Recombinant amebosite coagulation factors and their use

Abstract

Recombinant amebosite coagulation factors, their formulations, and their use in determining the presence and / or amount of microbial endotoxins in a sample are provided. A cartridge containing recombinant amebosite coagulation factors for determining the presence and / or amount of microbial endotoxins in a sample is also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications

[0001] This application claims priority and the benefit thereof to U.S. Provisional Patent Application No. 63 / 506,271, filed on January 5, 2023, the entire content of which is incorporated herein by reference.

[0002] Field

[0002] The present invention generally relates to methods and compositions for determining the presence and / or amount of microbial endotoxins in a sample. More particularly, the present invention relates to recombinant amoebocyte clotting factors, and their formulation, and use in determining the presence and / or amount of microbial endotoxins in a sample.

Background Art

[0003] Background

[0003] For example, microbial contamination by Gram - negative bacteria can cause severe diseases in humans and in some cases even death. Manufacturers in certain industries, such as the pharmaceutical, medical device, and food industries, need to meet strict criteria to ensure that their products do not contain levels of microbial contaminants that can harm the health of the recipient. In these industries, frequent, accurate, and sensitive tests for the presence of such microbial contaminants are required to meet specific criteria, such as those imposed by the U.S. Food and Drug Administration (USFDA) or the Environmental Protection Agency. As an example, the USFDA requires certain manufacturers of pharmaceuticals and invasive medical devices to confirm that their products do not contain detectable levels of Gram - negative bacterial endotoxins.

[0004]

[0004] Endotoxins are dangerous, and can even be fatal, to humans and animals. Symptoms of bacterial endotoxin infections can range from mild fever to sepsis and even death. Humans or animals can be exposed to bacterial endotoxins through infection with Gram-negative bacteria, thereby releasing endotoxins from the bacterial cell wall during death, mechanical injury, or growth / division. Humans and animals can also be exposed to endotoxins through the environment, as Gram-negative bacteria, and therefore environmental endotoxins, are everywhere.

[0005]

[0005] To date, various assays have been developed to determine the presence and / or amount of microbial endotoxins in a sample of interest. One family of assays uses hemolysates prepared from the hemolymph of crustaceans, such as horseshoe crabs. These assays typically utilize the coagulation cascade that occurs when hemolysates are exposed to endotoxins in some way. Currently preferred hemolysates are amebocytic lysates (AL) produced from the hemolymph of horseshoe crabs, such as the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), the southern horseshoe crab (Tachypleus gigas), and the round-tailed horseshoe crab (Carcinoscorpius rotundicauda). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006]

[0006] These assays typically use blood collected from horseshoe crabs, which raises concerns about the ecological sustainability of this method. However, to date, fully synthetic or recombinant amebosite lysate reagents have not performed comparably to natural amebosite reagents (Dubczak et al., (2021) Eur. J. Pharm. Sci.159:105716). Therefore, there is a need for new reagents and test methods that adequately detect endotoxins with the same level of sensitivity and accuracy as naturally derived amebosite lysate, while being independent of blood collected from horseshoe crab populations. [Means for solving the problem]

[0007] overview

[0007] The present invention is partly based on the discovery that, under certain conditions, isolated factor C (FC), for example recombinant factor C (rFC), and isolated factor B (FB), for example recombinant factor B (rFB), can be unstable when mixed together, even in the absence of exogenously added endotoxin. Based on this discovery, it is possible to prepare a stable FC and FB-containing composition, for example, a stable rFC and rFB-containing composition, for use in endotoxin detection. This is achieved, for example, by keeping the isolated FB and FC components separate from each other before use in an endotoxin assay or before drying in one or more suitable containers, for example, an endotoxin detection / quantification cartridge or one or more vials.

[0008]

[0008] In one embodiment, the present disclosure provides a cartridge for testing bacterial endotoxins of a test specimen. An exemplary cartridge comprises (a) a housing defining a test specimen inlet area (also referred herein to as a test specimen inlet port); (b) a first composition comprising a first recombinant factor disposed on a first area of ​​the cartridge; and (c) a second composition comprising a second recombinant factor disposed on a second area of ​​the cartridge. The second area is spaced apart from the first area. Within the cartridge, the first area is in fluid communication with the test specimen inlet area, and the first and second areas are in fluid communication with each other, allowing mixing of the first and second compositions in the presence of a test specimen supplied onto the test specimen inlet area. Furthermore, the first composition comprises recombinant factor B or recombinant factor C, and the second composition comprises recombinant factor B or recombinant factor C, but not both the first and second compositions contain recombinant factor B or recombinant factor C. The cartridge may further include an optical cell that is in fluid communication with the test sample inlet region, the first region, and / or the second region.

[0009]

[0009] In another embodiment, the present disclosure provides a cartridge for bacterial endotoxin testing. The cartridge comprises (a) a housing defining a fluid inlet port, an optical cell, and a conduit having a fluid contact surface that provides fluid communication between the fluid inlet port and the optical cell; (b) a first composition disposed on a first region of the fluid contact surface of the conduit; and (c) a second composition disposed on a second region of the fluid contact surface of the conduit. The first region is spaced apart from the second region so that when a sample is applied to the fluid inlet port, the sample passes through the first and second regions during transport to the optical cell, dissolving the first and second compositions. The first and second compositions are selected from the group consisting of recombinant factors B and C, wherein the first composition is not the same as the second composition.

[0010]

[0010] In these exemplary cartridges, factor C and / or factor B may remain substantially inactive until they come into contact with microbial endotoxins in the liquid sample introduced into the cartridge via the fluid inlet port or the test sample inlet region.

[0011]

[0011] In one cartridge configuration, the first composition on the first region contains recombinant factor C but does not contain recombinant factor B, and the second composition on the second region contains recombinant factor B but does not contain recombinant factor C. In another cartridge configuration, the first composition on the first region contains recombinant factor B but does not contain recombinant factor C, and the second composition on the second region contains recombinant factor C but does not contain recombinant factor B.

[0012]

[0012] In yet another cartridge configuration, the first composition further comprises recombinant procoagulant. Alternatively, or in addition, the second composition further comprises recombinant procoagulant. In another configuration, the third composition containing recombinant procoagulant is positioned on the fluid contact surface of a third region of the cartridge or conduit, spaced apart from the first and second regions, where the third region is in fluid communication with the first and / or second regions. The third composition may further comprise a chromogenic substrate. However, in some configurations, the third composition containing recombinant procoagulant does not comprise a chromogenic substrate. In another configuration, the third composition containing a chromogenic substrate is positioned on the fluid contact surface of a third region of the cartridge or conduit, spaced apart from the first and second regions, where the third region is in fluid communication with the first and / or second regions. In some configurations, the third composition containing a chromogenic substrate does not comprise recombinant procoagulant.

[0013]

[0013] In other configurations, the fourth composition containing the color-developing substrate is placed on a fourth region of the cartridge or on the fluid contact surface of a conduit, where the fourth region is spaced apart from the first, second, and third regions and is in fluid communication with the first, second, and / or third regions.

[0014]

[0014] In other configurations, the fourth composition comprising recombinant procoagulant is placed on a fourth region of the cartridge or on the fluid contact surface of a conduit, wherein the fourth region is spaced apart from the first, second, and third regions and is in fluid communication with the first, second, and / or third regions.

[0015]

[0015] In a particular configuration, the fluid contact surface of the third region of the cartridge or conduit is located between the sample inlet region or fluid inlet port and the first region, and the second region is located on the fluid contact surface of the cartridge or conduit between the first region and the optical cell. For example, in such a configuration, the third composition on the third region includes a chromogenic substrate, the first composition on the first region includes recombinant factor B (but not recombinant factor C), and the second composition on the second region includes recombinant factor C (but not recombinant factor B) and recombinant procoagulant. In another exemplary configuration, the third composition on the third region includes a chromogenic substrate, the first composition on the first region includes recombinant factor C (but not recombinant factor B) and recombinant procoagulant, and the second composition on the second region includes recombinant factor B (but not recombinant factor C). For example, in some configurations, if the third composition on the third region contains a chromogenic substrate, it does not contain any recombinants, and the recombinant procoagulant is contained in only one region, for example, together with recombinants B or C in the first composition on the first region or the second composition on the second region.

[0016]

[0016] In another embodiment, a cartridge for detecting bacterial endotoxins in a test sample is disclosed. The cartridge includes: a housing defining (a) a conduit having a fluid inlet port, an optical cell, and a fluid contact surface providing fluid communication between the fluid inlet port and the optical cell; (b) a chromogenic substrate disposed on a first region of the fluid contact surface of the conduit; (c) a first recombinant amebosite factor disposed on a second region of the fluid contact surface of the conduit; and (d) a second recombinant amebosite factor disposed on a third region of the fluid contact surface. The second region is located downstream of the first region in the direction of fluid flow along the conduit, and the third region is located downstream of the second and first regions in the direction of fluid flow along the conduit. (ii) The first recombinant amebocyte factor contains recombinant factor B, the second recombinant amebocyte factor contains recombinant factor C, and the recombinant procoagulant enzyme is located on the third region together with recombinant factor C; or (ii) the first recombinant amebocyte factor contains recombinant factor C, the second recombinant amebocyte factor contains recombinant factor B, and the recombinant procoagulant enzyme is located on the second region together with recombinant factor C. For example, in some configurations, the first recombinant amebocyte factor contains recombinant factor B, the second recombinant amebocyte factor contains recombinant factor C, and the recombinant procoagulant enzyme is located on the third region together with recombinant factor C. In other configurations, the first recombinant amebocyte factor contains recombinant factor C, the second recombinant amebocyte factor contains recombinant factor B, and the recombinant procoagulant enzyme is located on the second region together with recombinant factor C. In some configurations, recombinant factors C and B are not located in the same region on the cartridge.

[0017]

[0017] In some cartridge configurations, the substrate may include a -Gly-Arg-chromophore-containing moiety or a -Gly-Lys-chromophore-containing moiety. For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0018]

[0018] In some cartridge configurations, the composition (e.g., the first, second, and, if appropriate, third and fourth compositions) is a dry composition and is dried, for example, on the fluid contact surface of the cartridge conduit.

[0019]

[0019] In another embodiment, the disclosed cartridge can be used in a method for determining the absence, presence, or amount of bacterial endotoxin in a test sample. The method comprises applying the test sample to the test sample inlet region of the cartridge; contacting the test sample with the first and second compositions in the presence of a chromogenic substrate; and determining the absence, presence, and / or amount of bacterial endotoxin in the test sample based on a chemically detectable change in the chromogenic substrate. During the method, recombinant procoagulant enzyme may be provided in the first composition, the second composition, or different compositions. The substrate may comprise a -Gly-Arg-chromophore-containing moiety or a -Gly-Lys-chromophore-containing moiety. For example, the chromogenic substrate is Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0020]

[0020] In one embodiment, the present disclosure provides a method for producing a bacterial endotoxin test composition. The method comprises (a) providing a first composition comprising recombinant factor C without factor B; (b) providing a second composition comprising recombinant factor B without factor C; and (c) mixing the first composition with the second composition in the presence of recombinant procoagulant to form a third composition. During the method, the first and second compositions remain separated until (i) are mixed together immediately before contact with the test sample, or (ii) are mixed with the test sample. In one example, the first and second compositions remain separated until they are mixed together immediately (e.g., within about 30 minutes) before contact with the test sample. In another example, the first and second compositions remain separated until they are mixed with the test sample.

[0021]

[0021] In some embodiments, the chromogenic substrate may be mixed with the third composition immediately before contact with the test sample, or the chromogenic substrate may be mixed with the first and second compositions simultaneously with or after mixing with the test sample. In some embodiments, the method includes providing a composition comprising a chromogenic substrate which remains separated from the first and second compositions until it is mixed with the first and second compositions in step (c) to form the third composition. In further embodiments, the first composition further comprises a chromogenic substrate, or the second composition further comprises a chromogenic substrate. The substrate may comprise a -Gly-Arg-chromophore-containing moiety or a -Gly-Lys-chromophore-containing moiety. For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0022]

[0022] The first and second compositions provided in steps (a) and (b) may be provided as dry compositions. For example, the first and second compositions may be redissolved before mixing in step (c), for example, with a buffer or with a liquid test sample. Depending on the circumstances, the first and second compositions may be dried in a test cartridge as described herein, where the first and second compositions are mixed when the liquid test sample is applied to the cartridge. Alternatively, the first and second compositions provided in steps (a) and (b) may be provided as buffer solutions.

[0023]

[0023] In some embodiments, the first composition further comprises recombinant procoagulant before the mixing step (c). In other embodiments, the second composition further comprises recombinant procoagulant before the mixing step (c). Optionally, a fourth composition comprising recombinant procoagulant may be provided. In one embodiment, the first and second compositions are mixed with the fourth composition immediately, for example, within about 30 minutes, before contact with the test sample, and in another embodiment, the first, second, and fourth compositions are mixed together upon contact with the test sample.

[0024]

[0024] The fourth composition may further include a chromogenic substrate. The substrate may include a -Gly-Arg-chromophore-containing portion or a -Gly-Lys-chromophore-containing portion. For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0025]

[0025] In some embodiments, factor C provided in step (a) remains substantially inactive in the absence of endotoxin exogenously added from the test sample or control. Similarly, factor B provided in step (b) also remains substantially inactive in the absence of endotoxin exogenously added from the test sample or control.

[0026]

[0026] In another aspect, the present disclosure provides a method for detecting bacterial endotoxin in a test sample. The method includes contacting a bacterial endotoxin test composition generated according to the method for generating a bacterial endotoxin test composition disclosed herein, and determining the absence, presence, and / or amount of bacterial endotoxin in the sample based on a chemically detectable change in the chromogenic substrate. The substrate may include a -Gly-Arg-chromophore-containing portion or a -Gly-Lys-chromophore-containing portion. For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0027]

[0027] In certain embodiments, step (c) of the method is performed on a cartridge. For example, the test sample is applied to the test sample inlet region or fluid inlet port of the cartridge as described herein. The first and second compositions on the cartridge contact the test sample in the presence of the chromogenic substrate; the presence, absence, and / or amount of bacterial endotoxin in the test sample is determined based on a chemically detectable change in the chromogenic substrate.

[0028]

[0028] In yet another embodiment, the present disclosure provides a kit for determining the absence, presence and / or amount of bacterial endotoxin in a test sample. The kit comprises a first composition comprising recombinant factor C, which is not recombinant factor B; and a second composition comprising recombinant factor B, which is not recombinant factor C. The first and second compositions remain physically separated from each other. The kit further comprises means for mixing the first and second compositions together in the presence of recombinant procoagulant and a chromogenic substrate immediately before (e.g., within about 30 minutes) or simultaneously with contact with the test sample. In one example, the first and second compositions are dry compositions, and the kit further comprises one or more buffer solutions for redissolving each of the first and second compositions.

[0029]

[0029] In the cartridges, kits, and methods disclosed herein, recombinant factor B and / or recombinant factor C and / or recombinant procoagulant may be recombinant horseshoe crab (Limulus polyphemus) factor B and / or recombinant horseshoe crab (Limulus polyphemus) factor C and / or recombinant horseshoe crab (Limulus polyphemus) procoagulant. For example, recombinant factor C comprises the amino acid sequence of SEQ ID NO: 1; recombinant factor B comprises the amino acid sequence of SEQ ID NO: 3; and / or recombinant procoagulant comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, recombinant factor C lacks a (α-2,3) bond-terminal sialic acid and / or GnTI - It is expressed in HEK cell lines.

[0030]

[0030] In the cartridges, kits, and methods disclosed herein, the chromogenic substrate may include a -Gly-Arg-chromophore-containing moiety or a -Gly-Lys-chromophore-containing moiety. For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

[0031]

[0031] These and other aspects and features of the present disclosure are described in more detail in the following drawings, detailed description and claims.

[0032] Brief explanation of the drawing

[0032] The purposes and features of this disclosure can be better understood by referring to the drawings described below. [Brief explanation of the drawing]

[0033] [Figure 1]

[0033] This is a schematic diagram of the coagulation system present in amebosite. [Figure 2A]

[0034] This is a perspective view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 2B]

[0034] This is a top view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 2C]

[0034] This is a side view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 2D]

[0034] This is an end view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 3A]

[0035] This is a perspective view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 3B]

[0035] This is a top view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 3C]

[0035] This is a side view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 3D]

[0035] This is an end view of an exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 4A]

[0036] This is a schematic diagram of an exemplary cartridge, a top view of the exemplary cartridge showing the positions of immobilized recombinant amebosite factors (e.g., factors B and C) and chromogenic substrate. [Figure 4B]

[0036] This is a schematic diagram of an exemplary cartridge, and is a cross-sectional view of the manufactured cartridge passing through sections A-A' and B-B' in Figure 4A. [Figure 4C]

[0036] This is a schematic diagram of an exemplary cartridge, and is a top view of the exemplary cartridge showing the positions of the immobilized recombinant factor and the chromogenic substrate. [Figure 4D]

[0036] This is a schematic diagram of an exemplary cartridge, and is a cross-sectional view of a fabricated cartridge passing through sections A-A', B-B', and C-C' in Figure 4C. [Figure 4E]

[0036] This is a schematic diagram of an exemplary cartridge, and is a top view of another exemplary cartridge for use with the recombinant amebosite factor described herein. [Figure 5A]

[0037] Figure 5A is a graph showing the onset times (in seconds) over time (at 0, 2, 4, 6, and 24 hours) of endotoxin samples (0 EU / mL (negative control "NC"), 0.01 EU / mL, 0.1 EU / mL, or 1.0 EU / mL) prepared by mixing the two solutions prepared in Example 2 and contacted with the cascade reagent stored at 5°C. Figure 5A shows the onset times when all three factors (rFC, rFB, and rPCE) are combined together, and the substrates are kept separate before subsequent mixing with the recombinant factors. [Figure 5B]

[0037] This graph shows the onset times (in seconds) over time (at 0 hours, 2 hours, 4 hours, 6 hours, and 24 hours) of endotoxin samples (0 EU / mL (negative control "NC"), 0.01 EU / mL, 0.1 EU / mL, or 1.0 EU / mL) prepared by mixing the two solutions prepared in Example 2 and contacted with the Cascade reagent stored at 5°C. Figure 5B shows the onset times at different time intervals when rFC was maintained separately from rFB, rPCE, and substrate, and then mixed with rFB, rPCE, and substrate before contact with the test sample. [Figure 6]

[0038] This graph shows the onset time (in minutes) over time (at 0, 10, 20, 30, and 60 minutes) of a negative control (NC) sample, which was kept separate from the rFB and rPCE solutions and maintained at 25°C until the solutions were mixed together before performing the assay. [Figure 7]

[0039] This graph shows the onset time (in seconds) over time (at 0, 20, 40, 60, 120, and 180 minutes) of NC samples maintained at 10°C, with the rFC solution kept separate from the rFB and rPCE solutions until the solutions were mixed together before performing the assay. [Figure 8]

[0040] This graph shows the onset time (in minutes) over time (at 0, 5, 10, 20, and 30 minutes) of NC samples prepared by keeping the rFC solution separate from the rFB solution until the solutions were mixed together before performing the assay, and maintaining the rPCE at 37°C. [Figure 9]

[0041] This graph shows the onset time (in minutes) at different time intervals (0, 10, 20, 30, 60, and 135 minutes) of NC samples maintained at 25°C, with the rFB solution kept separate from the rFC and rPCE solutions until the solutions were mixed together before performing the assay. [Figure 10]

