in an aqueous solution, and the order of addition is not particularly limited. For example, glucose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD +The reaction can be started by preparing an aqueous solution containing lactose, the lactose-containing capture liquid obtained in the capture step, and adding the resulting aqueous solution to the aqueous solution. The enzymatic reaction steps may be carried out individually or partly or entirely together, and the reagents required for each enzymatic step may be added simultaneously or sequentially.

[0027] The reaction schemes for enzymatic reaction steps 1 to 7 are shown in FIG. It is not necessary to carry out all of the enzymatic reaction steps 1 to 7. For example, the D-glucose produced in the enzymatic reaction step 1 may be used without carrying out the next enzymatic reaction (glucose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD) required for the enzyme-substrate reaction with D-glucose). + ), the subsequent enzymatic reaction can be carried out only on the D-galactose produced in enzymatic reaction step 1. That is, in the above-mentioned case, enzymatic reaction steps 1, 3, 5, and 7 can be carried out without carrying out enzymatic reaction steps 2, 4, and 6. Alternatively, enzymatic reaction steps 1, 2, 4, and 6 may be carried out. The enzymatic reaction steps 1 to 7 can be carried out based on known enzyme-substrate reaction methods. For example, a series of reactions for D-glucose produced in the enzymatic reaction step 1 can be carried out using a glucose assay kit (eg, Glucose-Glo-Assay manufactured by Promega).

[0028] There are no particular limitations on the reaction vessel as long as the enzyme reaction proceeds in each enzyme reaction step, and there are no particular limitations on the reaction vessel in enzyme reaction step 6 and enzyme reaction step 7 as long as the bioluminescence resulting from the enzyme reaction can be measured. Examples of the reaction vessel include glass, metal, plastic, etc. There are no particular limitations on the shape of the reaction vessel, and examples of the shape include a slide glass shape, a microplate shape, and a disk shape. Among these, a microplate is preferable since it is possible to measure a large number of samples. Examples of the microplate include those in which any number of wells are arranged. Examples of the number of wells per plate include, for example, 6, 12, 24, 96, 384, and 1,536. The reaction vessel may be a microchannel device equipped with a fine channel. The size of the reaction vessel may be within a range that can be applied to the device to be used.

[0029] In the enzyme reaction step 1, the collected lactose aqueous solution is brought into contact with lactase (β-galactosidase). For example, a predetermined amount of the collected lactose aqueous solution is added to a microplate, and a predetermined amount of lactase (β-galactosidase) is added thereto, thereby bringing the two into contact with each other. As the lactase (β-galactosidase) used in the enzyme reaction step 1, any kind of enzyme may be used as long as it catalyzes the hydrolysis reaction of lactose with lactose contained in the collection liquid obtained in the collection step. Examples of the enzyme include lactase (β-galactosidase) derived from plants, yeasts, and bacteria. The temperature at which the enzyme reaction step 1 is carried out is not particularly limited as long as the enzymes involved in each enzyme reaction step exhibit catalytic activity. However, since the optimum temperature for lactase is said to be around 48°C, for example, a temperature of 20 to 60°C is preferable, and 20 to 50°C is more preferable.

[0030] In the enzymatic reaction step 2, D-glucose produced in the enzymatic reaction step 1 is reacted with glucose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + For example, the enzyme reaction step 1 is carried out, and glucose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + ) is added in a predetermined amount. As the glucose dehydrogenase used in the enzymatic reaction step 2, any type of enzyme may be used as long as it catalyzes an enzymatic reaction with the D-glucose produced in the enzymatic reaction step 1, and examples thereof include glucose dehydrogenase derived from bacteria. In the aqueous solution in the enzyme reaction step 2, oxidized nicotinamide adenine dinucleotide (NAD + The concentration of is not particularly limited. It is preferably 0.1 mM or more, more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. The upper limit is not particularly limited, but is usually 5 mM or less.