[0042] This graph shows the stability of a solution in which rFC has been separated from other factors at 25°C (in the absence or presence of the substrate). The lines indicated by white squares (□) show the onset time over time of the solutions in the absence of the substrate, when the solutions for rFB and rPCE were prepared separately from the rFC solution. For comparison, data from Figure 6 (black circles, ●) is included when rFC was prepared in a solution containing the substrate. [Figure 11]

[0043] This graph shows the onset time (minutes) over time for samples (NC, 0.02 EU / mL endotoxin, and 0.2 EU / mL endotoxin) when assay reagents were prepared using three separate solutions (one containing rFC, one containing rFB and rPCE, and one containing the substrate). The solutions were kept separately at 25°C until they were mixed together before contact with the test samples. The stable onset times over different time intervals indicate that the solutions remain stable and that the separation of rFC from, for example, rFB and rPCE, as well as from, for example, the substrate, is effective in stabilizing the solutions. [Figure 12]

[0044] This graph shows the onset times (in minutes) over time (at 0, 60, 120, and 220 minutes) for the samples (NC, 0.005 EU / mL endotoxin, and 0.05 EU / mL endotoxin), where the assay reagents were prepared using three separate solutions (one containing rFC and rPCE, one containing rFB, and one containing the substrate). The solutions were kept separately at 25°C until they were mixed together before contact with the test samples. The stable onset times over different time intervals indicate that the solutions remain stable and that the separation of rFB, for example, from rFC and rPCE, and from the substrate, is effective in stabilizing the solutions. [Figure 13]

[0045] This graph shows the onset time (minutes) of the samples (0 EU / mL endotoxin, NC, 0.002 EU / mL endotoxin, or 0.02 EU / mL endotoxin) over time (0 hours, 1 hour, and 2 hours), where the assay reagents were prepared using three solutions (one containing rPCE, one containing a mixture of rFC and rFB, and the other containing the substrate). The solutions were kept separately at 25°C until they were mixed together before contact with the test samples. The decrease in onset time over time indicates that the separation of rPCE from other factors was not effective in stabilizing the solution. [Figure 14]

[0046] This graph shows the onset time (in seconds) of NC samples from exemplary cartridges selected from a series of manufacturing trays (tray numbers). The cartridges were prepared using solutions containing rFC, rFB, and rPCE, which were then supplied to the same areas of the cartridge. The gradually decreasing onset times for manufacturing trays 1 through 5 indicate the activation of the solutions and that the combination of all three factors is unsuitable for cartridge manufacturing. [Figure 15]

[0047] This graph shows the onset times (in seconds) of exemplary cartridges selected from a series of manufacturing trays (tray numbers) containing samples (0.05, 0.5, and 5 EU / mL). The cartridges were prepared with different solutions: a first solution containing rFC supplied to one area of ​​the cartridge, and a second solution containing rFB and rPCE supplied to separate areas of the cartridge. The onset times are stable across the entire manufacturing tray, indicating that the separation of rFC from rFB and rPCE is effective in producing stable cartridges. [Figure 16]

[0048] This graph shows the onset times (in seconds) of exemplary cartridges selected from a series of manufacturing trays (tray numbers) containing samples (0(NC), 0.01, 0.1, and 1 EU / mL). The cartridges were prepared using different solutions: a first solution containing rFB supplied to a first region of the cartridge, and a second solution containing rFB and rPCE supplied to a second region of the cartridge. The onset times are stable across the entire manufacturing tray, indicating that the separation of rFB from rFC and rPCE is effective in producing stable cartridges. [Figure 17]

[0049] This graph shows the onset time (in seconds) of an exemplary cartridge selected from a series of manufacturing trays (tray number) containing a sample (0.01 EU / mL). The cartridges were prepared using different solutions: a first solution containing the substrate was supplied to a first region of the cartridge; a second solution containing rFB was supplied to a separate second region of the cartridge; and a solution containing rFC and rPCE was supplied to a separate third region of the cartridge. The onset time is stable across the entire manufacturing tray, indicating that the separation of rFC and rPCE from rFB is effective in producing stable cartridges. [Modes for carrying out the invention]

[0034] Detailed explanation

[0050] The present invention is partly based on the discovery that, under certain conditions, isolated factor C (FC), e.g., recombinant factor C (rFC), and isolated factor B (FB), e.g., recombinant factor B (rFB), can be unstable when mixed together, even in the absence of exogenously added endotoxin. Based on this discovery, it is possible to prepare stable FC and FB-containing compositions, e.g., stable rFC and rFB-containing compositions, for use in endotoxin detection. This is achieved, for example, by maintaining the isolated FB and FC components separately from each other before use in an endotoxin assay or before drying in one or more suitable containers, e.g., one or more vials or endotoxin detection / quantification cartridges.

[0035]

[0051] Various features and embodiments of the present invention will be discussed in more detail below.

[0036] I. Definition

[0052] To facilitate understanding of the present invention, several terms and phrases are defined below.

[0037]

[0053] As used herein, the terms “amebocytic lysate factor” and “hemocytic lysate factor” refer to one or more coagulation factors present in the lysed blood of horseshoe crabs (e.g., Limulus sp. or Tachypleus sp.), such as factor B, factor C and / or procoagulant enzymes, and include natural versions of factors that can be isolated from horseshoe crab blood.

[0038]

[0054] As used herein, the terms “recombinant amebocytic factor” and “recombinant factor” refer to one or more coagulation factors (e.g., factor B, factor C, and / or procoagulant enzymes) which are recombinant versions of factors present in the blood of horseshoe crabs (e.g., Limulus sp. or Tachypleus sp.), including, for example, recombinant factor B ("rFB"), recombinant factor C ("rFC"), and recombinant procoagulant enzyme ("rPCE" or "rPE").

[0039]

[0055] As used herein, the term “activator” refers to activator B, activator C, and coagulation enzymes, and the term “activator” refers to a combination of one or more of activator B, activator C, and coagulation enzymes. In this context, “activation” refers to the state in which the enzyme precursor form of a protein is converted into an active enzyme.

[0040]

[0056] As used herein, the term “immediately” refers to the occurrence of an action within a specific time frame and may depend on the context of a given method. For example, in the context of performing an endotoxin detection assay using the cartridge disclosed herein, “immediately” may mean within 10 seconds, 30 seconds, 45 seconds, 1 minute, 3 minutes, 5 minutes, 8 minutes, or 10 minutes. The assay performed in the cartridge is typically carried out at 37°C. In the context of performing an endotoxin detection assay using a liquid or reconstitution reagent containing recombinant factors B and C prepared as described herein, which is stored on ice or refrigerated at 2–8°C, for example 4°C, “immediately” may mean within approximately 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.

[0041]

[0057] As used herein, the term “isolated” is understood to mean, when used in conjunction with a specific article (e.g., a protein or peptide), (1) the article has been separated or purified from other components (e.g., other proteins, peptides, nucleic acids, or cellular material); (2) the article can be separated or purified from an environment in which it may naturally occur (e.g., a tissue or body fluid sample); or (3) the article can be separated or purified from an environment in which it may originally occur (e.g., a crude amebosite lysate, or a protein or peptide isolated from a cell culture containing an expression host). As used herein, the term “isolated” may also refer to a molecule that substantially does not contain other molecules of the same species. For example, a protein or peptide can be “isolated” from other proteins or peptides having different amino acid sequences. The purity or homogeneity of a desired article can be assayed using techniques well known in the art, including gel electrophoresis, high-performance liquid chromatography, or mass spectrometry.

[0042]

[0058] As used herein, the term “recombinant” refers to a factor produced using recombinant nucleic acid, such as recombinant DNA, for example, when referring to an amebosite coagulation factor. Generally, the DNA or other nucleic acid encoding the factor is inserted into a suitable expression vector, which is then introduced into a host cell to produce a heterologous protein within the host cell.

[0043]

[0059] As used herein, the terms “sample” and “test sample” are interchangeable and refer, for example, to a sample containing or suspected to contain bacterial contaminants and / or endotoxins. In some examples, a sample or test sample may include environmental samples such as water, soil, or dust, or may be from the surface of a manufacturing apparatus or medical device, or may be a pharmaceutical composition, or an article or composition used to manufacture a pharmaceutical composition, which is suspected to contain bacterial endotoxin contamination. In other examples, a sample may be a biological sample such as blood, serum, plasma, urine, saliva, lung fluid or lung aspirate, nasal mucus, vaginal secretions, ocular secretions, cerebrospinal fluid, lymph, tissue or homogenized tissue, pus or exudate from a wound, feces, semen, or any other bodily fluid, secretion, or exudate from the body of a human or animal.

[0044]

[0060] Throughout this specification, where a composition is described as having, encompassing, or containing a particular component, or where a process and method is described as having, encompassing, or containing a particular step, it is intended that there exist compositions disclosed herein that are essentially composed of or consist of the listed components, and processes and methods disclosed herein that are essentially composed of or consist of the listed processing steps.

[0045]

[0061] In this specification, when an element or component is said to be included in a list of enumerated elements or components, and / or selected from a list of enumerated elements or components, it should be understood that the element or component may be any one of the enumerated elements or components, or the element or component may be selected from a group consisting of two or more of the enumerated elements or components.

[0046]

[0062] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound or molecule is referred to, that compound or molecule may be used in various embodiments of the compositions and / or methods of the disclosure unless otherwise understood from the context. In other words, while embodiments within this application have been described and shown in a manner that allows for clear and concise understanding of those embodiments, it is intended and understood that embodiments can be combined or separated in various ways without departing from the claimed invention.

[0047]

[0063] The articles “a” and “an” are used in this disclosure to refer to one or more (i.e., at least one) grammatical objects of the article, unless the context makes it inappropriate. For example, “an element” means one or more elements.

[0048]

[0064] In this disclosure, the terms “and / or” are used to mean either “and” or “or” unless otherwise indicated.

[0049]

[0065] The expression "at least one of ~" should be understood to include each of the enumerated objects that follow this phrase, and any various combinations of two or more of the enumerated objects, unless otherwise understood from the context and usage. The expression "and / or" relating to three or more enumerated objects should be understood to have the same meaning unless otherwise understood from the context.

[0050]

[0066] The use of the terms “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing” (including their grammatical equivalents) should generally be understood to be open-ended and non-restrictive, for example, not excluding further unlisted elements or steps, unless specifically indicated or understood from the context.

[0051]

[0067] Where the term “about” is used before a quantitative value, the present invention also includes the specific quantitative value itself unless otherwise specified. As used herein, the term “about” refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred from the context.

[0052]

[0068] It should be understood that the order of the steps or the sequence in which a particular action is performed is not important, as long as the invention remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0053]

[0069] In various parts of this specification, variables or parameters are disclosed in groups or ranges. The description is particularly intended to include any individual partial combination of the components of such groups and ranges. For example, integers in the range of 0 to 40 are particularly intended to be disclosed individually as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and integers in the range of 1 to 20 are particularly intended to be disclosed individually as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0054]

[0070] Any use of any example or illustrative term herein, such as “etc.” or “including,” is intended merely to better illustrate the invention and, unless otherwise claimed, does not impose any limitation on the scope of the invention. No term herein should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0055]

[0071] As a general issue, unless otherwise specified, percentages for compositions are based on weight.

[0056]

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. Abbreviations used herein have their conventional meanings in chemical and biological art.

[0057] II. Horseshoe Crab Hemolymphatic Coagulation Cascade

[0073] As shown in Figure 1, the coagulation system of hemolymph collected from horseshoe crabs includes at least two coagulation cascades, including the endotoxin-mediated pathway (factor C pathway) and the (1→3)-β-D glucan-mediated pathway (factor G pathway).

[0058]

[0074] When bacterial endotoxins come into contact with amebosite lysates, the endotoxins initiate a series of enzymatic reactions known in the art as the factor C pathway, involving three serine protease enzyme precursors referred to as factor C, factor B, and procoagulant enzymes (see Figure 1). Briefly, exposure to endotoxins activates factor C, an endotoxin-sensitive factor. Subsequently, activated factor C hydrolyzes and activates factor B, which then activates procoagulant enzymes to produce coagulation enzymes. These coagulation enzymes then hydrolyze specific sites, such as coagulogens, invertebrates, and the fibrinogen-like coagulating proteins Arg18-Thr19 and Arg46-Gly47, to produce insoluble coaguling gel. See, for example, U.S. Patent No. 5,605,806.

[0059]

[0075] (1→3)-β-D glucans and other amebosite lysate-reactive glucans produced by microorganisms such as yeasts and molds can also activate the amebosite lysate coagulation cascade via a different enzymatic pathway, referred to in this art as the factor G pathway (see Figure 1). Factor G is a serine protease enzyme precursor activated by (1→3)-β-D glucan or other LAL-reactive glucans. For example, exposure to (1→3)-β-D glucan activates factor G, producing activator G. Subsequently, activator G converts procoagulase into coagulase, which in turn converts coagulogen into insoluble coagulin.

[0060]

[0076] This disclosure relates to a synthetic or recombinant factor C pathway. Surprisingly, unlike natural horseshoe crab lysates, it has been discovered that in order to produce a stable synthetic factor C pathway using isolated factors, e.g., isolated recombinant factors, it is necessary to maintain factors C and B separately until use in an endotoxin assay, until the factors are combined for use in an endotoxin assay, or until they are dried by other means, e.g., on a solid surface (e.g., in a vial or other container). The synthetic factor C pathway may include isolated factor C (e.g., recombinant factor C), isolated factor B (e.g., recombinant factor B), and isolated procoagulant (e.g., recombinant procoagulant), which can be used to determine the presence and / or amount of microbial (e.g., bacterial) endotoxin in a sample.

[0061] III. Method for producing recombinant amebosite coagulation factors A. Factor C

[0077] As used herein, the term “factor C” refers to an enzyme precursor or functional fragment thereof that can be activated by endotoxin and can cleave (e.g., enzymatically cleave) factor B to form activator factor B. The term factor C includes mutants having one or more amino acid substitutions, deletions, or insertions to the wild-type factor C sequence, and / or fusion proteins or complexes comprising factor C protein or polypeptide. As used herein, the term “functional fragment” of factor C refers to, for example, a fragment of full-length factor C that retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the enzymatic activity of the corresponding full-length naturally occurring factor C. The enzymatic activity of factor C can be assayed by any method known in the art, including measuring the cleavage of the chromogenic substrate Z-Val-Pro-Arg-pNA, for example, as described in Example 1 of International Publication No. 2022 / 174082. In certain embodiments, the functional fragment contains at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 consecutive amino acids present in full-length naturally occurring factor C.

[0062]

[0078] Recombinant factor C ("rFC") is a factor C produced by recombinant nucleic acid (e.g., recombinant DNA) technology, in which the DNA or other nucleic acid encoding factor C is inserted into a suitable expression vector, which is then introduced into a host cell to produce a heterologous protein within the host cell. Exemplary rFCs and their synthesis are described in International Publication No. 2022 / 174082.

[0063]

[0079] It is intended that factor C expressed from a DNA sequence encoding wild-type factor C from any horseshoe crab, or a DNA sequence having modifications such as those disclosed herein, derived from a wild-type DNA sequence, may be used herein. For example, DNA sequences encoding factor C from the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), the southern horseshoe crab (Tachypleus gigas), or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda) may be used.

[0064]

[0080] Exemplary horseshoe crab (Limulus polyphemus) factor C amino acid sequences are shown in SEQ ID NOs: 1 and 2. SEQ ID NO: 1 is the mature form, while SEQ ID NO: 2 includes a signal sequence as residues 1-25. Exemplary horseshoe crab (Tachypleus tridentatus) factor C amino acid sequences are shown in SEQ ID NOs: 7 and 8. SEQ ID NO: 7 is the mature form, while SEQ ID NO: 8 includes a signal sequence as residues 1-21. In certain embodiments, factor C includes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8. In certain embodiments, factor C includes a conserved substitution for the wild-type factor C sequence or a factor C sequence disclosed herein.

[0065]

[0081] As used herein, the term “conservative substitution” refers to a substitution by structurally similar amino acids. For example, conservative substitutions may include those in the following groups: Ser and Cys; Leu, Ile, and Val; Glu and Asp; Gln and Asn; Lys, Arg, and His; Phe, Tyr, and Trp. Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., the BLOSUM62 matrix), or the PAM substitution:p matrix (e.g., the PAM250 matrix).

[0066]

[0082] As used herein, “identity percentage” between a protein sequence and a reference sequence is defined as the percentage of amino acid residues in the protein sequence that are identical to amino acid residues in the reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible sequence identity percentage. Similarly, percentage “identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to nucleotides in the reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible sequence identity percentage. Alignment for determining sequence identity percentage (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways within the art, such as using publicly available computer software, including, for example, BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of the fundamental problems in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129 (fully incorporated herein by reference). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0067]

[0083] In certain embodiments, recombinant horseshoe crab factor C, for use alone or in combination with other horseshoe crab hemolymph enzyme precursors (e.g., factor B, procoagulant, which is naturally supplied or recombinantly produced) as an endotoxin detection reagent, is expressed in mammalian cells, e.g., Chinese hamster ovary (CHO) or human fetal kidney (HEK) cells. In certain embodiments, factor C may be synthesized by recombinant gene editing cell lines such as GnTI-HEK cells (e.g., HEK cells lacking N-acetylglucosaminyltransferase I (GnTI) activity) that produce factor C protein lacking (α-2,3)-binding sialic acid, e.g., recombinant factor C, e.g., recombinant American horseshoe crab (Limulus polyphemus) factor C. The expression of factor C (e.g., recombinant factor C, e.g., recombinant horseshoe crab (Limulus polyphemus) factor C) in GnTI-HEK cells is described in U.S. Patent Application Publication 2023 / 0258647 (the contents of which are incorporated herein by reference). The use of such readily available gene-edited cell lines may be desirable for high-yield expression of homogeneously glycosylated recombinant proteins.

[0068] B. Factor B

[0084] As used herein, the term “Factor B” refers to an enzyme precursor or functional fragment thereof that can be activated upon cleavage by Factor C and is capable of cleaving (e.g., enzymatically cleaving) procoagulase to form active coagulase. The term Factor B includes mutants having one or more amino acid substitutions, deletions, or insertions to the wild-type Factor B sequence, and / or fusion proteins or complexes comprising Factor B protein or polypeptide. As used herein, the term “functional fragment” of Factor B refers to, for example, a fragment of full-length Factor B that retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the enzymatic activity of the corresponding full-length naturally occurring Factor B. The enzymatic activity of factor B can be assayed by any method known in the art, including, for example, measuring the cleavage of the chromogenic substrate HD-Leu-Thr-Arg-pNA, as described in Example 1 of International Publication No. 2022 / 174082. In certain embodiments, the functional fragment contains at least 100, 150, 200, 250, 300, 350, 360, 370, 380, or 390 consecutive amino acids present in full-length naturally occurring factor B.

[0069]

[0085] Recombinant factor B ("rFB") is factor B produced by recombinant nucleic acid (e.g., recombinant DNA) technology, in which the DNA or other nucleic acid encoding factor B is inserted into a suitable expression vector, which is then introduced into a host cell to produce a heterologous protein within the host cell. Exemplary rFBs and their synthesis are described in International Publication No. 2022 / 174082.

[0070]

[0086] It is intended that factor B expressed from a DNA sequence encoding wild-type factor B from any horseshoe crab, or a DNA sequence having a modification as disclosed herein derived from a wild-type DNA sequence, may be used in the practice of the present invention. For example, DNA sequences encoding factor B from the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), the southern horseshoe crab (Tachypleus gigas), or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda) may be used in the methods and compositions described herein.

[0071]

[0087] Exemplary American horseshoe crab (Limulus polyphemus) factor B amino acid sequences are shown in SEQ ID NOs: 3 and 4. SEQ ID NO: 3 is the mature form, while SEQ ID NO: 4 includes a signal sequence as residues 1-25. Exemplary horseshoe crab (Tachypleus tridentatus) factor B amino acid sequences are shown in SEQ ID NOs: 9 and 10. SEQ ID NO: 9 is the mature form, while SEQ ID NO: 10 includes a signal sequence as residues 1-22. In certain embodiments, factor B includes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to the amino acid sequences of SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 9, or SEQ ID NOs: 10.

[0072]

[0088] In certain embodiments, factor B, for example recombinant factor B ("rFB"), includes a conservative substitution for a wild-type factor B sequence or a factor B sequence disclosed herein.

[0073] C. Procoagulation enzyme

[0089] As used herein, the term “procoagulant” refers to an enzyme precursor or functional fragment thereof that can be activated upon cleavage by activator B and can cleave (e.g., enzymatically cleave) coagulogen to form coagulin. The term “procoagulant” includes a mutant having one or more amino acid substitutions, deletions, or insertions to the wild-type procoagulant sequence, and / or a fusion protein or complex comprising a procoagulant protein or polypeptide. As used herein, the term “functional fragment” of a procoagulant refers to, for example, a fragment of a full-length procoagulant that retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the enzymatic activity of the corresponding full-length naturally occurring procoagulant. The enzymatic activity of the procoagulant can be assayed by any method known in the art, including, for example, measuring the cleavage of the chromogenic substrate Ac-Ilu-Glu-Gly-Arg-pNA, as described in Example 1 of International Publication No. 2022 / 174082. In certain embodiments, the functional fragment contains at least 100, 150, 200, 250, 300, 320, 330, 340, 350, 360, or 370 consecutive amino acids present in the full-length naturally occurring procoagulant.

[0074]

[0090] Recombinant procoagulants ("rPCE" or "rPE") refer to procoagulants produced by recombinant nucleic acid (e.g., recombinant DNA) technology, where the DNA or other nucleic acid encoding the procoagulant is inserted into a suitable expression vector, which is then introduced into a host cell to produce a heterologous protein within the host cell. Exemplary rPCEs and their synthesis are described in International Publication No. 2022 / 174082.

[0075]

[0091] It is intended that procoagulants expressed from DNA sequences encoding wild-type procoagulants from any horseshoe crab, or from wild-type DNA sequences having modifications as disclosed herein, may be used in the implementation of the present invention. For example, DNA sequences encoding procoagulants from the American horseshoe crab (Limulus polyphemus), horseshoe crab (Tachypleus tridentatus), southern horseshoe crab (Tachypleus gigas), or round-tailed horseshoe crab (Carcinoscorpius rotundicauda) procoagulants may be used. Exemplary amino acid sequences of the American horseshoe crab (Limulus polyphemus) procoagulant are shown in SEQ ID NOs: 5 and 6. SEQ ID NO: 5 is the mature form, while SEQ ID NO: 6 includes a signal sequence as residues 1-28. Exemplary amino acid sequences of the horseshoe crab (Tachypleus tridentatus) procoagulant are shown in SEQ ID NOs: 11 and 12. SEQ ID NO: 11 is the mature form, while SEQ ID NO: 12 includes a signal sequence as residues 1-21. In certain embodiments, the procoagulant enzyme comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, or SEQ ID NO: 12, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identity with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, or SEQ ID NO: 12. In certain embodiments, the procoagulant enzyme comprises a conservative substitution of a wild-type procoagulant enzyme sequence or a procoagulant enzyme sequence disclosed herein.

[0076] D. Methods for producing recombinant proteins

[0092] Methods for generating recombinant proteins are known in the art. For example, DNA molecules encoding the target protein (e.g., factor C, factor B, and / or procoagulant, as disclosed herein) can be synthesized chemically or by recombinant DNA methodologies. The resulting DNA molecule encoding the target protein can be ligated to other suitable nucleotide sequences, for example, including expression regulatory sequences, to generate a conventional gene expression construct (i.e., an expression vector) encoding the desired protein. The generation of defined gene constructs is within the realm of routine art in the art.

[0077]

[0093] Nucleic acids encoding the desired protein (e.g., factor C, factor B, and / or procoagulants as disclosed herein) can be incorporated (ligated) into an expression vector, which can then be introduced into host cells by conventional transfection or transformation techniques. Typical host cells include Escherichia coli (E. coli) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and myeloma cells. Transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the protein of interest.

[0078]

[0094] Specific expression and purification conditions will vary depending on the expression system used. For example, when the gene is expressed in Escherichia coli (E. coli), the gene is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein can be secreted. The expressed protein can accumulate in refractive inductors or inclusion bodies that can be recovered after cell disruption by French pressing or sonication. The refractive inductors can then be lysed, and the protein can be refolded and / or cleaved by methods known in the art.

[0079]

[0095] When the manipulated gene is to be expressed in eukaryotic host cells, such as CHO or HEK cells, it is first inserted into an expression vector containing an appropriate eukaryotic promoter, secretory signal, poly(A) sequence, and stop codon. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0080]

[0096] The target polypeptide or protein (e.g., factor C, factor B, and / or procoagulant enzymes as disclosed herein) can be produced by growing (culturing) host cells transfected with an expression vector encoding such polypeptide or protein under conditions that enable polypeptide or protein expression. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, such as affinity tags, e.g., glutathione-S-transferase (GST) or histidine tags.

[0081]

[0097] Nucleic acids encoding the polypeptide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 can be spliced ​​into a suitable expression vector and operably linked to a promoter for use in a desired expression host using standard recombinant DNA methodologies. Expression host cells (e.g., mammalian host cells) containing such expression vectors are also provided, which can then be used to express proteins encoded by one or more of the aforementioned nucleic acid sequences. Exemplary methods for producing recombinant factor B, procoagulant, and factor C are described in Example 1 of this specification.

[0082]

[0098] It is understood that recombinantly expressed polypeptides or proteins may have different molecular weights and / or be glycosylated differently compared to their corresponding native polypeptides or proteins. Similarly, a protein or polypeptide recombinantly expressed in a first host cell type may have different molecular weights and / or be glycosylated differently compared to its corresponding protein or polypeptide expressed in a second, different host cell type. Glycosylation of recombinant proteins produced in mammalian host cells is described in, for example, Lis et al. (1993) EUR. J. BIOCHEM. 218:1-27, Parodi (2000) ANNU.REV. BIOCHEM. 69:69-93, Viswanathan et al. (2005) BIOCHEM. 44:7526-7534, Tomiya et al. (2004) GLYCOCONJUGATE JOURNAL 21: 343-360, Tomiya et al. (2003) ACC. CHEM. RES. 36:613-620, Gerngros (2004) NAT. BIOTECHNOL.22:1409-1414, and Demain et al. (2009) BIOTECHNOLOGY ADVANCES This is described in 27:297-306.

[0083] IV. Method for preparing a natural horseshoe crab mebosite lysate and isolation of coagulation factors from the lysate.

[0099] In addition to recombinant factors, isolated native coagulation factors may also be used in the methods and compositions disclosed herein. Native factors can be isolated from crude amebosite lysates of wild horseshoe crabs using methodologies known in the art.

[0084]

[0100] Natural horseshoe crab amebosite lysate refers to any lysate or fraction thereof (e.g., components of the factor C-mediated cascade) produced by lysis, extrusion, or extraction of cellular contents from amebosite extracted from horseshoe crabs. Natural amebosite lysate contains naturally occurring components of the enzyme cascade (e.g., as shown in Figure 1) produced by lysis, extrusion, or extraction of cellular contents from amebosite extracted from horseshoe crabs. Depending on the components present in or mixed with the lysate, it may form blood clots in the presence of endotoxins, e.g., Gram-negative bacterial endotoxins and / or glucans, e.g., (1→3)-β-D-glucan produced by yeast or mold. Amebosite lysates may be derived from horseshoe crabs belonging to the genus Limulus, e.g., the American horseshoe crab (Limulus polyphemus); horseshoe crabs belonging to the genus Tachypleus, e.g., the horseshoe crab (Tachypleus tridentatus) and the southern horseshoe crab (Tachypleus gigas); and horseshoe crabs belonging to the genus Carcinoscorpius, e.g., the round-tailed horseshoe crab (Carcinoscorpius rotundicauda). The term “natural” refers to factors derived from natural sources (e.g., amebosite lysates) as opposed to factors produced using recombinant means (e.g., recombinant DNA technology).

[0085]

[0101] Crude amebosite lysates can be produced by modifying Levin et al. (1968) THROMB. DIATH. HAEMORRH. HAEMORRH. 19: 186, or by the method described in Prior 1990 “Clinical Applications of the Limulus Amebocyte Lysate Test” CRC PRESS 28-36 and 159-166, and U.S. Patent No. 4,322,217. Other lysates may include, for example, those described in U.S. Patents No. 6,270,982 and No. 6,391,570. In certain embodiments, the crude lysate is produced as described in Example 1 of International Publication No. 2022 / 174082.

[0086]

[0102] Crude lysates prepared according to the methods discussed above or methods known to those skilled in the art may be further processed to isolate inactive natural coagulation factors, such as natural factor C, natural factor B, or natural procoagulant enzymes. For example, natural factor B can be isolated from T. tridentatus crude amebosite lysates according to the method disclosed in Nakamura et al. (1986) J. BIOCHEM 99: 847-57. Natural factor C can be isolated from L. polyphemus according to the method disclosed in Nakamura et al. (1986) EUR. J. BIOCHEM 154: 511-21. Natural procoagulant enzymes can be isolated from T. tridentatus according to the method disclosed in Nakamura et al., J. BIOCHEM. 97:1561-1574 (1985).

[0087]

[0103] The endotoxin assay is intended to be performed using recombinant factors, isolated native factors, or combinations of recombinant factors and isolated native factors. For example, in certain embodiments, in the endotoxin detection assay method of the present invention, rFC may be substituted with a native isolated factor C, rFB may be substituted with a native isolated factor B, and / or rPCE may be substituted with a native isolated procoagulant enzyme.

[0088] V. Substrate

[0104] To detect the presence of endotoxins, substrates for coagulation enzymes can be used.

[0089]

[0105] Crude or natural amebosite lysates contain coagulogens, and the conversion of coagulogens to coaguling gel by coagulation enzymes can be observed by measuring the optical properties of the assay reaction. However, in certain embodiments, for example, when only recombinant rFC, rFB, and rPCE are combined, coagulogens are absent. In such embodiments, if the coagulation cascade is activated by exogenous endotoxins, the formation of coaguling gel cannot be observed. Therefore, in certain embodiments, when recombinant factors, for example recombinant rFC, rFB, and / or rPCE are used, the synthetic substrate is, for example, a chromogenic substrate for coagulation enzymes. For example, the activated coagulation enzyme cleaves the substrate to produce a detectable product, such as a colored or fluorescent product. The resulting product can be detected, for example, using a suitable detector, such as an optical or fluorescence detector.

[0090]

[0106] In some embodiments, recombinant coagulogen (rCoagulogen) can be used as a substrate, such as a chromogenic substrate, to measure the optical properties of the assay reaction by the formation of a coaguling gel (gel coagulation assay) or a change in turbidity (endpoint or kinetic turbidimetry assay) when the coagulation cascade is activated by exogenous endotoxin. rCoagulogen can be used, for example, with rPCE, rFB, and rFC.

[0091]

[0107] In one embodiment, the chromogenic substrate of the coagulation enzyme contains a para-nitroaniline (pNA) chromophore. For example, the pNA chromophore is linked to a short, colorless peptide that is cleaved by an activator and releases a pNA group that can be detected using an optical detector. The peptide may include a -Gly-Arg-containing peptide or a -Gly-Lys-containing peptide. Exemplary substrates include, for example, Ac-Ile-Glu-Gly-Lys-pNA (available from Pentapharm AG, Aesch BL, Switzerland), also known as S2834, and Ac-Ile-Glu-Gly-Arg-pNA (available from Pentapharm AG, Aesch BL, Switzerland), also known as S2423, where Ac represents the acetyl moiety and PNA represents the para-nitroaniline chromophore. Other exemplary chromogenic substrates are described in Harada-Suzuki et al. (1982) J. BIOCHEM. 92: 783-800 and Nakamura et al., J. BIOCHEM., 81, 1567-1569 (1977).

[0092] VI. Considerations for detection methods and sample preparation

[0108] The recombinant amebocyte factors disclosed herein can be used in a variety of assays to determine the presence and / or amount of microbial endotoxins, such as bacterial endotoxins, in a sample (e.g., biopharmaceutical compositions, parenteral drugs, medical devices, solutions used in the manufacture of biopharmaceuticals and parenteral drugs, environmental samples, blood or body fluid samples, etc.). The method includes contacting an amebocyte coagulation factor (e.g., recombinant factor C, recombinant factor B, and / or recombinant procoagulant) with a sample (e.g., a sample suspected of containing endotoxins), reacting the factor with the sample to detect a detectable product (e.g., gel, increased turbidity), or a colored product (e.g., a chromophore) or a luminescent product (e.g., a fluorophore), and detecting the detectable product (e.g., visually or by the use of an optical detector).

[0093]

[0109] Amebosite coagulation factors disclosed herein, such as recombinant amebosite coagulation factors, such as rFC, rFC and / or rPCE, can be used, for example, to detect microbial contaminants (e.g., endotoxins) using kinetic assays or endpoint assays. An example kinetic assay is a one-step kinetic assay. An example endpoint assay is an endpoint colorimetric assay. Each of the assays is discussed in more detail below. Furthermore, it is understood that the assays can be modified to be performed in a specific assay format, for example, in a cartridge or in a plate, for example, in the wells of a 96-well plate.

[0094] A. Kinetic assay

[0110] An exemplary kinetic assay is a one-step kinetic assay, for example, a one-step colorimetric assay described in U.S. Patent No. 5,310,657. Briefly, a kinetic colorimetric assay includes the steps of (i) dissolving a recombinant amebosite lysate factor together with the sample and substrate to be analyzed, such as a chromogenic substrate; (ii) incubating the resulting mixture at a temperature of about 0°C to about 40°C, preferably about 25°C to about 40°C, for a predetermined time range; and (iii) measuring the time required for the colorimetric change to reach a pre-selected value, or the change in the colorimetric reading, using a conventional spectrophotometer.

[0095]

[0111] This type of assay can be performed in cartridge or well-type format.

[0096] (i) Cartridge-based format

[0112] Kinetic assays are understood to be performed in cartridge form. The cartridge is preferably used with an optical detector, such as a handheld optical detector as shown and described in U.S. Patent No. 390,661. Kinetic assays can also be performed in one or more wells, for example, in a multi-well plate.

[0097]

[0113] The cartridges disclosed herein can be used in methods for detecting microbial contamination in a sample, such as bacterial endotoxins, and / or amounts thereof. For example, the method may include (a) introducing a liquid sample into a cartridge, for example, the sample inlet port of the cartridge disclosed herein; (b) moving the sample into an optical cell; and (c) measuring the optical properties of the sample in the optical cell, wherein a change in optical properties indicates the presence and / or amount of microbial endotoxins in the sample. The change in optical properties may be an increase in the absorbance of light at a pre-selected wavelength. For example, the cartridges disclosed herein may be inserted into an instrument (e.g., a handheld detector) that measures the intensity of a color produced by a substrate in an optical cell and compares the resulting color to a standard curve to provide a coconcentration of endotoxins in a test sample.

[0098]

[0114] As disclosed herein, factor B (e.g., recombinant factor B) and factor C (e.g., recombinant factor C) should be maintained separately on the cartridge to maintain the stability of each factor. Therefore, factors B and C are arranged separately on the cartridge, for example, maintained in separate regions or areas of the cartridge, and are mixed together only in the presence of a liquid sample passing through the cartridge. In other words, a region or area of ​​the cartridge in which factor B (e.g., recombinant factor B) is present will also not contain factor C (e.g., recombinant factor C), and vice versa.

[0099]

[0115] The cartridges disclosed herein can be used in a method for determining the presence or amount of microbial contamination in a sample, such as bacterial endotoxin. For example, the method may include: (a) introducing a liquid sample into a cartridge, for example, the sample inlet port of a cartridge disclosed herein; (b) moving the sample into an optical cell; (c) measuring the time over which a pre-selected change occurs in the optical properties of the sample in the optical cell; and (d) determining the amount of microbial endotoxin in the sample by comparing the time measured in step (c) with a predetermined standard curve. The change in optical properties may be an increase in absorbance of light at a pre-selected wavelength. The change in optical properties may be a decrease in absorbance or transmittance of light at a pre-selected wavelength. The change in optical properties may be an increase in fluorescence of light at a pre-selected wavelength. The presence or amount of endotoxin indicates bacterial infection or bacterial contamination in the sample.

[0100]

[0116] For example, as shown in Figures 2A-2D and 3A-3D, the cartridge 1 has a substantially planar housing made of, for example, a moldable biocompatible material. The housing can be made of any material, but transparent and / or translucent glass or polymer is preferred. Preferred polymers include, for example, polystyrene, polycarbonate, acrylic, polyester, optical-grade polymer, or any plastic such that the optical cell is substantially transparent. The housing includes at least one fluid inlet port 4, at least one optical cell 6, and at least one conduit 8 having a fluid contact surface for providing fluid communication between the fluid inlet port 4 and the optical cell 6. The only requirement of the optical cell 6 is that it defines a void in which the sample to be tested can be accommodated, and that a portion of the optical cell 6 is transparent to light. The cartridge 1 may also have at least one pump port 12 for mounting the cartridge 1 to a pump, which is in fluid communication with the fluid inlet port 4 and the optical cell 6. Next, the pump can apply negative pressure through the pump port 12 to draw the sample from the fluid inlet port 4 to the optical cell 6.

[0101]

[0117] In the configuration shown in Figures 2A-D, a first amebosite factor, e.g., rFC, is located on a first region 14 of the fluid contact surface of the conduit 8. As a result, when a sample is applied to the fluid inlet port 4, the sample passes through region 14 and dissolves or reconstitutes the amebosite factor in the sample as it moves toward the optical cell 6. A second region 16 of the fluid contact surface of the conduit 8 is spaced apart from the first region 14 and located downstream of the first region 14 (i.e., away from the fluid inlet port 4 and in the direction of fluid flow along the conduit 8). A second amebosite factor, e.g., rFB, is located in the second region 16. As a result, after the sample comes into contact with the first factor in region 14, the sample factor mixture passes through the conduit 8 and comes into contact with the second factor in region 16. The substrate and procoagulant enzyme may also be located in the first and second regions, or may be divided between the first and second regions. Next, the mixture of sample, factor, and substrate passes through the conduit 8 to the optical cell 6. Before the optical properties of the sample are measured, forward and reverse pumping actions facilitated by the pump port 12 may be used to enable proper mixing of the sample, factor, and substrate mixture.

[0102]

[0118] Referring to Figures 2A-D, in an alternative configuration, the amebocytic factor located in the first region 14 is, for example, rFB, and the amebocytic factor located in the second region 16 is, for example, rFC. The substrate and procoagulant are also intended to be located in the first region 14, the second region 16, or to be divided between the first and second regions. In one configuration, rFB and rPCE are located on the first region 14, and rFC and the substrate (e.g., a chromogenic substrate) are located on the second region 16. In another embodiment, rFC and rPCE are located on the first region 14, and rFB and the substrate (e.g., a chromogenic substrate) are located on the second region 16.