[0031] In the enzymatic reaction step 3, D-galactose produced in the enzymatic reaction step 1 is reacted with galactose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + For example, the enzyme reaction step 1 is carried out, and in the wells of the microplate in which a certain amount of D-galactose is produced, galactose dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD + Alternatively, a predetermined amount of glucose dehydrogenase, galactose dehydrogenase, and oxidized nicotinamide adenine dinucleotide (NAD) can be added to the wells of the microplate in which the enzyme reaction step 1 is carried out and D-glucose and D-galactose are generated. + ) is added in a predetermined amount. The galactose dehydrogenase used in enzymatic reaction step 3 may be any type of enzyme that catalyzes an enzymatic reaction with D-galactose produced in enzymatic reaction step 1, and examples of such enzymes include plant-derived and bacterial-derived galactose dehydrogenases. In the aqueous solution in the enzymatic reaction step 3, oxidized nicotinamide adenine dinucleotide (NAD + The concentration of is not particularly limited. It is preferably 0.1 mM or more, more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. The upper limit is not particularly limited, but is usually 5 mM or less.

[0032] In the enzymatic reaction step 4, the reduced nicotinamide adenine dinucleotide (NADH) produced in the enzymatic reaction step 2 is contacted with proluciferin and a reductase required for the reduction reaction of proluciferin. As the reductase required for the reduction reaction of proluciferin used in enzymatic reaction step 4, any type of enzyme may be used as long as it catalyzes the reduction reaction of proluciferin together with the reduced nicotinamide adenine dinucleotide (NADH) produced in enzymatic reaction steps 2 and 3. For example, a reductase required for the reduction reaction of proluciferin derived from a plant or a bacterium may be used. Any type of proluciferin may be used in the enzymatic reaction step 4, so long as the luciferin produced therefrom generates bioluminescence through an enzymatic reaction with luciferase. Examples of proluciferins include D-luciferin-6-O bD-galactopyranoside and D-luciferin-myo-inositol-1-phosphate.

[0033] In the enzymatic reaction step 5, the reduced nicotinamide adenine dinucleotide (NADH) produced in the enzymatic reaction step 3 is contacted with proluciferin and a reductase required for the reduction reaction of proluciferin. As the reductase required for the reduction reaction of proluciferin used in the enzymatic reaction step 5, any type of enzyme may be used as long as it catalyzes the reduction reaction of proluciferin together with the reduced nicotinamide adenine dinucleotide (NADH) produced in the enzymatic reaction steps 2 and 3. For example, a reductase required for the reduction reaction of proluciferin derived from a plant or a bacterium may be used. Any type of proluciferin may be used in the enzymatic reaction step 5, so long as the luciferin produced therefrom generates bioluminescence through an enzymatic reaction with luciferase. Examples of proluciferins include D-luciferin-6-O bD-galactopyranoside and D-luciferin-myo-inositol-1-phosphate.

[0034] In the enzymatic reaction step 6, the luciferin produced in the enzymatic reaction step 4, luciferase, adenosine 5'-triphosphate (ATP), and magnesium ions (Mg 2+ ) in the presence of oxygen. The luciferase used in the enzymatic reaction step 6 is adenosine 5'-triphosphate (ATP) and magnesium ions (Mg 2+ Any type of enzyme may be used as long as it catalyzes the enzymatic reaction of the luciferase 100 produced in the enzymatic reaction steps 4 and 5 in the presence of the luciferase 100 and oxygen. Examples of the enzyme include luciferase derived from bacteria and fireflies. The concentration of adenosine 5'-triphosphate (ATP) in the aqueous solution in the enzyme reaction step 6 is not particularly limited, but is preferably 0.1 mM or more, more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. The upper limit is not particularly limited, but is usually 50 mM or less. In the aqueous solution in the enzyme reaction step 6, magnesium ions (Mg 2+ The concentration of ) is not particularly limited. It is preferably 0.1 mM or more, more preferably 0.5 mM or more, and even more preferably 1 mM or more. The upper limit is not particularly limited, but is usually 50 mM or less. In addition, other divalent metal ions in the aqueous solution in the enzyme reaction step 6 include, for example, Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+etc. can be used. The solution containing divalent metal ions is not particularly limited as long as it contains a salt or hydrate containing the above divalent cation dissolved therein. Furthermore, it may be diluted with a buffer solution such as physiological saline or distilled water. The concentration of the divalent metal ions in the solution may be, for example, 0.1 mM or more and 100 mM or less, 1 mM or more and 50 mM or less, or 5 mM or more and 30 mM or less.