[0103]

[0119] Referring to Figures 2A-D, in yet another alternative configuration, a first amebosite lysate factor, e.g., rFC, is located on the first region 14 along with a fluorescent substrate or chromogenic substrate, and a second amebosite lysate factor, e.g., rFB, is located on the second region 16. The substrate and procoagulant are also intended to be located in the first region 14 and the second region 16, or to be divided between the first region 14 and the second region 16.

[0104]

[0120] In the configuration shown in Figures 3A-D, a first amebosite factor, e.g., rFC, is placed on a first region 14 of the fluid contact surface of the conduit 8, so that when a sample is applied to the fluid inlet port 4, the sample passes through region 14 and moves toward the optical cell 6, dissolving or reconstituting the amebosite factor in the sample. A second region 16 of the fluid contact surface of the conduit 8 is spaced apart from the first region 14 and located downstream of the first region 14 (i.e., in the direction of fluid flow along the conduit 8). A third region 18 is spaced apart from the second region 16 and located further downstream of the second region 16 (and downstream of the first region 14). A second amebosite factor, e.g., rFB, is placed on the second region 16, and a chromogenic substrate or fluorescent substrate is placed on the third region 18. After the sample comes into contact with the first amebocyte factor in the first region 14, the sample-factor mixture passes through the conduit 8 and comes into contact with the second amebocyte factor located in the second region 16, and then the sample-factor mixture passes further through the conduit 8 and comes into contact with the chromogenic substrate or fluorescent substrate. The sample-factor-substrate mixture then passes through the conduit 8 to the optical cell 6. The procoagulant enzyme may be located in the first region 14, the second region 16, or the third region 18, or it may be divided between the first region 14 and the second region 16. Before the optical properties of the sample are measured, forward and reverse pumping action facilitated by the pump port 12 may be applied to allow proper mixing of the sample-factor-substrate mixture. In other words, the first region 14 is positioned spaced apart from the fluid inlet port 4 and the second region 16, the second region 16 is positioned spaced apart from the first region 14 and the third region 18, and the third region 18 is positioned spaced apart from the second region 16 and the optical cell 6. When a liquid sample is applied to the fluid inlet port 4, the sample passes through the first region 14, then the second region 16, then the third region 18, dissolving the substrate and / or factors in each region before reaching the optical cell 6.

[0105]

[0121] Referring to Figures 3A-D, in the alternative configuration, the amebocyte factor located in the first region 14 is rFB, and the amebocyte factor located in the second region 16 is rFC. The substrate can be located in the third region 18. The procoagulant enzyme can be located in the first region 14, the second region 16, or the third region 18, or it is intended to be divided between the first region 14 and the second region 16.

[0106]

[0122] Referring to Figures 3A-D, in yet another configuration, the substrate is placed on the first region 14, the first amebosite factor is placed on the second region 16, and the second amebosite factor is placed on the third region 18. Thus, when the sample is applied to the fluid inlet port 4, the sample moves along the conduit 8 and comes into contact with the substrate in the first region 14. The resulting sample-substrate mixture passes through the conduit 8 and comes into contact with the first amebosite factor in the second region 16. The sample-factor-substrate mixture then passes through the conduit 8 and comes into contact with the second amebosite factor in the third region 18. The sample-factor-substrate mixture then passes through the conduit 8 to the optical cell 6. Mixing can also be facilitated by moving the fluid and dissolved components back and forth along the conduit before measuring the solution in the optical cell. The first amebosite factor may be rFC, and the second amebosite factor may be rFB. Alternatively, the first amebocyte factor may be rFB, and the second amebocyte factor may be rFC. A procoagulant, such as rPCE, may be located in the first region 14, the second region 16, or the third region 18, or may be divided between the first region 14 and the second region 16. In one embodiment, the first amebocyte factor is rFC, the second amebocyte factor is rFB, and rPCE is located on the second region 16 together with RFC. In one embodiment, the first recombinant factor is rFC, the second recombinant factor is rFB, and rPCE is located on the third region 18 together with rFB. In one embodiment, the first recombinant factor is rFB, the second recombinant factor is rFC, and rPCE is located on the second region 16 together with rFB. In another embodiment, the first amebosite factor is rFB, the second amebosite factor is rFC, and rPCE is located on the third region 18 together with rFC. Thus, in some configurations, the first region 14 does not contain any recombinant factors.

[0107]

[0123] Referring to Figures 3A-D, in yet another exemplary configuration, the first amebocyte factor is located on the first region 14, the substrate is located on the second region 16, and the second amebocyte factor is located on the third region 18. The first amebocyte factor may be rFC, and the second amebocyte factor may be rFB. Alternatively, the first amebocyte factor may be rFB, and the second amebocyte factor may be rFC. The procoagulant enzyme may be located on the first region 14, the second region 16, or the third region 18, or it may be divided between the first region 14 and the second region 16 or the third region 18.

[0108]

[0124] Referring to Figures 4C-4D, which provide illustrative cross-sectional views of the cartridges shown in Figures 3A-D, the conduit 8 has a first surface 8' and a second surface 8''. Thus, as described with respect to Figures 3A-D, the amebocyte factor, substrate, and procoagulant enzyme may be placed on the first surface 8' of each region of the conduit 8 (e.g., 14, 16, or 18), on the second surface 8'' of each region of the conduit 8 (e.g., 14, 16, or 18), or on both the first surface 8' and the second surface 8'' of each region of the conduit 8 (e.g., 14, 16, or 18).

[0109]

[0125] Similarly, in the configuration shown in Figures 4A-B, the first amebosite factor (e.g., rFC 20) is positioned on the first surface 8' of the conduit 8 in the first region 14, and the second amebosite factor (e.g., rFB 22) is positioned on the second surface 8'' of the conduit 8 in the first region 14. Therefore, when the sample is applied to the fluid inlet port 4, as the sample passes through region 14 and moves toward the optical cell 6, it dissolves or reconstitutes the first and second amebosite factors within the sample. The second region 16 of the fluid contact surface 8 is separated from the first region 14 and positioned downstream of the first region 14 (i.e., in the direction of fluid flow along the conduit 8). After the sample has come into contact with the first and second amebosite factors in the first region 14, the sample-factor mixture passes through the conduit 8 and into region The substrate 24, for example, a fluorescent substrate or a chromogenic substrate, located on the second region 16, comes into contact with the substrate 24 located on the second region 16, either on the first surface 8' or the second surface 8'', or on both the first surface 8' and the second surface 8''. The sample-factor-substrate mixture then passes through the conduit 8 to the optical cell 6. The procoagulant enzyme may be located in the first region 14 or the second region 16, or it is intended to be located on the first surface 8', the second surface 8'', or both the first and second surfaces of the fluid contact surface 8 of either the first region 14 or the second region 16.

[0110]

[0126] Referring to Figure 4E, in another configuration, the first amebosite factor is located on the first region 14, the second amebosite factor on the second region 16, the third amebosite factor on the third region 18, and the substrate on the fourth region 20. The first amebosite factor may be rFC, the second amebosite factor may be rFB, and the third amebosite factor may be rPCE. Alternatively, the first amebosite factor may be rFB, the second amebosite factor may be rFC, and the third amebosite factor may be rPCE. When the sample is applied to the fluid inlet port 4, the sample passes through regions 14, 16, 18, and 20 and moves toward the optical cell 6, dissolving or reconstituting the amebosite factors and substrate within the sample. The pump action may be applied via the pump 12 to enable mixing of the sample, factor, and substrate, to promote the reaction between the factor, substrate, and any endotoxin in the sample, and to produce a detectable change that can be read by an analyzer capable of detecting changes in the optical properties of the sample in or generated by the sample within the optical cell 6.

[0111]

[0127] Cartridges can be designed and used according to the type and / or number of tests required. For example, a single sample may be tested, for example, for laboratory use, or for medical device and biopharmaceutical testing, for example, in double or triple doses. Alternatively, two or more different samples may be tested individually. Cartridges are preferably disposable cartridges that are discarded after a single use.

[0112]

[0128] In certain embodiments, one or more naturally isolated factors are replaced with corresponding recombinant amebosite factors on the cartridge. For example, in one embodiment, naturally isolated factor C is replaced with rFC. Alternatively, or in addition, naturally isolated factor B is replaced with rFB. Alternatively, or in addition, naturally isolated procoagulant enzyme may be replaced with rPCE.

[0113] (ii) Well-base format

[0129] While kinetic assays can be performed in the cartridges of the type described above, they may also be used in various other forms, such as in the wells of a microtiter plate or in one or more vials.

[0114]

[0130] With respect to assays performed in a well format, in one embodiment, the sample of interest may be combined with a first factor in a well, then a second factor, and then a substrate may be added to the well. A procoagulant enzyme may be included together with the first factor, the second factor, or the substrate. After mixing, the time required for a pre-selected change in optical properties is measured. The result can then be compared with one or more standard values ​​to determine the presence and / or amount of microbial endotoxin in the sample. The first factor may be rFC, and the second factor may be rFB. In an alternative embodiment, the first factor may be rFB, and the second factor may be rFC.

[0115]

[0131] Such assays are intended to be performed in multiple wells. For example, the first amebocyte factor may be placed in the first well, the second amebocyte factor in the second well, and the substrate in the third well. The procoagulant enzyme may be present in the first, second, or third well. The sample of interest may be added to the first well, the resulting mixture may be added to the second well, and the resulting mixture may be added to the third well. However, the order in which the samples are added to the wells is intended to vary. For example, the first amebocyte factor may be factor C, e.g., rFC, and the second amebocyte factor may be factor B, e.g., rFB. Alternatively, the first amebocyte factor may be factor B, e.g., rFB, and the second amebocyte factor may be factor C, e.g., rFB. Similarly, the sample may be added first to the second well, the resulting mixture to the first well, and then the resulting mixture to the third well. For example, other sequential steps may be considered, such as adding the sample to the third well, the resulting mixture to the first well, and then the resulting mixture to the second well.

[0116]

[0132] After mixing the first factor, the second factor, the procoagulant enzyme, and the substrate, the time it takes for a pre-selected change in optical properties to occur is measured. The result can then be compared with one or more standard values ​​to determine the presence and / or amount of microbial endotoxins in the sample.

[0117]

[0133] In the well-type format, an automated system, preferably such as a robot, is used to add the sample and reagents to each well, and the plate is processed by a microplate reader, which can be programmed to repeatedly and sequentially read the absorbance of each well. In some examples, when the well-type format is performed, for example, in a vial or tube, a tube reader is used to detect changes in the optical properties of the sample. The order of steps performed in the well-type format can also be adapted for use in microfluidic technology, such that the test sample is added in the same order as when performed in a well plate, and various reagents are present in the chamber of a microfluidic device and move through it. When using a microfluidic device, changes in the optical properties of the sample can be measured as the sample passes through the device.

[0118]

[0134] In certain embodiments, recombinant amebosite factor, rPCE, and substrate are dried, e.g., lyophilized, or otherwise coated onto the surface of each well, and reconstituted or dissolved when the sample is added to each well. It is also intended that a similar approach can be carried out when the factors are placed in vials instead of wells in a microtiter plate.

[0119]

[0135] In certain embodiments, recombinant factors (e.g., rFB, rFC, rPCE) are provided as lyophilized products, either together in a vial or in separate vials. For example, rFB, rFC, and rPCE may be provided as lyophilized products in a single vial, or rFB and rFC may be provided in separate vials, and rPCE may be provided separately or together with rFC or rFB. Substrates may be included in a vial together with one or more factors, or may be provided in a separate vial and lyophilized. The vials of lyophilized factors and / or substrates are reconstituted with appropriate liquid reagents, such as endotoxin-free water (e.g., LAL reagent water) or endotoxin-free buffer, and mixed together before performing an assay with a sample. One approach is to place the sample to be tested for endotoxin at the bottom of the well or vial, and then, for example, immediately before use, for example, within about 30 minutes, add the reconstituted factors and substrates to the well or vial. In another approach, the sample is added to a well or vial that already contains the factor and substrate. The well or vial is then analyzed with an automated analyzer equipped with endotoxin measurement software to detect changes in the optical properties of the sample.

[0120]

[0136] In certain embodiments performed in one or more wells or vials, one or more naturally isolated factors are replaced with corresponding recombinant amebocyte factors. For example, in one embodiment, naturally isolated factor C is replaced with rFC, naturally isolated factor B is replaced with rFC, and / or naturally isolated procoagulant enzyme is replaced with rPCE.

[0121] A. Endpoint assay

[0137] An exemplary endpoint assay is an endpoint colorimetric assay or fluorescence assay. An endpoint colorimetric assay or fluorescence assay may include the steps of (i) dissolving a recombinant factor in the sample to be analyzed; (ii) incubating the resulting mixture at a temperature of about 0°C to about 40°C, preferably about 25°C to about 40°C, for a predetermined time; (iii) contacting a substrate, such as a colorimetric or fluorescent substrate, with the incubated sample-factor mixture; (iv) optionally adding a reaction inhibitor, such as acetic acid; and (v) measuring the enzyme activity of the substance produced from the substrate, for example, by a colorimetric change. The endpoint assay measures how much of the substance has been produced by a predetermined specific time point.

[0122]

[0138] Such assays can be performed in a cartridge, one or more wells of a microtiter plate, or one or more vials, as described above for kinetic assays, and are intended to be performed using the same or similar steps. The only difference is that the change in optical properties is measured at a predetermined time rather than over time, as in kinetic assays.

[0123] (i) Cartridge

[0139] The cartridges disclosed herein can be used in a method for determining the presence and / or amount of microbial endotoxins in a sample. For example, the method may include (a) introducing a liquid sample into a cartridge, for example, the sample inlet port of a cartridge disclosed herein; (b) moving the sample into an optical cell; and (c) measuring the optical properties of the sample in the optical cell at a predetermined time, wherein a change in optical properties indicates the presence and / or amount of microbial endotoxins in the sample. The change in optical properties may be an increase or decrease in the absorbance of light at a pre-selected wavelength. The method may further include (d) determining the amount of microbial endotoxins in the sample by comparing the optical properties of the sample in the optical cell measured at a predetermined time (c) with a predetermined standard curve.

[0124]

[0140] As disclosed herein, factors B (e.g., recombinant factor B) and C (e.g., recombinant factor C) should be maintained separately on the cartridge to maintain the stability of each factor. Thus, factors B and C are kept separate within the cartridge until use, for example, maintained in separate regions or areas of the cartridge, and mixed together only in the presence of a liquid sample passing through the cartridge. In other words, a region or area of ​​the cartridge containing factor B (e.g., recombinant factor B) also does not contain factor C (e.g., recombinant factor C), and vice versa.

[0125]

[0141] When an endpoint colorimetric assay or fluorescence assay is performed in cartridge 1 (see Figures 2A-2D), the sample is moved, for example, to a first region 14 of conduit 8 containing a first recombinant factor, where it is dissolved by the sample, for example, by cycling forward and reverse pumping. The sample-factor mixture is then moved, for example, by pumping, to a second region 16 of conduit 8 containing a second recombinant factor and a colorimetric or fluorescent substrate, where they are dissolved, for example, by cycling forward and reverse pumping. The sample-factor-substrate mixture is then transferred to optical cell 6 for measurement of the optical properties of the sample, such as absorbance or transmittance, using an optical detector. However, when performing an endpoint colorimetric or fluorescence assay in cartridge, it is intended that there is no need to stop the reaction using a reaction inhibitor. Under this type of assay, the final optical reading (endpoint reading) is recorded at a predetermined time.

[0126]

[0142] Referring to Figures 2A-D, in one configuration, the recombinant factor located in the first region 14 is rFB, and the recombinant factor located in the second region 16 is rFC. Depending on the situation, for example, a chromogenic substrate or a fluorescent substrate may be located in the first or second region, but can be optionally separated from rFB or rFC. This can be achieved, for example, when the substrate is coated on the upper surface of the conduit 8 and the factor is coated on the lower surface of the conduit 8. rPCE may be located on the first region 14, or optionally on the second region 16. In one embodiment, rFB and rPCE are located on the first region 14, and rFC and the substrate (e.g., a chromogenic substrate) are located on the second region.

[0127]

[0143] Referring to Figures 2A-D, in another configuration, the recombinant factor located in the first region 14 is rFC, and the second factor located in the second region 16 is rFB. Depending on the situation, for example, a chromogenic substrate or a fluorescent substrate may be located in the first or second region, but can be separated from rFB or rFC by optional choice. This can be achieved, for example, when the substrate is coated on the upper surface of the conduit 8 and the factor is coated on the lower surface of the conduit 8. rPCE may be located on the first region 14, or optionally on the second region 16.

[0128]

[0144] Referring to Figures 2A-D, in yet another alternative configuration, the recombinant factor rFB is located on the first region 14 together with a fluorescent substrate or chromogenic substrate, and rFC is located on the second region 16. rPCE may be located on the first region 14, or rPCE may be located on the second region 16.

[0129]

[0145] Referring to Figures 2A-D, in yet another alternative configuration, the recombinant factor rFC is located on the first region 14 together with a fluorescent substrate or chromogenic substrate, and rFB is located on the second region 16. rPCE may be located on the first region 14, or rPCE may be located on the second region 16.

[0130]

[0146] Referring to Figures 3A-3D, the endpoint colorimetric assay or fluorescence assay is performed in cartridge 1. The sample is moved, for example, to a first region 14 of conduit 8 containing a first recombinant factor (e.g., rFC), where it is dissolved by the sample, for example, by cycling forward and reverse pumping. The sample-factor mixture is then moved, for example, by pumping, to a second region 16 of conduit 8 containing a second recombinant factor (e.g., rFB), where they are dissolved by the sample-factor mixture, for example, by cycling forward and reverse pumping. The sample-factor mixture is then moved, for example, by pumping, to a third region 18 of conduit 8 containing a colorimetric or fluorescent substrate, where they are dissolved, for example, by cycling forward and reverse pumping. The sample-factor-substrate mixture is then transferred to optical cell 6 for measurement of the optical properties of the sample, for example, absorbance or transmittance properties, using an optical detector. The final optical reading (endpoint reading) is recorded at a predetermined time.

[0131]

[0147] Referring to Figures 3A-D, in one configuration, the recombinant factor located in the first region 14 is rFB, and the recombinant factor located in the second region 16 is rFC. rPCE may be located on the first region 14 or within the second region 16. Alternatively, rPCE may be located on the third region 18 together with the substrate.

[0132]

[0148] Referring to Figures 3A-D, in one configuration, the recombinant factor located in the first region 14 is rFC, and the recombinant factor located in the second region 16 is rFB. rPCE may be located on the first region 14 or within the second region 16. Alternatively, rPCE may be located on the third region 18 together with the substrate.

[0133]

[0149] Referring to Figures 3A-D, in yet another configuration, the substrate is located on the first region 14, the first recombinant factor is located on the second region 16, and the second recombinant factor is located on the third region 18. The first recombinant factor may be an rFC, and the second recombinant factor may be an rFB. Alternatively, the first recombinant factor may be an rFB, and the second recombinant factor may be an rFC. The rPCE can be located on the first region 14, the second region 16, or the third region 18. In one embodiment, the first recombinant factor is an rFC, the second recombinant factor is an rFB, and the rPCE is located on the second region 16 together with the rFC. In another embodiment, the first recombinant factor is an rFC, the second recombinant factor is an rFB, and the rPCE is located on the third region 18 together with the rFB. In one embodiment, the first recombinant factor is rFB, the second recombinant factor is rFC, and rPCE is located on the second region 16 together with rFB. In another embodiment, the first recombinant factor is rFB, the second amebocytic factor is rFC, and rPCE is located on the third region 18 together with rFC.

[0134]

[0150] Referring to Figures 3A-D, in yet another configuration, the first recombinant factor is located on the first region 14, the substrate is located on the second region 16, and the second recombinant factor is located on the third region 18. The first recombinant factor may be an rFC, and the second recombinant factor may be an rFB. Alternatively, the first recombinant factor may be an rFB, and the second recombinant factor may be an rFC. The rPCE can be located on the first region 14, the second region 16, or the third region 18.

[0135]

[0151] The assay can be calibrated by measuring optical properties, such as absorbance or transmittance, when a specific amount of microbial contaminant (e.g., endotoxin) is introduced into the assay. The presence or amount of microbial endotoxin in the test sample can be determined by comparing the results produced by the test sample with one or more results produced by a known amount of microbial endotoxin.

[0136]

[0152] In some embodiments, one or more naturally isolated factors can be used to replace the corresponding recombinant factors on the cartridge. For example, depending on the situation, naturally isolated factor C can replace rFC, naturally isolated factor B can replace rFB, or naturally isolated procoagulant can replace rPCE.

[0137] (ii) Well

[0153] As discussed, this type of assay can be used in various other forms, such as in the wells of a microtiter plate. In this type of assay, the sample of interest is mixed with recombinant factors and substrates and incubated for a pre-selected period. Optionally, a reaction inhibitor, such as acetic acid, is added to the sample, and the optical properties of the sample, such as absorbance or transmittance, are measured. The results can then be compared to standard values ​​to determine the presence or amount of microbial endotoxins in the sample of interest.

[0138]

[0154] As described above for kinetic assays, it is intended that the assay can be performed in a single well or multiple wells of a microtiter plate. However, the optical properties are measured at a predetermined time, rather than over time as in kinetic assays. Similarly, it is intended that such assays can be performed in one or more vials, as described above for kinetic assays.

[0139]

[0155] In some embodiments, one or more naturally occurring isolated factors can be used to replace the corresponding recombinant factors in a well or vial. For example, depending on the context, naturally occurring isolated factor C can replace rFC, naturally occurring isolated factor B can replace rFB, or naturally occurring isolated procoagulant can replace rPCE.

[0140] VII. Preparation of Compositions, Cartridges, and Vials

[0156] All reagents and materials used to prepare the cartridges, well plates, or vials disclosed herein preferably do not contain microbial contaminants, such as endotoxins, that the cartridges will ultimately be used to test.

[0141]

[0157] Cartridges, well plates, or vials may be prepared using recombinant factor B, recombinant factor C, or procoagulant enzymes disclosed herein. For example, rFB and / or rFC and / or rPCE may be derived from the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda).

[0142]

[0158] In some embodiments, the cartridge may be prepared using a combination of recombinant amebosite coagulation factors and naturally isolated coagulation factors. For example, one of the natural or recombinant factor B, one of the natural or recombinant factor C, and one of the natural or recombinant procoagulant enzymes may be used to prepare the cartridge. For example, naturally isolated factor B and / or factor C and / or PCE may be factor B and / or factor C and / or PCE from the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda).

[0143] A. Endotoxin test composition

[0159] A method for producing a bacterial endotoxin test composition is disclosed herein. The composition can be used with a cartridge, for example, the cartridge disclosed herein, or in a well or vial-based assay disclosed herein. The composition may be a liquid, such as a buffer solution, or it may be lyophilized or freeze-dried depending on the application.

[0144]

[0160] In one embodiment, the present disclosure provides a method for producing a stabilized composition for detecting bacterial endotoxins in a sample. The method includes (a) providing a first solution containing recombinant amebocyte factor C (rFC) and a second different solution containing recombinant amebocyte factor B (rFB), wherein rFC and rFB are substantially inactive; and (b) combining the first and second solutions to produce a third solution containing a mixture of rFC and rFB, under conditions such that rFC and rFB remain substantially inactive in the mixture in the absence of a test sample (or control) containing endotoxins. In some embodiments, the mixture is dehydrated immediately after mixing the first solution containing rFC and the second solution containing rFB, for example, within about 30 minutes. For example, in some examples, dehydration of the mixture provides the conditions necessary for rFB and rFC to remain substantially inactive. Dehydration can be achieved by lyophilization or freeze-drying. In some embodiments, the first solution further comprises recombinant procoagulant (rPCE), and in other embodiments, the second solution further comprises rPCE. The method may further comprise a further step (c) of providing a third solution comprising rPCE, and in step (b), further comprising mixing the first, second, and third solutions to produce a mixture of rFC, rFB, and rPCE. rFC may be Limulus polyphemus factor C, Tachypleus tridentatus factor C, or Carcinoscorpius rotundicauda factor C. rFB may be Limulus polyphemus factor B, Tachypleus tridentatus factor B, or Carcinoscorpius rotundicauda factor B. rPCE may be procoagulant from the American horseshoe crab (Limulus polyphemus), the horseshoe crab (Tachypleus tridentatus), or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda).The method may further include the step of contacting the mixture with a sample suspected of containing microbial endotoxins and determining the presence and / or amount of endotoxins in the sample, if any. If the mixture is lyophilized, it may be redissolved by the addition of the sample or by the addition of a liquid such as a buffer. With respect to factor C, the term “substantially inactive” refers to factor C (e.g., rFC) that is not substantially converted into an active enzyme that activates factor B (e.g., rFB) and procoagulant (e.g., rPCE). In other words, factor C (e.g., rFC) remains inactive to the lowest level of endotoxin detectable in a given endotoxin detection assay, as determined, for example, by the standard curve of the assay. With respect to factor B, the term “substantially inactive” refers to factor B (e.g., rFB) that is not substantially converted into an active enzyme that activates factor C (e.g., rFC) and procoagulant (e.g., rPCE). In other words, factor B (e.g., rFB) remains inactive for the lowest detectable levels of endotoxin in a given endotoxin detection assay, as determined, for example, by the assay's standard curve.

[0145]

[0161] In another embodiment, the disclosure provides a method for producing bacterial endotoxin test compositions, wherein a first composition is provided containing a first amebosite factor, e.g., factor C without factor B (e.g., rFC), and a second composition is provided containing a second amebosite factor, e.g., factor B without factor C (e.g., rFB). In one embodiment, the first and second compositions are mixed together in the presence of a procoagulant (e.g., rPCE) to form a third composition, and the first and second compositions remain separated until they are mixed together immediately before contact with the test sample (e.g., within about 30 minutes). This approach may be used, for example, in wells or vials. In another embodiment, the first and second compositions are mixed together in the presence of a procoagulant (e.g., rPCE) to form a third composition, and the first and second compositions remain separated until they are mixed with the test sample. This approach may be used, for example, with a cartridge.

[0146]

[0162] In some embodiments of the method, the first and second compositions provided in steps (a) and (b) of the method are provided as dry compositions. These dry compositions may be redissolved before mixing in step (c) of the method. For example, the first and second compositions may be redissolved in one or more buffer solutions. Alternatively, the first and second compositions may be redissolved by the test sample. If the first and second compositions are provided as solutions, they may be mixed together in a container (e.g., vial, test tube, or well) immediately before the test sample is added to the container (e.g., within about 30 minutes).

[0147] B. Cartridge

[0163] Exemplary cartridges useful for carrying out the disclosed method, and methods for manufacturing the same, can be found in U.S. Patent No. 7,329,538 and U.S. Design Patent No. D472,324.

[0148]

[0164] In one embodiment, the present disclosure provides a cartridge for bacterial endotoxin testing. The cartridge includes a housing defining a test sample inlet region, a first region in which a first composition containing a first recombinant factor is disposed, and a second region in which a second composition containing a second recombinant factor is disposed. The first and second regions are spaced apart from each other and are in fluid communication with each other. The first region is in fluid communication with the sample inlet region. Therefore, when a test sample is added to the sample inlet region, the sample mixes with the first and second compositions. In some embodiments, a pump is applied to the cartridge to move the sample back and forth across the first and second regions, enabling the sample to mix with the first and second compositions.

[0149]

[0165] In one embodiment, the first composition contains recombinant factor B or recombinant factor C, and the second composition contains recombinant factor B or recombinant factor C, but not both the first and second compositions contain recombinant factor B or recombinant factor C. For example, in one configuration, the first composition on the first region contains recombinant factor C but does not contain recombinant factor B, and the second composition on the second region contains recombinant factor B but does not contain recombinant factor C.

[0150]

[0166] In other embodiments, the first composition further comprises recombinant procoagulant, or the second composition further comprises recombinant procoagulant. In other configurations, the cartridge includes a third region separate from the first and second regions and in fluid communication with the first and / or second regions of the cartridge; the third composition is placed on the third region. In some configurations, the third composition may include a chromogenic substrate; in other configurations, the third composition may include recombinant procoagulant; and in other configurations, the third composition may include both recombinant procoagulant and a chromogenic substrate. In some embodiments, the third region is placed between the sample inlet region and the first region such that the sample passes through the third region, then the first region, and then the second region. For example, when the third region is placed between the sample inlet region and the first region, the third composition includes a chromogenic substrate but does not include any recombinant factor.

[0151]

[0167] In some embodiments, the cartridge includes a fourth region separate from the first, second, and third regions, and in fluid communication with the first, second, and / or third regions. In one configuration, the fourth region includes a fourth composition comprising a recombinant procoagulant disposed thereon, and in another configuration, the fourth composition comprises a chromogenic substrate.

[0152]

[0168] An exemplary cartridge containing the first, second, third, and fourth regions is shown in Figure 4E. The sample inlet 4 is upstream of the first region 14, the first region 14 is upstream of the second region 16, the second region 16 is upstream of the third region 18, and the third region 18 is upstream of the fourth region 20. When the test sample is placed at the sample inlet 4 on the cartridge, it flows downstream from the sample inlet 4 toward the optical cell 6. A pump port 12 is provided for mounting a pump that can draw the sample from the fluid inlet port 4 toward the optical cell 6 under negative pressure. The pump moves the sample back and forth across the first, second, third, and fourth regions under positive and negative pressure, ensuring that the sample is thoroughly mixed with the first, second, third, and fourth compositions before the results are read in the optical cell 6.

[0153]

[0169] In one embodiment, the cartridge includes a housing defining a sample inlet region, a first region where a first composition is placed, a second region where a second composition is placed, and a third region where a third composition is placed. Each region is spaced apart from the others and is in fluid communication with one another. The first region is in fluid communication with the sample inlet region and the second region and is located between the sample inlet region and the second region. The second region is in fluid communication with the first region and the third region and is located between the first region and the third region. The third region is in fluid communication with the second region, is located next to the second region, and optionally is located next to an optical cell and is in fluid communication with the optical cell. Thus, when a sample is added to the sample inlet region, the sample is first mixed with the first composition in the first region, then moves to the second composition in the second region and is mixed, then moves to the third composition in the third region and is mixed, and then optionally moves to the optical cell. In some embodiments, a pump is applied to the cartridge to move the sample back and forth across first, second, and third regions, allowing the sample to mix with the first, second, and third compositions. In one configuration, the first composition includes a substrate, e.g., a chromogenic substrate, but no recombinant factor; the second composition includes recombinant factor B, but no recombinant factor C or procoagulant; and the third composition includes recombinant factor C and recombinant procoagulant, but no recombinant factor B. In another configuration, the first composition includes a substrate, e.g., a chromogenic substrate, but no recombinant factor; the second composition includes recombinant factor C and recombinant procoagulant, but no recombinant factor B; and the third composition includes recombinant factor B, but no recombinant procoagulant or recombinant factor C.

[0154]

[0170] An exemplary cartridge containing the first, second, and third regions is shown in Figures 3A-B. The sample inlet 4 is located upstream of the first region 14, the first region 14 is located upstream of the second region 16, and the second region 16 is located upstream of the third region 18. When the test sample is placed in the sample inlet 4 on the cartridge, it flows downstream from the sample inlet 4 towards the optical cell 6, through the first region 14, then the second region 16, and then the third region. A pump port 12 is provided for attachment to a pump that can draw the sample from the fluid inlet port 4 into the optical cell 6 under negative pressure. The pump moves the sample back and forth across the first, second, and third regions under positive and negative pressure, ensuring that the sample is thoroughly mixed with the first, second, and third compositions before the results are read in the optical cell 6.

[0155]

[0171] Also disclosed is an exemplary cartridge for determining the presence and / or amount (if present) of microbial (e.g., bacterial) endotoxins in a liquid sample. The cartridge comprises (a) a housing defining a fluid inlet port, an optical cell, and a conduit having a fluid contact surface providing fluid communication between the fluid inlet port and the optical cell; (b) a first composition disposed on a first region of the fluid contact surface of the conduit; and (c) a second composition disposed on a second region of the fluid contact surface of the conduit. The first region is separated from the second region so that when the sample is applied to the fluid inlet port, the sample passes through the first and second regions during transport to the optical cell, dissolving the first and second compositions. The first and second compositions are selected from the group consisting of amebosite factor B and amebosite factor C, wherein the first composition is not the same as the second composition. In some embodiments, factor C and / or factor B remain substantially inert until they come into contact with microbial endotoxins in the liquid sample.

[0156]

[0172] In some embodiments, the second region of the cartridge is located between the first region and the optical cell. In other embodiments, the first region is a first circumferential section of the fluid contact surface of the conduit, and the second region is a second different circumferential region of the fluid contact surface of the conduit. For example, the first circumferential section is defined by at least a portion of the upper half of the conduit, and the second circumferential section is defined by at least a portion of the lower half of the conduit.

[0157]

[0173] In some configurations of the cartridge, the first composition contains factor C and the second composition contains factor B, while in other configurations, the first composition contains factor B and the second composition contains factor C. Factor B may be recombinant factor B, for example, American horseshoe crab (Limulus polyphemus) factor B, horseshoe crab (Tachypleus tridentatus) factor B, or round-tailed horseshoe crab (Carcinoscorpius rotundicauda) factor B. Factor C may be recombinant factor C, for example, American horseshoe crab (Limulus polyphemus) factor C, horseshoe crab (Tachypleus tridentatus) factor C, or round-tailed horseshoe crab (Carcinoscorpius rotundicauda) factor C.

[0158]

[0174] In some configurations of the cartridge, the first or second region further contains a procoagulant. In yet other configurations, the procoagulant is located in a third region of the fluid contact surface of the conduit, spaced apart from the first and second regions. For example, the procoagulant is a recombinant procoagulant such as the American horseshoe crab (Limulus polyphemus) procoagulant, the horseshoe crab (Tachypleus tridentatus) procoagulant, or the round-tailed horseshoe crab (Carcinoscorpius rotundicauda) procoagulant.

[0159]

[0175] In some cartridge configurations, the chromogenic substrate is positioned on a first, second, third, or fourth region of the fluid contact surface, spaced apart from the first, second, or third region. The third region may be downstream of the first and / or second region. The fourth region, if present, may also be downstream of the first and / or second region. However, in certain configurations, the third region is positioned upstream of the first and second regions, and upstream of any fourth region, if present. For example, the chromogenic substrate is positioned on a third region located upstream of the first and second regions, and the fourth region, if present. The fourth region may be downstream of the first, second, and third regions.

[0160]

[0176] An exemplary method for manufacturing a cartridge is described with reference to Figures 4A and 4B, where Figure 4A provides a top view showing one configuration of a cartridge according to the present disclosure, and Figure 4B provides a cross-sectional view of the cartridge at positions A-A' and B-B' in Figure 4A. As shown in cross-section A-A' of Figure 4B, the cartridge 1 has a first (e.g., lower) half 2 and a second (e.g., upper) half 3. Once manufactured, the two halves of the cartridge 1 are joined to each other by adhesive, solvent bonding, ultrasonic welding, snap-fit ​​joint, etc. It is intended that the cartridges of Figures 2 and 3 can be manufactured by similar principles.

[0161]

[0177] As shown in Figures 4A-B, cartridge 1 comprises a first (e.g., lower) half 2 and a second (e.g., upper) half 3. The first half 2 of cartridge 1 defines one half of each conduit 8 (each having a first region 14 and a second region 16), so that the conduit 8 has a first (e.g., lower) surface 8' on the first (e.g., lower) half of cartridge 1 and a second (e.g., upper) surface 8'' on the second (e.g., upper) half of cartridge 1. During preparation, a first reagent, e.g., amebocyte factor 20, can be applied to the first (e.g., lower) surface 8' of the first region 14' of the conduit 8, and a third reagent 24 can be applied to the cartridge This applies to the first (e.g., lower) surface 8' of the second region 16' of the conduit 8 on the first (e.g., lower) half 2 of the cartridge 1. The direction of fluid flow on the cartridge is away from the fluid inlet port 4 and towards the optical cell 6 through the conduit. Thus, the first region 14' is downstream from the fluid inlet port 4 and upstream of the second region 16'. The second region 16' is downstream of the first region 14'. The first region 14' and the second region 16' are in fluid communication with each other, and the first region 14' is in fluid communication with the fluid inlet port.

[0162]

[0178] During preparation, a second reagent, for example, amebosite factor 22, is applied to the second (e.g., upper) surface 8" of the first region 14" of the conduit 8 on the second (upper) half 3 of cartridge 1. Cartridge 1 also has a fluid inlet port 4 and at least one pump port 12 that is in fluid communication with the optical cell 6 for mounting cartridge 1 and applying negative pressure through the pump port 12 to draw the sample from the fluid inlet port 4 into the optical cell 6.

[0163]

[0179] Referring further to Figures 4A-4B, in one configuration, the first reagent contains factor B, e.g., rFB, and the second reagent contains factor C, e.g., rFC. The third reagent contains a substrate for the procoagulant (e.g., rPCE), such as a chromogenic substrate or a fluorescent substrate. In another configuration, the first reagent contains factor C, e.g., rFC, and the second reagent contains factor B, e.g., rFB. The third reagent contains a substrate for the procoagulant (e.g., rPCE), such as a chromogenic substrate or a fluorescent substrate. In one configuration, the procoagulant, e.g., rPCE, is located on the first face 8', the second face 8'', or both the first face 8' and the second face 8'' of the first region 14, and in another configuration, the procoagulant, e.g., rPCE, is located on the first face 8', the second face 8'', or both the first face 8' and the second face 8'' of the second region 16. After applying reagents to the upper three halves and lower two halves of cartridge 1, cartridge halves 2 and 3 are dried under conditions that allow the reagents, e.g., amebocyte factors (e.g., rFC, rFB, rPCE) and substrates to maintain their activity and allow the factors and substrates to be dissolved or reconstituted in order to perform assays for detecting microbial endotoxins.

[0164]

[0180] In another configuration shown in Figures 4A-B, the first amebosite factor 20 (e.g., rFC) is located on the first surface 8' of the fluid contact surface 8 of the first region 14, and the second amebosite factor 22 (e.g., rFB) is located on the second surface 8'' of the fluid contact surface 8 of the first region 14. The procoagulant enzyme may be located in the first region 14 or the second region 16, and may be located on the first surface 8', the second surface 8'', or both the first and second surfaces of the fluid contact surface 8 of either the first region 14 or the second region 16. The substrate 24 may be located on the second region 16 on either the first surface 8' or the second surface 8'' of region 16, or on both the first and second surfaces.

[0165]

[0181] Another exemplary method of manufacturing the cartridge is described with reference to Figures 4C–4D, where Figure 4C provides a top view showing one configuration of a cartridge according to the present disclosure, and Figure 4D provides a cross-sectional view of the cartridge at positions A–A', B–B', and C–C' in Figure 4C. As shown in the cross-sections at A–A', B–B', and C–C' in Figure 4D, the cartridge 1 has a first (e.g., lower) half 2 and a second (e.g., upper) half 3. Once manufactured, the two halves of the cartridge 1 are joined to each other by adhesive, solvent bonding, ultrasonic welding, snap-fit ​​joint, etc.