[0035] In the enzymatic reaction step 7, the luciferin produced in the enzymatic reaction step 5, luciferase, adenosine 5'-triphosphate (ATP), and magnesium ions (Mg 2+ ) in the presence of oxygen. The luciferase used in the enzymatic reaction step 7 is adenosine 5'-triphosphate (ATP) and magnesium ions (Mg 2+ Any type of enzyme may be used as long as it catalyzes the enzymatic reaction of the luciferase 102 with the luciferin produced in the enzymatic reaction steps 4 and 5 in the presence of the luciferase 102 and oxygen. Examples of the enzyme include luciferase derived from bacteria and fireflies. The concentration of adenosine 5'-triphosphate (ATP) in the aqueous solution in the enzyme reaction step 7 is not particularly limited. It is preferably 0.1 mM or more, more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. The upper limit is not particularly limited, but is usually 50 mM or less. In the aqueous solution in the enzyme reaction step 7, magnesium ions (Mg 2+ The concentration of ) is not particularly limited, but is preferably 0.1 mM or more, more preferably 0.5 mM or more, and even more preferably 1 mM or more. The upper limit is not particularly limited, but is usually 50 mM or less. In addition, other divalent metal ions in the aqueous solution in the enzyme reaction step 7 include, for example, calcium ions (Ca 2+ ), zinc ion (Zn 2+ ), manganese ion (Mn 2+) or the like can be used. The solution containing a divalent metal ion is not particularly limited as long as it is a solution in which a salt or hydrate containing the above-mentioned divalent cation is dissolved. Furthermore, it may be a solution diluted with a buffer solution such as physiological saline or distilled water. The concentration of the divalent metal ion in the solution may be, for example, 0.1 mM or more and 100 mM or less, 1 mM or more and 50 mM or less, or 5 mM or more and 30 mM or less.

[0036] The lower limit of the pH of the aqueous solution in the enzymatic reaction steps 1 to 7 is 6.0 or more, preferably 6.5 or more, and more preferably 7.0 or more, and the upper limit is 10 or less, and preferably 9.5 or less. In this case, the pH is the value at the temperature during the enzyme reaction step. In general, the optimum pH for lactase is considered to be around 6.5. In contrast, the above-mentioned enzymatic reaction steps 1 to 7 may be carried out in a high pH range (for example, pH 7.0 to 9.5) slightly away from the optimum pH for lactase. This is because, in the pH range of 7.0 to 9.5, it is possible to suppress variations due to differences in background and reaction time when measuring the optical property values ​​(e.g., luminescence) of the enzyme reaction product. This allows the lower limit of the measurable concentration (quantitation limit) to be lowered, and accurate and easy quantification can be performed even for small amounts of samples that tend to have a short cell length when measuring optical properties, or samples containing target compounds such as low concentrations of phenols. In addition, since the measured values ​​are less likely to vary even when the enzyme reaction time is shortened, the time required for measurement can be shortened, making the entire test, including the measurement, more efficient.

[0037] In order to adjust the pH of the aqueous solution in each of the above-mentioned enzyme reaction steps to a predetermined range, the aqueous solution may contain a buffer solution. The type of buffer is not particularly limited, and any of the following can be used: phosphoric acid, Tris-HCl, CHES, MES, Bis-Tris, EDTA, TAPSO, Tricine, Bicine, TAPS, CAPS, etc.

[0038] The temperature at which each of the above-mentioned enzymatic reaction steps is carried out is not particularly limited as long as the enzyme involved in each enzymatic reaction step exhibits catalytic activity, but is preferably, for example, 10 to 60°C, more preferably 20 to 40°C.