[0166]

[0182] As shown in Figures 4C-D, cartridge 1 comprises a first (e.g., lower) half 2 and a second (e.g., upper) half 3. The first half 2 of cartridge 1 defines one half of each conduit 8 (each having a first region 14, a second region 16, and a third region 18), so that the conduit 8 has a first (e.g., lower) surface 8' on the first (e.g., lower) half of cartridge 1 and a second (e.g., upper) surface 8'' on the second (e.g., upper) half of cartridge 1. During preparation, in one configuration, a first reagent 20 (e.g., amebocyte factor or substrate) is applied to the first (e.g., lower) surface 8' of the first region 14 of the conduit 8 on the first (e.g., lower) half 2 of cartridge 1, and a second reagent is applied to the first (e.g., lower) surface 8' of the second region 16 of the conduit 8. Drug 22 (e.g., amebosite factor) is applied, and a third reagent 24 (e.g., amebosite factor) is applied to the first (e.g., lower) surface 8' of the third region 18 of the conduit 8. The direction of fluid flow on the cartridge is away from the fluid inlet port 4 and toward the optical cell 6 through the conduit. Thus, the first region 14 is downstream from the fluid inlet port 4 and upstream of the second region 16. The second region 16 is downstream of the first region 14 and upstream of the third region 18. The first region 14 is in fluid communication with the fluid inlet port, the first region 14 and the second region 16 are in fluid communication with each other, and the second region 16 is in fluid communication with the third region 18.

[0167]

[0183] The second (e.g., upper) half 3 of cartridge 1 defines the second (e.g., upper) surface 8" of the conduit 8, which in one embodiment may not contain any reagents. However, in some embodiments, during the preparation of the cartridge, a first reagent 20 (e.g., amebocyte factor or substrate) is applied to the second (e.g., upper) surface 8" of the first region 14 of the conduit 8 on the second (e.g., upper) half 2 of cartridge 1, a second reagent (e.g., amebocyte factor) 22 is applied to the second (e.g., upper) surface 8" of the second region 16 of the conduit 8, and / or a third reagent 24 (e.g., amebocyte factor) is applied to the second (e.g., upper) surface 8' of the third region 18 of the conduit 8. In one embodiment, the reagents applied to the first, second, and third regions of the lower surface 8' of the conduit 8 are also applied to the same regions of the upper surface 8" of the conduit 8. Cartridge 1 also has a fluid inlet port 4 and at least one pump port 12 that is in fluid communication with the optical cell 6 for attaching cartridge 1 and applying negative pressure through the pump port 12 to draw a sample from the fluid inlet port 4 into the optical cell 6.

[0168]

[0184] Referring further to Figures 4C-4D, in one configuration, the first reagent contains factor B, e.g., rFB, and the second reagent contains factor C, e.g., rFC. The third reagent contains a substrate for the procoagulant (e.g., rPCE), such as a chromogenic substrate or a fluorescent substrate. In another configuration, the first reagent contains factor C, e.g., rFC, and the second reagent contains factor B, e.g., rFB. The third reagent contains a substrate for the procoagulant (e.g., rPCE), such as a chromogenic substrate or a fluorescent substrate. In one configuration, the procoagulant, e.g., rPCE, is located on the first face 8', the second face 8'', or both the first face 8' and the second face 8'' of the first region 14, and in another configuration, the procoagulant, e.g., rPCE, is located on the first face 8', the second face 8'', or both the first face 8' and the second face 8'' of the second region 16.

[0169]

[0185] In an alternative configuration shown in Figures 4C-D, the first amebosite factor, e.g., rFC 20, is positioned on the first surface 8' (or the first surface 8' and the second surface 8") of the fluid contact surface 8 of the first region 14, and the second amebosite factor, e.g., rFB 22 is located on the first surface 8' (or the first surface 8' and the second surface 8") of the fluid contact surface 8 of the second region 16, and the substrate 24 is located on the first surface 8' (or the first surface 8' and the second surface 8") of the third region 18 of the fluid contact surface 8. The procoagulant may be located in the first region 14 or the second region 16, or on both the first surface 8', the second surface 8'', or the first surface 8' and the second surface 8'' of the fluid contact surface 8 of any of the first region 14, the second region 16, or the third region 18. The substrate may be located on the first surface 8' (or the first surface 8' and the second surface 8") of the fluid contact surface of the third region 16. In an alternative configuration, the first amebosite factor 20 is rFB and the second amebosite factor 22 is rFC. In a further configuration, the fluid contact surface has a first region 14, a second region 16, and a third region 18 on the fluid contact surface 8, the first amebosite factor, the second amebosite factor, and the substrate are each arranged in different regions of the fluid contact surface 8, and rPCE may be arranged in any of the first, second, or third regions of the fluid contact surface. However, rFC and rFB are not arranged in the same region.

[0170]

[0186] In some embodiments, the order in which reagents are applied to regions (e.g., 14, 16, or 18) varies, as long as factor B, e.g., rFB, is applied to one region (e.g., 14, 16, or 18), factor C, e.g., rFC, is applied to a second region (e.g., 14, 16, or 18), and the substrate is applied to a third region of conduit 8 (e.g., 14, 16, or 18), where factors C and B are not applied to the same region, and each region (14, 16, and 18) contains only one of factor C, factor B, and the substrate. A procoagulant, e.g., rPCE, can be applied to at least one of regions 14, 16, or 18. For example, in one particular configuration, the substrate is applied to the first region 14, rFC and rPCE are applied to the second region 16, and rFB is applied to the third region 18. In another configuration, the substrate is applied to the first region 14, rFB and rPCE are applied to the second region 16, and rFC is applied to the third region 18. In yet another configuration, the substrate, for example, a chromogenic substrate, is applied to the first region 14, rFB is applied to the second region 16, and rFC and rPCE are applied to the third region 18. For example, in one configuration, the first reagent 20 contains a substrate, for example, a chromogenic substrate, the second reagent 22 contains recombinant factor B, and the third reagent 24 contains recombinant factor C and recombinant procoagulant. In another embodiment, the first reagent 20 contains a substrate, for example, a chromogenic substrate, the second reagent 22 contains recombinant factor C and recombinant procoagulant, and the third reagent 24 contains recombinant factor B.

[0171]

[0187] After applying the reagent to the lower half 2 of cartridge 1 (or to both the lower half 2 and upper half 3 if the reagent is added to the upper half 3), cartridge halves 2 and 3 are dried under conditions that allow the reagent, e.g., amebocyte factors (e.g., rFC, rFB, rPCE) and substrates to remain active and allow the factors and substrates to be dissolved or reconstituted for assays to detect microbial endotoxins.

[0172]

[0188] Referring to Figure 4E, in one configuration, the first reagent is placed on the first region 14, the second reagent on the second region 16, the third reagent on the third region 18, and the fourth reagent on the fourth region 20. In one configuration, the first reagent contains factor B, e.g., rFB, the second reagent contains factor C, e.g., rFC, the third reagent contains procoagulant, e.g., rPCE, and the fourth reagent contains a chromogenic substrate. In another configuration, the first reagent contains factor C, e.g., rFC, the second reagent contains factor B, e.g., rFB, the third reagent contains procoagulant, e.g., rPCE, and the fourth reagent contains a chromogenic substrate.

[0173]

[0189] Another exemplary manufacturing process is described with reference to Figures 2A-D, where Figure 2A provides a perspective view of the cartridge, Figure 2B provides a top view, Figure 2C provides a cross-sectional view, and Figure 2D provides an end view. During manufacturing, a first reagent is applied to a first region 14 of the conduit 8, and a second reagent is applied to a second region 16 of the conduit 8. The reagents can be dried on the conduit. In one configuration, the first reagent contains factor B, e.g., rFB, and the second reagent contains factor C, e.g., rFC. In another configuration, the first reagent contains factor C, e.g., rFC, and the second reagent contains factor B, e.g., rFB. In some configurations, the first reagent also contains a chromogenic substrate or a fluorescent substrate, and in other embodiments, the second reagent contains a substrate. In some configurations, the first reagent also contains a procoagulant, e.g., rPCE, and in other embodiments, the second reagent contains a procoagulant, e.g., rPCE. In one exemplary configuration, the first reagent contains rFB and rPCE, and the second reagent contains rFC and a substrate. In another exemplary configuration, the first reagent contains rFC and rPCE, and the second reagent contains rFB and a substrate.

[0174]

[0190] To maintain the activity of the reagents during drying, cartridge halves 2 and 3 are placed in an environment with a temperature of about 4°C to about 40°C, more preferably about 10°C to about 35°C, more preferably about 15°C to about 30°C, and a relative humidity of about 0% to about 30%, more preferably about 2% to about 20%, more preferably about 4% to about 10%. Preferred drying conditions include a temperature of about 25°C and a relative humidity of about 5%. In an alternative approach, factors, such as recombinant factors, may be dried by freeze-drying, for example, lyophilization, under standard conditions, under vacuum, at about -30°C to about -40°C.

[0175]

[0191] The dimensions of a particular cartridge 1 may vary depending on the number and / or type of assay being performed. However, in one embodiment, as schematically shown in Figure 2A, for example, cartridge 1 has a length of approximately 10.16 cm (4.00 inches), a width of approximately 2.54 cm (1.00 inch), and a height of approximately 0.476 cm (0.188 inches). The inner diameter of the conduit 8 extending from the fluid inlet port 4 to the optical cell 6 is approximately 0.127 cm (0.050 inches), where the first reagent is dried in a region 14 of the conduit 8 approximately 2.381 cm (0.938 inches) from the fluid inlet port 4, and the second reagent is dried in a region 16 of the conduit 8 approximately 4.65 cm (1.831 inches) from the fluid inlet port 4. The optical cell 6 in this embodiment is sized to accommodate approximately 25 μL of sample.

[0176] C. well plate

[0192] The present invention also provides multiwell plates and vials for carrying out the assays disclosed herein.

[0177]

[0193] For example, in one embodiment, a well plate is prepared having a first reagent placed in a first well and a second reagent placed in a second well. In one embodiment, the first reagent contains factor B, e.g., rFB, and the second reagent contains factor C, e.g., rFC. In another configuration, the first reagent contains factor C, e.g., rFC, and the second reagent contains factor B, e.g., rFB. A procoagulant enzyme, e.g., rPCE, may be placed in the first or second well. A substrate for the coagulation enzyme, e.g., a chromogenic substrate or a fluorescent substrate, may be placed in the first or second well, or in the third well. For example, in another example, a well plate is prepared having a first reagent placed in the first well, a second reagent placed in the second well, and a third reagent placed in the third well. The first, second, and third reagents are each factor B (e.g., rFB), factor C (e.g., rFC), and one of the substrates; only one of these reagents is applied to each of the first, second, and third wells. However, a procoagulant, such as rPCE, can be applied to one or more of the first, second, and third wells.

[0178]

[0194] As the final step in preparing the well plate, the reagents are dried on the well plate, for example, by air drying or freeze drying under conditions that maintain the activity of the reagents, and then reconstituted with the sample to be applied when performing the endotoxin assay on the well plate.

[0179]

[0195] To maintain the activity of the reagents during drying, the wells are placed in an environment with a temperature of about 4°C to about 40°C, more preferably about 10°C to about 35°C, more preferably about 15°C to about 30°C, and a relative humidity of about 0% to about 30%, more preferably about 2% to about 20%, more preferably about 4% to about 10%. Preferred drying conditions include a temperature of about 25°C and a relative humidity of about 5%. In an alternative approach, factors, such as recombinant factors, may be dried by freeze-drying, for example, lyophilization, under standard conditions, under vacuum, at about -30°C to about -40°C.

[0180] D. vial

[0196] This invention describes the preparation of vials of amebosite factors (e.g., factor C, factor B) and procoagulants (e.g., rFC, rFB, and rPCE) for use in performing endotoxin detection assays.

[0181]

[0197] In one embodiment, a vial containing stabilized factor C, for example, stabilized rFC, is prepared. Neither the rFC nor the vial itself contains rFB. The rFC may also be substantially free of exogenous endotoxins, for example, containing only trace amounts of environmental endotoxins acceptable as contaminants. Thus, the rFC is substantially inert until it comes into contact with a test sample (or control) containing endotoxins. In another embodiment, a vial containing stabilized factor B, for example, stabilized recombinant factor B, is prepared. Neither the rFB nor the vial itself contains rFC. The rFB may also be substantially free of exogenous endotoxins. Thus, the rFB is substantially inert until it comes into contact with a test sample (or control) containing endotoxins.

[0182]

[0198] Stabilized amebosite factors, such as recombinant factors, may be present in aqueous solutions (e.g., pyrogen-free, e.g., endotoxin-free, prepared from water or a suitable buffer). The aqueous solution may be used in an endotoxin detection assay, or it may be dried in a vial, for example, by air drying, heating, or freeze-drying, and subsequently reconstituted in the vial with endotoxin-free water to produce a solution of the factor for use in an endotoxin detection assay. Vials containing dried amebosite factors may be stored preferably at -20°C to 25°C until ready for use.

[0183]

[0199] In another embodiment, a vial containing a stabilized recombinant factor is prepared. The vial is prepared by a method that provides (i) a first solution containing recombinant factor C and a second solution containing recombinant factor B. The rFC in the first solution and the rFB in the second solution are substantially inert; (ii) the first and second solutions are combined to produce a third solution under conditions that the rFC and rFB remain substantially inert in the mixture until they come into contact with endotoxin in, for example, a test sample (or control). Alternatively, after combining in step (ii), the third solution containing the rFC and rFB can be dried on the surface of a solid support, for example, by freeze-drying or lyophilization, such that the rFC and rFB remain substantially inert in a dehydrated form. For example, the third solution can be dried immediately on the surface of a solid support within about 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute after the third solution is produced.

[0184]

[0200] Examples of solid supports include cartridges or vials. In another embodiment, the rPCE is provided in a third solution, and step (ii) above includes mixing the first, second, and third solutions together to produce a mixture of rFC, rFB, and rPCE. The first or second solution may further contain a substrate for the rPCE. Alternatively, the third solution may further contain a substrate for the rPCE.

[0185]

[0201] The substrate may be a chromogenic substrate or a fluorescent substrate. For example, the substrate may be a chromogenic substrate having a p-nitroaniline group (pNA). For example, the substrate may be Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA. For example, factor C, factor B, or procoagulant may be factor C, factor B, or procoagulant of the American horseshoe crab (Limulus polyphemus), horseshoe crab (Tachypleus tridentatus), or round-tailed horseshoe crab (Carcinoscorpius rotundicauda), such as rFC, rFB, or rPCE.

[0186] E. General considerations

[0202] In manufacturing the cartridge, well plate, or vial of the present invention, it may be useful to combine factors, such as recombinant factors, and / or substrates (e.g., chromogenic substrates or fluorescent substrates) with one or more resolubilants (such as sugars or salts) and / or one or more anti-detachment agents (e.g., polymers) before drying the factors on the well, vial, or solid support (e.g., cartridge).

[0187]

[0203] The resolubilizing agent preferably stabilizes the dry form of the factor, such as recombinant factors, and promotes the resolving of the reagent during the assay. Useful sugar resolubilizing agents include, for example, mannitol, mannose, sorbitol, trehalose, maltose, dextrose, sucrose, and other monosaccharides and disaccharides.

[0188]

[0204] Anti-detachment agents can be used to prevent or reduce the possibility of factors (e.g., recombinant factors) and / or chromogenic substrates separating from a solid support in the form of dry flakes. Anti-detachment agents also preferably stabilize the factors (e.g., recombinant factors) and / or chromogenic substrates in their dry form. Useful anti-detachment agents include, for example, one or more polymers comprising polyethylene glycol, polyvinylpyrrolidone, dextran, mannitol, and proteins (e.g., serum albumin).

[0189]

[0205] Antifoaming agents may also be added to the factor (e.g., recombinant factor) before drying the factor on a well, vial, or solid support (e.g., cartridge). Certain polymers reduce the formation of bubbles (e.g., foaming) when the factor (e.g., recombinant factor) and / or chromogenic substrate are redissolved. Useful antifoaming agents include polyvinyl alcohol and polypropylene glycol.

[0190] VIII. Considerations for specimen collection and preparation

[0206] In general, materials used to collect, store, or otherwise come into contact with the sample being tested, as well as test reagents, must be free from microbial contamination, and for example, must be free from pyrogens. Materials can be made free of pyrogens, for example, by heating them at 250°C for 30 minutes. Appropriate precautions should be taken to protect the pyrogen-free materials from subsequent environmental contamination.

[0191]

[0207] The recombinant amebosite coagulation factors disclosed herein may be used to measure the presence or amount of microbial endotoxins in a sample of interest, such as a fluid, such as a fluid administered topically or systemically to a mammal, such as parenterally, or in a body fluid being tested for infection, such as blood, lymph, urine, serum, plasma, ascites, or lung aspirate. Furthermore, the assay may be used to detect microbial endotoxins present on a surface. For example, the surface of interest is swabbed, and then the swab is introduced into or dissolved in a liquid. The liquid can then be assayed as described herein.

[0192] IX. Kit

[0208] The present invention provides a kit of components used to perform an assay for determining the presence and / or amount of endotoxin in a sample of interest.

[0193]

[0209] This disclosure provides a kit for determining the presence and / or amount of bacterial endotoxin in a sample, comprising a first composition containing rFC but not rFB. The kit also comprises a second composition containing rFB but not rFC. The kit optionally further comprises means for mixing the first and second compositions together in the presence of recombinant procoagulant and a chromogenic substrate (e.g., vial or test tube, stirrer (e.g., spatula, spoon, etc.), dispensing tool (e.g., pipette tip, syringe, etc.)).

[0194]

[0210] In some embodiments, the kit may further include a third composition comprising a chromogenic substrate for procoagulant enzymes (rPCE), such as Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA.

[0195]

[0211] Depending on the circumstances, the first or second composition may further contain rPCE. Alternatively, the kit may contain a fourth composition containing rPCE.

[0196]

[0212] In some embodiments, the rFCs in the first composition are substantially inactive and / or the rFBs are substantially inactive until they come into contact with endotoxins, for example, from a test sample (or control).

[0197]

[0213] In one embodiment, the first and second compositions, and the third and fourth compositions (if provided), are dried on a solid support, for example, on one or more fluid contact surfaces of a cartridge. In another embodiment, the first and second compositions, and the third and fourth compositions (if provided), are dried on the surface of the corresponding first, second, third, or fourth container, for example, a well or vial. The kit may further include pyrogen-free, for example, endotoxin-free water, for reconstituting the dried compositions in each container.

[0198]

[0214] Alternatively, a kit for determining the presence and / or amount of bacterial endotoxin in a sample may comprise a composition comprising: a first stabilizing solution containing rFC (wherein rFC is substantially inert); a second stabilizing solution containing rFB (wherein rFB is substantially inert); and a chromogenic substrate for rPCE. In some embodiments, the first or second stabilizing solution further comprises rPCE. In other embodiments, the kit further comprises a third stabilizing solution containing rPCE. In some embodiments, the kit has a shelf life of at least 6 months.