[0039] The length of time for reacting the enzyme with the substrate in the enzyme reaction steps 1 to 7 is arbitrary. The enzyme reaction time may be the time from the start of the reaction by contact between the enzyme and the substrate (usually, when the enzyme is added to the substrate or when the substrate is added to the enzyme) to the start of the reaction in the next enzyme reaction step, or the time when the enzyme reaction is stopped by any method. In the enzyme reaction step 7, it may be the time from the start of the reaction by contact between the enzyme and the substrate to the measurement of the optical property value.

[0040] Following the enzyme reaction step, a measurement step is performed to measure the optical property value of the sample obtained in the enzyme reaction step. Examples of the optical property value include absorbance, reflectance, and transmittance. In view of the high detection sensitivity of the measurement, it is preferable to measure the intensity of the bioluminescence generated in the enzyme reaction step 7 using a spectrofluorophotometer. The optical property value may be the coloration degree of the enzyme reaction product, or a colorimetric method in which the coloration degree after the enzyme reaction is measured visually or the like. In the case of a colorimetric method, the coloration degree may be measured in an aqueous solution state, or may be applied to an embodiment in which an enzyme reaction is performed on a test paper such as a urine test paper and the coloration degree on the test paper is measured.

[0041] The measurement step is carried out during or after the enzyme reaction step, and is preferably carried out within 48 hours, more preferably within 24 hours, even more preferably within 8 hours, and even more preferably within 2 hours from the start of the enzyme reaction step. The shortest timing for carrying out the measurement step is not particularly limited, and may be immediately after the start of the reaction (usually, when the enzyme is added to the substrate or when the substrate is added to the enzyme). By setting the pH of the reaction system within the above-mentioned range, the measured value is unlikely to fluctuate even if the enzyme reaction time is shortened, so that the time required for the enzyme reaction and measurement can be shortened. From the viewpoint of shortening the time, it is desirable to determine the lactose concentration and the amount of lactose contained in the sample using the measured value, preferably 2 hours after the start of the luminescence reaction using luciferase and luciferin.

[0042] Next, a quantification step is performed to quantify the amount of lactose in the sample from the measured optical property values ​​obtained in the measurement step. The quantification step can usually be performed by converting the measured optical property values ​​into the target compound concentration based on a calibration curve prepared using a standard sample containing the target compound at a known concentration. For example, bioluminescence generated by the luminescence reaction using luciferase and luciferin in the enzyme reaction step 6 or enzyme reaction step 7 can be measured using a spectrofluorometer, and simulated powders such as highly pharmacologically active drugs can be quantitatively analyzed based on the correlation between the amount of luminescence and the amount of lactose. From the viewpoint of accuracy and stability of the measured value, the measurement is preferably performed within a period of 2 to 48 hours from the start of the luminescence reaction using luciferase and luciferin.

[0043] In the containment performance evaluation method of this embodiment, the amount of lactose contained in a sample can be quantified based on the change in the detection intensity of luminescence or fluorescence over time. That is, after a predetermined time has elapsed since the start of the enzyme substrate reaction, the detection intensity of luminescence or fluorescence is continuously measured for a predetermined time. The change in the detection intensity over time is obtained from this measurement result. Alternatively, after a predetermined time has elapsed since the start of the enzyme substrate reaction, the detection intensity of luminescence or fluorescence after the predetermined time has elapsed is measured. The change in the detection intensity over time is obtained from this measurement result.

[0044] For example, a sample specimen containing lactose of known concentration is prepared, and the detection intensity of luminescence or fluorescence generated by the enzyme substrate reaction in this sample specimen is continuously measured as described above. Based on the obtained measurement results, a characteristic line showing the relationship between measurement time and detection intensity (hereinafter also referred to as time characteristic) is created, and the least square method is applied to this characteristic line to approximate this characteristic line to a straight line. The slope of this straight line indicates the amount of change in detection intensity over time, and depends on the state of the enzyme substrate reaction in the sample solution, that is, the amount of biological material in the sample solution. Therefore, using multiple samples of known concentration, a calibration curve showing the relationship between the slope of the time characteristic and the concentration of the measured material is created, and the slope of the time characteristic of a sample containing an unknown amount of lactose is measured, and the amount of lactose in the sample can be quantified from the calibration curve. In addition, based on a calibration curve created using a standard solution containing lactose of known concentration, the lactose concentration contained in the sample and the amount of lactose contained in the sample can be obtained from the measured value of bioluminescence intensity.

[0045] The enzymes that undergo an enzyme-substrate reaction with lactose contained in the sample may include lactase (β-galactosidase), glucose dehydrogenase, and galactose dehydrogenase. One molecule of glucose and one molecule of galactose are generated by hydrolysis of one molecule of lactose via lactase (β-galactosidase). The resulting one molecule of glucose and one molecule of galactose can undergo an enzyme-substrate reaction with glucose dehydrogenase or galactose dehydrogenase, respectively. As a result, each of the subsequent enzyme-substrate reactions (enzyme reaction steps 4 and 6, or enzyme reaction steps 5 and 7) can be further performed, and each can generate bioluminescence. In other words, it is possible to measure both the bioluminescence resulting from the enzyme-substrate reaction with glucose and the bioluminescence resulting from the enzyme-substrate reaction with galactose, thereby achieving detection sensitivity that is roughly twice as high as that achieved when the enzyme reaction is performed using only glucose or galactose as the substrate for the next reaction (enzyme reaction step 2 or 3). That is, according to this embodiment, even a sample containing a lower concentration of lactose can be detected.

[0046] According to this embodiment, it is possible to evaluate the containment performance on-site while maintaining the same or higher measurement sensitivity as the conventional method (high performance liquid chromatography). Furthermore, since it has the feature of using lactose powder, which is an alternative sample recommended by SMEPAC, additional risk assessment and thorough cleaning are not required, and it can be performed in the same procedure as the conventional containment performance evaluation.

[0047] The concentration of lactose in the sample is not particularly limited, and can be measured even at a low concentration, for example, preferably 1 ng / mL or more, more preferably 5 ng / mL or more. The upper limit is also not particularly limited, but is preferably 1 μg / mL or less. When the concentration of lactose in the sample to be measured is high, the concentration in the aqueous solution in which the enzyme reaction is performed may be appropriately adjusted, such as by diluting.

[0048] The sample to be measured in this embodiment may be one that "may contain" lactose. In other words, it is sufficient that the sample is suspected of containing lactose before the measurement, and the purpose of the containment performance evaluation method of this embodiment is met even if the measurement result shows that the target compound is not contained.

[0049] The evaluation method of this embodiment can be applied to known powder handling facilities equipped with a space for handling powder, and the size of the space is not particularly limited. Specific examples of powder handling facilities include a powder handling room, a powder handling booth, a safety cabinet, a glove box, a powder storage room, etc. in a pharmaceutical manufacturing plant.

[0050] A second aspect of the present invention is a lactose detection and measurement kit (hereinafter referred to as lactose measurement kit) for use in the on-site containment performance evaluation method of the first aspect, which comprises a reagent for quantitatively or qualitatively measuring lactose contained in the collection liquid prepared by the measurement method. The reagent preferably contains one or more enzymes selected from lactase (β-galactosidase), glucose dehydrogenase, galactose dehydrogenase, proluciferin reductase, and luciferase.

[0051] The lactose measurement kit of the present embodiment more preferably further contains proluciferin as a substrate that generates luciferin through a reduction reaction, and further contains a reductase necessary for the reduction reaction of proluciferin.

[0052] The lactose measurement kit of this embodiment further includes oxidized nicotinamide adenine dinucleotide (NAD + ) and a divalent metal ion.

[0053] The lactose measurement kit of this embodiment may contain, in addition to the above-mentioned reagents, appropriate reagents selected appropriately according to the measurement method, such as a sample pretreatment reagent and a standard reagent.