[0199]

[0215] Alternatively, a kit for determining the presence and / or amount of bacterial endotoxin in a sample may include a first dehydration composition containing rFC (wherein rFC is substantially inactive, for example, when endotoxin is absent in the test sample or control), a second dehydration composition containing rFB (wherein rFB is substantially inactive, for example, when endotoxin is absent in the test sample or control), and a third dehydration composition containing a chromogenic substrate for procoagulant enzyme (rPCE). In some embodiments, the first or second dehydration composition further comprises rPCE, and in other embodiments, the kit further comprises a fourth dehydration composition containing rPCE. In some embodiments, each dehydration composition is placed in a separate container. In yet another embodiment, each dehydration composition is placed in a cartridge for determining the presence and / or amount of bacterial endotoxin in the sample of interest. [Examples]

[0200] Examples

[0216] The following examples are for illustrative purposes only and are not intended to limit the scope or content of the present invention.

[0201] I. Example 1 - Preparation of Recombinant Lysate Factors

[0217] Recombinant Limulus polyphemus factors rFB, rFC, and rPCE were prepared as follows. DNA sequences encoding Limulus polyphemus factors C, B, and procoagulant (with codon usage optimized for expression in mammalian cells) were cloned into the expression plasmid BD609 (ATUM). The amino acid sequence of Limulus polyphemus factor C is shown in SEQ ID NO: 2. The amino acid sequence of Limulus polyphemus factor B is shown in SEQ ID NO: 4. The amino acid sequence of Limulus polyphemus procoagulant is shown in SEQ ID NO: 6. The expression plasmids were transfected into HEK-293 cells using the FreeStyle 293 Expression System (Thermo Fisher) to generate stable clonal cell lines.

[0202]

[0218] For expression and purification, HEK-293 cells were thawed and added to FreeStyle 293 Expression Media in a flask. The cells were grown at 37°C, 5–7% CO2, at 120 rpm, and subcultured every 24–72 hours. Once the cells reached the desired volume and density, they were used to seed a total of 20 L of culture in a WAVE bioreactor. After 72 hours, the supernatant was collected by centrifugation at 4,000 × g for 15 minutes, followed by sterile filtration. The supernatant was concentrated to less than 2 L and buffered by tangential flow filtration (TFF, GE Life Sciences). The TFF system was equilibrated with 20 mM Tris-HCl buffer pH 8.0 containing 20 mM NaCl. The supernatant can also be used directly without further purification in various experiments, for example, for factor formulation or to study factor performance.

[0203]

[0219] To mitigate endotoxin exposure, all materials used were single-use. Water for injection was used for all buffer solutions, and all buffer solutions were prepared on the day of use.

[0204]

[0220] Each factor was formulated separately in a buffer solution until ready for use.

[0205]

[0221] International Publication No. 2022 / 174082 describes the production of recombinant elements (e.g., rFB, rFC, and rPCE) from horseshoe crabs (Tachypleus tridentatus), as described below.

[0206]

[0222] In particular, DNA sequences encoding horseshoe crab (Tachypleus tridentatus) factors C, B, and procoagulant (with codon usage optimized for expression in mammalian cells) were cloned into the expression plasmid BD609 (ATUM). The amino acid sequences of horseshoe crab (Tachypleus tridentatus) factor C are shown in SEQ ID NOs. 7 and 8, where SEQ ID NOs. 7 is the mature form and SEQ ID NOs. 8 is the factor C protein still containing the signal sequence as residues 1-21. The amino acid sequences of horseshoe crab (Tachypleus tridentatus) factor B are shown in SEQ ID NOs. 9 and 10, where SEQ ID NOs. 9 is the mature form and SEQ ID NOs. 10 is the factor B protein still containing the signal sequence as residues 1-22. The amino acid sequences of the horseshoe crab (Tachypleus tridentatus) procoagulase are shown in SEQ ID NOs. 11 and 12, where SEQ ID NOs. 11 represents the mature form, and SEQ ID NOs. 12 represents the procoagulase still containing the signal sequence as residues 1-21. The expression plasmid was transfected into HEK-293 cells using the FreeStyle 293 Expression System (Thermo Fisher) to generate stable clonal cell lines.

[0207]

[0223] For expression and purification, HEK-293 cells were thawed and added to FreeStyle 293 Expression Media in a flask. The cells were grown at 37°C, 5–7% CO2, at 120 rpm, and subcultured every 24–72 hours. Once the cells reached the desired volume and density, they were used to seed a total of 20 L of culture in a WAVE bioreactor. After 72 hours, the supernatant was collected by centrifugation at 4,000 × g for 15 minutes, followed by sterile filtration. The supernatant was concentrated to less than 2 L and buffered by tangential flow filtration (TFF, GE Life Sciences). The TFF system was equilibrated with 20 mM Tris-HCl buffer pH 8.0 containing 20 mM NaCl. The supernatant can also be used directly without further purification in various experiments, for example, for factor formulation or to study factor performance.

[0208]

[0224] To mitigate endotoxin exposure, all materials used were single-use. Water for injection was used for all buffer solutions, and all buffer solutions were prepared on the day of use.

[0209]

[0225] Each factor was formulated separately in a buffer solution until ready for use.

[0210] VIII. Evaluation of the stability of mixtures containing factors (rFC, rFB, and rPCE) in Example 2-3

[0226] Numerous assays were performed using recombinant factors to determine the effect of various combinations of factors on the overall stability of the formulation. Tests were conducted on multi-well plates using solutions of recombinant factors and other reagents, as well as in test tubes, to simulate the planned reaction sequence on the cartridge.

[0211]

[0227] Similar to commercially available bacterial endotoxin tests prepared from natural amebosite lysates, the stability of mixtures of rFB, rFC, and rPCE was evaluated at 5°C to assess whether recombinant factors could be formulated together in a synthetic cascade reagent solution (assay reagent).

[0212]

[0228] Using supernatants containing rFC, rFB, and rPCE from horseshoe crab (Limulus polyphemus) cell cultures produced as described in Example 1 and not subjected to TFF purification, aqueous solutions containing 12% rFC, 12% rFB, and 12% rPCE (each in volume ratio) were prepared. To stabilize the factors in the solution, the pH of the solution was lowered to approximately pH 6 by adding 1 mM HEPES (pH 5.3), and the solution was maintained at 5°C.

[0213]

[0229] To measure the onset time after different incubation periods, the above solution was mixed with 100 mM HEPES buffer (pH 7.6) containing the substrate (10 mM S2423, Ac-Ile-Glu-Gly-Arg-pNA) and 2% NaCl and 24 mM MgSO4 within 15 minutes of performing the experiment described below. The final synthesis cascade reagent contained 5% rFC, 5% rFB, 5% rPCE, 0.75 mM substrate, 12 mM MgSO4, and 1% NaCl in 50 mM HEPES buffer pH 7.6. Samples (0.1 mL each of water for negative control (NC), and endotoxin standards of 0.01 EU / mL, 0.1 EU / mL, and 1 EU / mL) were dispensed into a 96-well microplate (M9005, Charles River), and the same amount of the final reagent was added to each well. Absorbance at 405 nm was monitored at 37°C using a microplate reader (BioTek Elx 808®, BioTek Instruments, Inc., Winooski, VT), and the onset time for each sample was measured. Onset time was defined as the reaction time until a predetermined absorbance (onset OD, 0.05 in this experiment) was reached. Therefore, the higher the activity of the sample, the shorter the onset time.

[0214]

[0230] Figure 5A shows the stability of the solution tested at 0, 2, 4, 6, and 24 hours after preparation of the final synthetic cascade (assay) reagent solution. The onset times for NC and 0.01 EU / mL endotoxin were shortened at 2 hours, and there was no difference between NC and 0.1 EU / mL endotoxin. This indicates that the solution was activated, and the reagent could not detect 0.1 EU / mL endotoxin at 2 hours. The solution was fully activated at 24 hours. Contrary to expectations, the results showed that the synthetic cascade reagent solution containing all three factors was not stable even at low pH and 5°C.

[0215] IX. Example 3. Separating rFC from rFB improves the stability of the factor solution used in the preparation of assay reagents.

[0231] This experiment demonstrates the importance of separating rFCs from rFBs.

[0216]

[0232] To investigate the effect of rFC separation on stability, two solutions were prepared. The first solution contained 15.4% rFC in 1 mM HEPES (pH 5.3). The second solution contained 7.4% rFB, 7.4% rPCE, 1.1 mM substrate, 1.4% NaCl, 17.8 mM MgSO4, and 74.1 mM HEPES buffer pH 7.6. The horseshoe crab (Limulus polyphemus) factor used to prepare the solutions was the supernatant from the cell culture, i.e., the factor obtained before the TFF concentration step described in Example 1. Within 15 minutes before contact with the endotoxin standard, the two solutions were mixed in a ratio of 0.65:1.35 (first solution (rFB): second solution (rFC)) to form a synthetic cascade (assay) reagent. Therefore, the final reagent consisted of 5% rFC, 5% rFB, 5% rPCE, 0.75 mM substrate, 12 mM MgSO4, and 1% NaCl in 50 mM HEPES buffer pH 7.6. The final concentrations were the same as in Example 2. Onset times after exposure to endotoxin standards of various concentrations were measured at different time points (0 hours, 2 hours, 4 hours, 6 hours, and 24 hours) after the preparation of the first two solutions, as in Example 2.

[0217]

[0233] Figure 5B shows the effect of rFC separation on solution stability. The results clearly show that the solution was far more stable than the solution in Example 2, where rFCs and rFBs were mixed together in the initial solution. This is evident from the fact that the onset time was generally consistent for all samples over the tested period. This indicates that separating rFCs from other factors in the initial solution results in a stable synthetic cascade assay reagent.

[0218] X. Example 4 - Effect of temperature on the stability of factor solutions used to prepare assay reagents (25°C)

[0234] Two solutions were prepared to test the effect of temperature on the stability of the solutions used to prepare the synthetic cascade assay reagents. The first solution contained 0.6% rFC and 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The second solution contained 1.4% rFB, 5.8% rPCE, 100 mM MgSO4, and 72.8% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. At different time points (i.e., 0, 10, 20, 30, and 60 minutes from the preparation time of the first and second solutions), 0.1 mL of sample (NC) was added to a glass tube containing 0.05 mL each of the two solutions and 0.05 mL of a solution containing 0.1% polyvinyl alcohol and 1% mannitol. For each time point measurement, the glass tube was set in a tube reader (Fujifilm Wako, Japan), and endotoxin was measured by a kinetic colorimetric assay at 37°C. Onset time was defined as the reaction time until a specific absorbance (onset OD, 95% of the initial light intensity in this experiment) was reached. The first and second solutions were kept at 25°C, and mixtures of the solutions in the glass tubes to form the synthesis cascade (assay) reagents were prepared within 15 minutes before each assay.

[0219]

[0235] Figure 6 shows the stability of the solution over time. As can be seen, unacceptable levels of solution inactivation were observed during the first 10 minutes at 25°C. Therefore, it was found that maintaining the factor solution at a temperature of 25°C before the formation of the assay reagents adversely affects the stability of the solution used to prepare the synthetic cascade assay reagents.

[0220] XI. Example 5 - Effect of temperature on the stability of factor solutions used to prepare assay reagents (10°C)

[0236] To further investigate the effect of temperature on the stability of the factor solutions used to prepare the synthetic cascade reagents, two solutions were prepared. The first solution contained 0.6% rFC, 100 mM MgSO4, and 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The second solution contained 1.4% rFB, 5.8% rPCE, and 82.8% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. At different time points, 0.1 mL of sample (NC) was added to glass tubes containing 0.05 mL each of the two solutions and 0.05 mL of a solution containing 0.1% polyvinyl alcohol and 1% mannitol. Onset times were measured at different time points as described in Example 4, except that the time of use was measured from the time the first and second solutions were prepared, at 0, 20, 40, 60, 120, and 180 minutes. The first and second solutions were maintained at 10°C and mixed together to form the synthetic cascade (assay) reagent within 15 minutes prior to performing the assay.

[0221]

[0237] Figure 7 shows the time-dependent stability of the solution at 10°C, as demonstrated by the relatively stable onset times across the tested time points. No significant inactivation was observed after 3 hours at 10°C, indicating that the solution remained stable before the assay reagent formed.

[0222] XII. Example 6 - Effect of temperature on the stability of factor solutions used to prepare assay reagents (37°C)

[0238] To further investigate the effect of temperature on the stability of the factor solutions used to prepare the synthetic cascade reagents, two solutions were prepared. The first solution contained 0.6% rFC and 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The second solution contained 1.4% rFB, 5.8% rPCE, 300 mM MgSO4, and 52.8% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After the preparation of the first and second solutions, 0.1 mL of sample (NC) was added to a glass tube containing 0.05 mL each of the two solutions and 0.05 mL of a solution containing 0.1% polyvinyl alcohol and 1% mannitol at different time points (i.e., 0 min, 5 min, 10 min, 20 min, and 30 min). The measurement was performed in the same manner as in Example 4. The first and second solutions were maintained at 37°C and mixed together to form the synthetic cascade (assay) reagent within 15 minutes before performing the assay.

[0223]

[0239] Figure 8 shows the stability of the solution. Inactivation of the solution was observed during the first 5 minutes. The results from Examples 4-6 suggest that inactivation is temperature-dependent, and that under conditions where the substrate is contained in the rFC solution, a lower storage temperature is required for the stability of the first and second solutions.

[0224] XIII. Example 7 - Effect of temperature on the stability of the rFB separation solution used to prepare the assay reagent (25°C)

[0240] Two solutions were prepared to investigate the effect of separating rFB from rFC and rPCE on the stability of the factor solution used to prepare the synthetic cascade reagent. The first solution contained 1.4% rFB and 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The second solution contained 0.6% rFC, 5.8% rPCE, 100 mM MgSO4, and 68.6% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After the preparation of the first and second solutions, 0.1 mL of sample (NC) was added to a glass tube containing 0.05 mL each of the two solutions and 0.05 mL of a solution containing 0.1% polyvinyl alcohol and 1% mannitol at different time points (i.e., 0, 10, 20, 30, 60, and 135 minutes). The measurement was performed in the same manner as in Example 4. The solutions were maintained at 25°C and mixed together within 15 minutes prior to performing the assay to form the synthetic cascade (assay) reagent.

[0225]

[0241] Figure 9 shows the stability of the solution. In the rFB separation solution (containing the substrate), gradual and unacceptable inactivation was observed at 25°C.

[0226] XIV. Example 8 - Effect of substrate on the stability of factor solution used to prepare assay reagents (25°C)

[0242] To investigate the potential causes of solution inactivation, three solutions were prepared. The first solution was 1.2% rFC. The second solution was 6.6 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The third solution contained 1.4% rFB, 5.8% rPCE, 100 mM MgSO4, and 72.8% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After the preparation of the first, second, and third solutions, at different time points, 0.1 mL of sample (NC) was added to a glass tube containing 0.025 mL each of the first and second solutions, 0.05 mL of the third solution, and 0.05 mL of a solution containing 0.1% polyvinyl alcohol and 1% mannitol. The composition of the reagent mixture was the same as that of Example 4. The glass tubes were set in a tube reader (Fujifilm Wako, Japan), and endotoxin was measured by kinetic colorimetric assay at 37°C. The solutions were maintained at 25°C and mixed together within 15 minutes before performing each assay to prepare the synthetic cascade assay reagents.

[0227]

[0243] Figure 10 shows the stability of the solution (white square) when the substrate was kept separate from the factors. The results of Example 4 (Figure 6), in which rFC and the substrate were mixed in the initial solution, are shown for comparison (black circle) because the reagent composition produced by the mixture of the two solutions prepared in Example 4 had the same components as the reagent composition produced by the mixture of the three solutions in this example. Unlike Example 4, no significant inactivation of the solution was observed, suggesting that the potential cause of solution inactivation at higher temperatures is the presence of the substrate together with the factors in the solution. Separation of the substrate from each factor was effective in stabilizing the solution.

[0228] XV. Example 9 - Effect of rFC separation on the stability of factor solutions used to prepare assay reagents under substrate separation conditions (25°C)

[0244] Three solutions were prepared to examine the stability of the factor solutions containing isolated rFCs and isolated substrates. The first solution contained 1.2% rFCs, 63 mM MgSO4, and 43.3% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The second solution contained 1.4% rFBs, 5.8% rPCEs, 63 mM MgSO4, and 40.7% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The third solution contained 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA), 0.1% polyvinyl alcohol, and 1% mannitol. The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After preparing the first, second, and third solutions, 0.1 mL of the sample (NC, 0.02 EU / mL, or 0.2 EU / mL) was added to glass tubes containing 0.05 mL of each of the three solutions at different time points (0 hours, 1 hour, and 3 hours). Endotoxin was measured by a kinetic colorimetric assay at 37°C, and the measurement was performed in the same manner as in Example 4. The three solutions were maintained separately at 25°C and mixed together within 15 minutes before performing each assay to prepare the synthetic cascade assay reagent. Figure 11 shows the stability of the solutions over time. The solutions were stable at 25°C for at least 3 hours. This suggests that the separation of rFC from other factors (e.g., from rFB) is effective in stabilizing the solutions.

[0229] XVI. Example 10 Effect of rFB separation on the stability of factor solutions used to prepare assay reagents under substrate separation conditions (25°C)

[0245] Three solutions were prepared to examine the stability of the factor solutions containing the isolated rFB and isolated substrate. The first solution contained 1.4% rFB, 63 mM MgSO4, and 50.0% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The second solution contained 0.6% rFC, 5.8% rPCE, 63 mM MgSO4, and 50.0% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The third solution contained 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA), 0.1% polyvinyl alcohol, and 1% mannitol. The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After preparing the first, second, and third solutions, 0.1 mL of sample (NC, 0.005 EU / mL, or 0.5 EU / mL) was added to glass tubes containing 0.05 mL of each of the three solutions at different time points (0 min, 60 min, 120 min, and 220 min). Endotoxin was measured by a kinetic colorimetric assay at 37°C, as described in Example 4. The three solutions were maintained separately at 25°C and mixed together within 15 minutes before performing each assay to prepare the synthetic cascade assay reagent. Figure 12 shows the stability of the solutions. The three solutions were stable at 25°C for at least 3 hours. This suggests that the separation of rFB from other factors (e.g., from rFC and rPCE) is effective in stabilizing the solutions.

[0230] XVII. Example 11: Effect of rPCE separation on the stability of factor solutions used to prepare assay reagents under substrate separation conditions (25°C)

[0246] To investigate the effect of the factor solution from which rPCE was isolated on stability, three solutions were prepared. The first solution contained 5.8% rPCE, 63 mM MgSO4, and 41.5% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The second solution contained 1.2% rFC, 1.4% rFB, 63 mM MgSO4, and 43.0% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The third solution contained 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA), 0.1% polyvinyl alcohol, and 1% mannitol. The horseshoe crab (Limulus polyphemus) factor was prepared as described in Example 1, including TFF purification and concentration. After preparing the first, second, and third solutions, 0.1 mL of the sample (NC, 0.002 EU / mL, or 0.02 EU / mL) was added to glass tubes containing 0.05 mL of each of the three solutions at different time points (0 hours, 1 hour, and 2 hours). Endotoxin was measured by a kinetic colorimetric assay at 37°C, as described in Example 4. The three solutions were maintained separately at 25°C and mixed together within 15 minutes prior to performing the assay to prepare the synthetic cascade (assay) reagent.

[0231]

[0247] As shown in Figure 13, the solution was gradually activated over a 2-hour test period. This suggests that the separation of rPCE from other factors was not effective in stabilizing the solution.

[0232] XVIII. Example 12 - Application of Recombinant Factors to Cartridge Technology

[0248] The endotoxin detection cartridge was prepared by applying three solutions to different stations within the cartridge channel. The solution in the first station was 0.1% polyvinyl alcohol and 1% mannitol. The solution in the second station contained 8.9% rFC, 4.2% rFB, 7.4% rPCE, 10 mM MgSO4, 4.5% NaCl, 1.4% dextran, and 224 mM HEPES buffer pH 7.4. The solution applied to the third station was 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). The horseshoe crab (Limulus polyphemus) factor was prepared according to Example 1, including TFF purification and concentration.