[0054] According to the lactose measurement kit of this embodiment, the amount of lactose in a sample can be measured with high sensitivity and ease.

[0055] In the lactose measurement kit of this embodiment, the divalent metal ion used in the luminescence reaction using luciferase and luciferin is magnesium ion (Mg 2+ Other examples include the same as those mentioned above, and calcium ions (Ca 2+ ), zinc ion (Zn 2+ ), manganese ion (Mn 2+ ) etc. can be used. In the lactose measurement kit of this embodiment, magnesium ions (Mg 2+ The concentration of is not particularly limited, but is preferably 0.1 mM or more, more preferably 0.5 mM or more, and even more preferably 1 mM or more. The upper limit is not particularly limited, but is usually 50 mM or less. The solution containing the divalent metal ion is not particularly limited as long as it contains a salt or hydrate containing the divalent cation. Furthermore, it may be diluted with a buffer solution such as physiological saline or distilled water. The concentration of the divalent metal ion in the solution may be, for example, 0.1 mM or more and 100 mM or less, 1 mM or more and 50 mM or less, or 5 mM or more and 30 mM or less.

[0056] In the lactose measurement kit of this embodiment, lactose is not particularly limited in terms of origin, etc., as long as it can undergo an enzymatic reaction with lactase (β-galactosidase) as a substrate, but is preferably D-lactose. Lactose may also be a salt or a hydrate. Furthermore, lactose may be in the form of a powder or a solution diluted with an appropriate solvent (e.g., a buffer solution such as physiological saline, distilled water, etc.).

[0057] The specific method of using the lactose measurement kit of this embodiment is explained in the measurement step of the first embodiment, so duplicated explanations will be omitted here.

[0058] According to the lactose measurement kit of this embodiment, the amount of lactose can be measured with a measurement sensitivity equivalent to that of a high-performance liquid chromatograph. In addition, since lactose is an alternative sample recommended by SMEPAC, the lactose measurement kit of this embodiment is also suitable for application in known powder handling facilities equipped with a space for handling powder. Specific powder handling facilities include, for example, powder handling rooms, powder handling booths, safety cabinets, glove boxes, powder storage rooms, etc. in pharmaceutical manufacturing plants. In these facilities, on-site lactose concentration measurement is realized, and additional risk assessment and thorough cleaning are not required, and the same procedure as conventional containment performance evaluation can be performed.

[0059] The configurations and combinations thereof in each of the embodiments described above are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention.

Claims

1. An on-site containment performance evaluation method for evaluating a powder dispersion state in an evaluation target space, comprising: A step of dusting and scattering lactose as the powder in the evaluation target space; A step of collecting scattered lactose at a predetermined position inside or outside the evaluation space; A step of preparing a collection liquid containing the collected lactose, water, and an enzyme, contacting the lactose with the enzyme in the collection liquid, carrying out an enzyme-substrate reaction using the lactose as a substrate, and quantitatively or qualitatively analyzing a product generated by the enzyme-substrate reaction; An on-site containment performance evaluation method having the above-mentioned features.

2. The on-site containment performance evaluation method according to claim 1 , wherein the enzyme comprises lactase.

3. The on-site containment performance evaluation method according to claim 2 , wherein the enzyme further includes at least one of glucose dehydrogenase and galactose dehydrogenase.

4. The on-site containment performance evaluation method according to claim 3 , wherein the enzyme further comprises a proluciferin reductase and a luciferase.

5. The on-site containment performance evaluation method according to claim 1 , wherein the analysis is carried out within a period of 2 hours from the start of the enzyme substrate reaction.

6. A lactose detection kit for use in the on-site containment performance evaluation method according to claim 1, A reagent for measuring lactose contained in the collection liquid is provided, A lactose detection kit, wherein the reagent contains, as the enzyme, one or more selected from lactase, glucose dehydrogenase, galactose dehydrogenase, proluciferin reductase, and luciferase.

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