[0233]

[0249] Cartridges similar to those shown in Figures 4C and 4D were manufactured by supplying a first solution to a first station 14, a second solution to a second station 16, and a third solution to a third station 18 onto the surface of the conduit 8' of the lower half of the cartridge. The solutions were dried on the conduit. The upper half 3 of the cartridge was bonded to the cartridge bottom 2 to manufacture a complete cartridge 1, and the resulting cartridge housing defines the fluid inlet port 4 and the optical cell 6. Further details regarding the manufacture of the cartridge can be found in U.S. Patent No. 7,329,538.

[0234]

[0250] The resulting cartridges were sampled from each manufacturing tray, with lower tray numbers corresponding to earlier manufacturing times and higher tray numbers corresponding to later manufacturing times. Water for injection (negative control) was assayed on the sampled cartridges. Figure 14 shows the onset times for each tray. Activation was observed at the start of manufacturing (trays 1-5). This indicates that factor solutions containing rFC, rFB, and rPCE in a mixture are not suitable for cartridge manufacturing.

[0235] XIX. Example 13 - Application of rFC Isolation to Cartridge Technology

[0251] rFC separation was applied to cartridge technology. Cartridges were prepared in the same manner as in Example 12, except that the solution for the first station was 0.1% polyvinyl alcohol and 1% mannitol. The solution for the second station contained 1.7% rFB, 5.7% rPCE, 50 mM MgCl2, and 82.1% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The solution for the third station contained 48% rFC and 3.3 mM substrate (S2834, Ac-Ile-Glu-Gly-Lys-pNA). rFC, rFB, and rPCE were prepared from cell culture supernatant as described in Example 1.

[0236]

[0252] The resulting cartridges were sampled from each tray. Endotoxin dilutions (0.05, 0.5, and 5 EU / mL) were measured in the sampled cartridges. Figure 15 shows the onset time for each tray. The onset time was stable, suggesting that the factor solution was stable during production. Therefore, the separation of rFC from rFB and rPCE was effective in producing stable cartridges.

[0237] XX. Example 14 - Application of rFB isolation to cartridge technology

[0253] rFB separation was applied to cartridge technology. The cartridge was prepared in the same manner as in Example 12, except that the solution for the first station contained 3.3 mM substrate (S2423, Ac-Ile-Glu-Gly-Arg-pNA), 0.1% polyvinyl alcohol, and 1% mannitol. The solution for the second station contained 6.0% rFC, 5.8% rPCE, 63 mM MgSO4, and 50.0% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The solution for the third station contained 1.4% rFB, 63 mM MgSO4, and 50.0% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). Recombinant horseshoe crab (Limulus polyphemus) factors were prepared according to Example 1, including TFF purification and concentration.

[0238]

[0254] The resulting cartridges were sampled from each tray. Endotoxin dilutions (0.05, 0.5, and 5 EU / mL) were measured in the sampled cartridges. Figure 16 shows the onset time for each tray. The onset time was stable, suggesting that the factor solution was stable during production. Therefore, the separation of rFB from rFC and rPCE was effective in producing stable cartridges.

[0239] XXI. Overview of Examples 1-14

[0255] Table 1 summarizes the results regarding the stability of the factor solutions (Examples 2-11). When the substrate was separated from the factor, stable factor solutions were obtained even at 25°C by separating rFC or rFB (Examples 8-10). When the substrate was contained in the factor solution (i.e., rFC or rFB), low temperatures (below 10°C) were required to obtain a stable solution containing the formulation from which rFC or rFB had been separated. When the solution contained all three factors (i.e., rFC, rFB, and rPCE) or rFC and rFB together, the factor solution was unstable. These results clearly suggest that the separation of rFC from rFB can provide a stable solution. When the substrate is separated from the factor, the temperature restriction is not significant. When the substrate is in the solution together with the factor, low temperatures are desirable to obtain solution stability.

[0240] [Table 1]

[0241]

[0256] Applying these observations to the preparation of endotoxin detection cartridges demonstrated the stabilizing effect of separating rFCs from rFBs when preparing the initial factor solution. Table 2 shows the results of cartridge preparation (Examples 12-14). The results indicate that when preparing endotoxin detection cartridges containing recombinant factors, it is preferable for rFCs and rFBs to be separated from each other.

[0242] [Table 2]

[0243]

[0257] In summary, the data from these examples demonstrate that rFC, rFB, and rPCE should be mixed together to form the cascade assay reagent immediately before performing the endotoxin detection assay, and should not be combined together on the cartridge during preparation. Rather, it is possible to mix rFC and rFB during the assay on the cartridge. However, a stable cascade assay reagent was achieved when rFC was separated from rFB.

[0244]

[0258] For example, when designing an endotoxin detection cartridge, it is beneficial to separate rFCs from rFBs at different locations on the cartridge to prepare a stable assay reagent. rPCEs may be mixed with other factors, such as rFBs or rFCs, and factors not mixed with rPCEs must be placed separately on the cartridge. rPCEs may also be placed on the cartridge separately from rFBs and rFCs. If necessary, separating substrates from recombinant factors during cartridge manufacturing can provide further temperature-independent stability of the solution for cartridge production or use in endotoxin detection assays.

[0245] XXII. Example 15 - Application of rFB separation to cartridge technology

[0259] rFB separation was applied to cartridge technology. The cartridges were prepared in the same manner as in Example 14, except that the solutions in the second and third stations were replaced. Thus, the first solution for the first station contained 3.3 mM substrate (S2423, Ac-Ile-Glu-Gly-Arg-pNA), 0.1% polyvinyl alcohol, and 1% mannitol. The second solution for the second station contained 1.4% rFB, 63 mM MgSO4, and 50% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). The third solution for the third station contained 6.0% rFC, 5.8% rPCE, 63 mM MgSO4, and 50% 250 mM HEPES buffer pH 7.4 (containing 1.6% dextran and 5% NaCl). Recombinant factors were prepared according to Example 1, including TFF purification and concentration.

[0246]

[0260] The obtained cartridges were sampled from each tray. The endotoxin diluent (0.01 EU / mL) was measured with the sampled cartridges. Figure 17 shows the onset time of each tray. The onset time was stable across the entire manufacturing tray, demonstrating that the factor solution was stable during manufacturing. Therefore, the separation of rFB from rFC and rPCE was effective for manufacturing stable cartridges.

[0247] Incorporation by reference

[0261] The entire disclosure of each patent and scientific document referred to herein is incorporated by reference for all purposes.

[0248] Equivalents

[0262] The present invention can be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered illustrative rather than limiting of the invention described herein in every respect. Accordingly, the scope of the present invention is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning and range of equivalents of the claims are intended to be embraced therein.

[0249] Sequence listing SEQ ID NO: 1

Chem.

Chem.

Chem.

Chem.

Claims

1. A cartridge for bacterial endotoxin testing of test samples, (a) Housing defining the inlet area of ​​the test sample; (b) A first composition comprising a first recombinant factor disposed on a first region of the cartridge; (c) A second composition comprising a second recombinant factor disposed on the second region of the cartridge. Includes, The second region is separated from the first region. The first region is in fluid communication with the test sample inlet region, and the first and second regions are in fluid communication with each other, enabling the mixing of the first and second compositions in the presence of the test sample supplied onto the test sample inlet region. A cartridge in which the first composition contains recombinant factor B or recombinant factor C, and the second composition contains recombinant factor B or recombinant factor C, but neither the first composition nor the second composition contains recombinant factor B or recombinant factor C.

2. The cartridge according to claim 1, further comprising an optical cell having fluid communication with the test sample inlet region, the first region, and / or the second region.

3. A cartridge for bacterial endotoxin testing, (a) A housing defining a fluid inlet port, an optical cell, and a conduit having a fluid contact surface that provides fluid communication between the fluid inlet port and the optical cell; (b) A first composition disposed on a first region of the fluid contact surface of the conduit; and (c) A second composition disposed on the second region of the fluid contact surface of the conduit. Includes, When a liquid sample is applied to the fluid inlet port, the first region is spaced apart from the second region so that the sample passes through the first region and the second region during transport to the optical cell and dissolves the first and second compositions. The first and second compositions are selected from the group consisting of recombinant factor B and recombinant factor C, however, the first composition is not the same as the second composition, in a cartridge.

4. The cartridge according to any one of claims 1 to 3, wherein factor C and / or factor B remain substantially inactive until they come into contact with microbial endotoxins in a liquid sample introduced into the cartridge via a test sample inlet region or a fluid inlet port.

5. The cartridge according to any one of claims 1 to 4, wherein the first composition further comprises a recombinant procoagulant.

6. The cartridge according to any one of claims 1 to 4, wherein the second composition further comprises recombinant procoagulant.

7. The cartridge according to any one of claims 1 to 4, further comprising a third composition comprising a recombinant procoagulant disposed spaced apart from the first and second regions on the third region of the cartridge or on the fluid contact surface of the conduit, wherein the third region is in fluid communication with the first and / or second regions.

8. The cartridge according to claim 7, wherein the third composition further comprises a color-developing substrate.

9. The cartridge according to claim 7, further comprising a fourth composition comprising a chromogenic substrate disposed on a fourth region of the cartridge or on the fluid contact surface of the conduit, spaced apart from the first, second, and third regions, wherein the fourth region is in fluid communication with the first, second, and / or third regions.

10. The cartridge according to any one of claims 1 to 6, further comprising a third composition comprising a chromogenic substrate disposed spaced apart from the first and second regions on a third region of the cartridge or on the fluid contact surface of the conduit, wherein the third region is in fluid communication with the first and / or second regions.

11. The cartridge according to claim 10, further comprising a fourth composition comprising a recombinant procoagulant disposed on a fourth region of the cartridge or on the fluid contact surface of the conduit, spaced apart from the first, second, and third regions, wherein the fourth region is in fluid communication with the first, second, and / or third regions.

12. The cartridge according to any one of claims 1 to 11, wherein the first and second compositions are dry compositions.

13. The cartridge according to any one of claims 1 to 12, wherein the first composition on the first region contains recombinant factor C and does not contain recombinant factor B, and the second composition on the second region contains recombinant factor B and does not contain recombinant factor C.

14. The cartridge according to any one of claims 1 to 12, wherein the first composition on the first region contains recombinant factor B and does not contain recombinant factor C, and the second composition on the second region contains recombinant factor C and does not contain recombinant factor B.

15. The cartridge according to claim 10, wherein the third region or fluid contact surface of the cartridge is located between the sample inlet region or fluid inlet port and the first region, and the second region is located on the cartridge or fluid contact surface between the first region and the optical cell.

16. The cartridge according to claim 15, wherein the first composition on the first region contains recombinant factor B and does not contain recombinant factor C, and the second composition on the second region contains recombinant factor C and does not contain recombinant factor B and recombinant procoagulant.

17. The cartridge according to claim 15, wherein the first composition on the first region contains recombinant factor C but does not contain recombinant factor B and recombinant procoagulant, and the second composition on the second region contains recombinant factor B but does not contain recombinant factor C.

18. A cartridge for detecting bacterial endotoxins in a sample, (a) A housing defining a fluid inlet port, an optical cell, and a conduit having a fluid contact surface that provides fluid communication between the fluid inlet port and the optical cell; (b) A color-developing substrate placed on the first region of the fluid contact surface of the conduit; (c) A first recombinant amebosite factor disposed on a second region of the fluid contact surface of the conduit, and (d) comprising a second recombinant amebosite factor positioned on a third region of the fluid contact surface; The second region is located downstream of the first region in the direction of fluid flow along the conduit, and the third region is located downstream of the second region and the first region in the direction of fluid flow along the conduit. (i) The first recombinant amebosite factor comprises recombinant factor B, the second recombinant amebosite factor comprises recombinant factor C, and recombinant procoagulant enzyme is located on the third region together with recombinant factor C; or (ii) The cartridge comprises the first recombinant amebosite factor comprises recombinant factor C, the second recombinant amebosite factor comprises recombinant factor B, and recombinant procoagulant enzyme is located on the second region together with recombinant factor C.

19. The cartridge according to claim 18, wherein the first recombinant amebosite factor comprises recombinant factor B, the second recombinant amebosite factor comprises recombinant factor C, and recombinant procoagulant enzyme is arranged on the third region together with recombinant factor C.

20. The cartridge according to claim 18, wherein the first recombinant amebosite factor comprises recombinant factor C, the second recombinant amebosite factor comprises recombinant factor B, and recombinant procoagulant enzyme is arranged on the second region together with recombinant factor C.

21. The cartridge according to any one of claims 8 to 20, wherein the substrate is Ac-Ile-Glu-Gly-Arg-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

22. The cartridge according to any one of claims 8 to 20, wherein the substrate is Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

23. A method for producing a bacterial endotoxin test composition, (a) To provide a first composition that contains recombinant factor C but does not contain factor B; (b) To provide a second composition containing recombinant factor B but not factor C; and (c) Mixing the first composition with the second composition in the presence of recombinant procoagulant to form a third composition, (i) The first and second compositions remain separate until they are mixed together immediately before contact with the test sample; or (ii) A method wherein the first and second compositions remain separated until they are mixed with the test sample.

24. The method according to claim 23, wherein the first and second compositions remain separated until they are mixed together immediately before contact with the test sample.

25. The method according to claim 23 or 24, wherein the first and second compositions are mixed together within 30 minutes prior to contact with the test sample.

26. The method according to claim 23 or 24, wherein the chromogenic substrate is mixed with the third composition immediately before contact with the test sample.

27. The method according to claim 23, wherein the first and second compositions remain separated until they are mixed with the test sample.

28. The method according to claim 23 or 27, wherein the color-developing substrate is mixed with the first and second compositions simultaneously with or after mixing with the test sample.

29. The method according to any one of claims 23 to 28, wherein the first composition further comprises a recombinant procoagulant before the mixing step (c).

30. The method according to any one of claims 23 to 29, wherein the second composition further comprises a recombinant procoagulant prior to the mixing step (c).

31. The method according to any one of claims 23 to 25 or 27 to 30, wherein the first composition further comprises a chromogenic substrate.

32. The method according to any one of claims 23 to 25, 27, 29, or 30, wherein the second composition further comprises a chromogenic substrate.

33. The method according to any one of claims 23 to 26, further comprising providing a fourth composition comprising recombinant procoagulant, wherein the first and second compositions are mixed together with the fourth composition immediately before contact with the test sample.

34. The method according to any one of claims 23 to 27, further comprising providing a fourth composition comprising recombinant procoagulant, wherein the first, second and fourth compositions are mixed together upon contact with the test sample.

35. The method according to claim 33 or 34, wherein the fourth composition further comprises a chromogenic substrate.

36. The method according to any one of claims 23 to 30 or 33 to 35, further comprising providing a composition comprising a chromogenic substrate which remains separate from the first and second compositions until it is mixed with the first and second compositions in step (c) to form the third composition.

37. The method according to any one of claims 26, 28, 31, 32, 35, or 36, wherein the chromogenic substrate is Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

38. The method according to any one of claims 23 to 37, wherein the first and second compositions are provided as a dry composition in steps (a) and (b).

39. The method according to claim 38, wherein the first and second compositions are redissolved before mixing in step (c).

40. The method according to claim 39, wherein the first and second compositions are redissolved in a buffer solution.

41. The method according to claim 39, wherein the first and second compositions are redissolved by the test sample.

42. The method according to any one of claims 23 to 37, wherein the first and second compositions are provided as buffer solutions in steps (a) and (b).

43. The method according to any one of claims 23 to 42, wherein step (c) is performed on the cartridge.

44. The method according to any one of claims 23 to 43, wherein the factor C provided in step (a) is substantially inactive in the absence of endotoxin exogenously added from the sample or control.

45. The method according to any one of claims 23 to 44, wherein the factor B provided in step (b) is substantially inactive in the absence of endotoxin exogenously added from the sample or control.

46. A method for detecting bacterial endotoxins in a test sample, (a) Contacting a bacterial endotoxin test composition prepared by any one of claims 23 to 45 with the test sample in the presence of a chromogenic substrate; and (b) A method comprising determining the presence, absence, and / or amount of bacterial endotoxin in a sample based on a chemically detectable change in the chromogenic substrate.

47. The method according to claim 46, wherein the substrate is Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

48. A method for detecting bacterial endotoxins in a test sample, (a) Applying the test sample to the test sample inlet region or the fluid inlet port of the cartridge according to any one of claims 1 to 22; (b) Contacting the first and second compositions with the test sample in the presence of a chromogenic substrate; and (c) A method comprising determining the presence, absence, and / or amount of bacterial endotoxin in a test sample based on a chemically detectable change in the chromogenic substrate.

49. The method according to claim 48, wherein the chromogenic substrate is Ac-Ile-Glu-Gly-Arg-pNA or Ac-Ile-Glu-Gly-Lys-pNA, where Ac is an acetyl group and pNA is a para-nitroaniline group.

50. A kit for determining the presence and / or amount of bacterial endotoxins in a test sample, (a) A first composition containing recombinant factor C, but not containing recombinant factor B; (b) A second composition comprising recombinant factor B, which does not contain recombinant factor C, wherein the first and second compositions are physically separated; and (c) A kit comprising means for mixing the first and second compositions together in the presence of recombinant procoagulant and chromogenic substrate immediately before or simultaneously with contact with a test sample.

51. The kit according to claim 50, wherein the first composition and the second composition are dry compositions, and the kit further comprises a buffer for redissolving each of the first and second compositions.

52. The cartridge according to any one of claims 1 to 22, the method according to any one of claims 23 to 29, or the kit according to claim 50 or 51, wherein the recombinant factor C lacks an (α-2,3) terminal sialic acid.

53. The aforementioned recombinant factor C is GnTI － A cartridge according to any one of claims 1 to 22 or 52, expressed in a HEK cell line, a method according to any one of claims 23 to 29 or 52, or a kit according to any one of claims 50 to 52.

54. A cartridge according to any one of claims 1 to 22, 52, or 53, wherein the recombinant factor B and / or the recombinant factor C is recombinant horseshoe crab (Limulus polyphemus) recombinant factor B and / or recombinant horseshoe crab (Limulus polyphemus) recombinant factor C, a method according to any one of claims 23 to 29, 52, or 53, or a kit according to any one of claims 50 to 53.

55. A cartridge according to any one of claims 1 to 22 or 52 to 54, wherein the recombinant factor C comprises the amino acid sequence of SEQ ID NO: 1, a method according to any one of claims 23 to 49 or 52 to 54, or a kit according to any one of claims 50 to 54.

56. A cartridge according to any one of claims 1 to 22 or 52 to 55, wherein the recombinant factor B comprises the amino acid sequence of SEQ ID NO: 3; a method according to any one of claims 23 to 49 or 52 to 55; or a kit according to any one of claims 50 to 55.

57. A cartridge according to any one of claims 5 to 17, 19 to 22, or 52 to 56, wherein the recombinant procoagulant is recombinant horseshoe crab (Limulus polyphemus) procoagulant, a method according to any one of claims 20 to 46, or 52 to 56, or a kit according to any one of claims 50 to 56.

58. A cartridge according to any one of claims 5 to 17, 19 to 22, or 52 to 57, wherein the recombinant procoagulant enzyme comprises the amino acid sequence of SEQ ID NO: 5; a method according to any one of claims 20 to 46, or 52 to 57; or a kit according to any one of claims 50 to 57.

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