HK40007693B - Coagulogen-free clarified limulus amebocyte lysate - Google Patents

Description

Clear limulus amebocyte lysate without coagulogen

Technical Field

The present invention relates to compositions comprising clarified Limulus Amoebocyte Lysate (LAL), wherein the LAL is substantially free of coagulogen, and methods of making such compositions. The present invention also relates to a method for detecting endotoxin in a sample using a chromogenic assay, the method comprising: (a) Contacting the sample with a reagent comprising clarified LAL and a chromogenic substrate; and (b) measuring the chromogenic effect caused by a change in the chromogenic substrate in the presence of endotoxin in the sample, wherein the LAL is substantially free of coagulogen. The invention also relates to kits comprising clarified LAL substantially free of coagulogen, and methods of making the same.

Background

Gram-negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. Endotoxins, also called Lipopolysaccharides (LPS), are present in the outer membrane of gram-negative bacteria, such as Salmonella (Salmonella), escherichia coli (Escherichia coli), shigella (Shigella) and neisseria (Neisseira). Endotoxin-induced toxicity mechanisms are reported to be caused by the lipid fraction of lipopolysaccharides. For example, when bacteria in an organism undergo lysis, a response to lipids entering the bloodstream may occur through activation of the complement system. The lipid fraction leads to the release of different cytokines, such as interleukins 1 and 8. Tumor necrosis factor production can also be activated. The resulting infection is associated with an inflammatory process and may constitute a significant risk to the infecting organism. Interleukin 1 is a family of cytokines that organisms release as an immune response and against inflammation. This reaction causes neutrophils to migrate to the site where the infection occurred, creating chemotaxis. This facilitates the onset of phagocytosis; however, in some cases, depending on the status of the individual's immune system and the extent of infection, endotoxin may contribute to systemic sepsis, as well as the risk associated with sepsis. It has been reported that in many cases gram-negative bacteria cause multiple organ failure and even systemic infection leading to death in higher mammals. Early and sensitive detection of endotoxin is critical to the pharmaceutical industry and the medical community because of the adverse effects associated with endotoxin.

The Limulus Amebocyte Lysate (LAL) assay was introduced commercially in the 70's of the 20 th century for the detection of endotoxins. LAL is derived from blood cells of Limulus polyphemus (Limulus polyphemus) or amoeba-like cells. The initial LAL test constitutes a cascade of serine proteases, triggered by trace levels of endotoxin, reaching the gel clot at the end of the reaction. Factor C, usually present as a zymogen, is a primer for this coagulation cascade. In vivo, factor C is a perfect biosensor that alerts horseshoe crabs to the presence of a gram-negative invader. The hemostatic endpoint traps the invader, kills it and limits further infection.

The LAL test can be modified to measure the response of amoebic-like cells to endotoxin using different methods. These methods include the so-called gel-clot method, the turbidimetric method and the chromogenic method. These LAL tests are recommended in the international pharmacopoeia as a method for detecting bacterial toxins in raw materials for the production of pharmaceuticals and end products. These tests are also useful for the cosmetic industry and food production, as it is the FDA recommended method for detecting pyrogens.

Summary of The Invention

Provided herein are compositions comprising a clarified Limulus Amoebocyte Lysate (LAL), wherein the composition is substantially free of coagulogen. In embodiments, the composition comprising clarified LAL further comprises a buffer and a detergent. In an embodiment, the composition comprises 50% clarified LAL.

In an embodiment, the present invention provides a composition comprising (a) a clarified LAL, wherein the composition is substantially free of coagulogen, and (b) a chromogenic substrate.

Embodiments herein also relate to a composition comprising clarified LAL, a buffer, a detergent, and a chromogenic substrate, wherein said composition comprises 30% to 50% LAL (v/v) and 10% to 30% (v/v) chromogenic substrate.

In an embodiment, the present invention also provides a composition comprising clarified LAL, wherein the composition is substantially free of coagulogen, wherein the composition is prepared by a process comprising the steps of: (a) Centrifuging a solution of lysed amoebocytes from Limulus polyphemus (Limulus polyphemus) at 2000rpm for 8 minutes at 4 ℃ to produce a supernatant; (b) combining the supernatant from (a) with a buffer; (c) Tangential flow filtration of the pool from (b) using a 30kDa membrane filter to produce a retentate; (d) Centrifuging the retentate from (c) at 5,000rpm for 5 minutes at 4 ℃ to produce a supernatant, wherein the supernatant is clarified LAL, which is substantially free of coagulogen.

In an embodiment, the present invention also provides a composition comprising clarified LAL, wherein the composition is substantially free of coagulogen, wherein the composition is prepared by a process comprising the steps of: (a) Obtaining a solution of lysed amoeboid cells from a horseshoe crab (Limulus polyphemus); combining the solution of (a) with a buffer; (b) combining the solution from (a) with a buffer; (c) Performing continuous tangential flow filtration on the pool from (b) using a 20kDa to 50kDa membrane filter to produce a retentate; and (d) centrifuging the retentate from (c) at greater than 20,000Xg for more than 25 minutes to produce a supernatant, wherein the supernatant is clarified LAL, which is substantially free of coagulogen. In some embodiments, the continuous TFF of (c) comprises at least 4 diafiltration volumes. In some embodiments, the continuous TFF of (c) comprises at least 6 diafiltration volumes.

Embodiments herein relate to a method of detecting endotoxin in a sample using a chromogenic assay, the method comprising: (a) Contacting the sample with a reagent comprising a clarified Limulus Amebocyte Lysate (LAL) and a chromogenic substrate; and (b) measuring a chromogenic effect caused by a change in a chromogenic substrate in the presence of endotoxin in the sample, wherein the LAL is substantially free of coagulogen.

In some embodiments, the chromogenic substrate is p-nitroaniline covalently bonded to more than three amino acids. In some embodiments, the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the chromogenic substrate is altered as a result of an enzymatic reaction. In some embodiments, the enzymatic reaction is the cleavage of a chromophore from a polypeptide. In some embodiments, the chromogenic effect is measured by detecting absorbance at 380nm to 420 nm. In some embodiments, the chromogenic effect is measured by detecting absorbance at 405 nm.

In some embodiments, the reagent is a liquid. In some embodiments, the reagent is an aqueous liquid. In some embodiments, the reagents are lyophilized and then reconstituted in an aqueous liquid prior to contact with the sample.

In some embodiments, the LAL is lyophilized and then reconstituted prior to contact with the sample. In some embodiments, the LAL is frozen and then thawed prior to contact with the sample. In some embodiments, the chromogenic substrate is lyophilized and then reconstituted prior to contact with the sample.

In some embodiments, the sample is a biological sample. In some embodiments, the sample is selected from: parenteral dosage forms, vaccines, antibiotics, therapeutic proteins, therapeutic nucleic acids, therapeutic antibodies, and biological products. In some embodiments, the clarified LAL substantially free of coagulogen has less than 5% by weight coagulogen relative to total protein in the LAL as measured by SDS-PAGE and confirmed by Western blot. In some embodiments, the clarified LAL substantially free of coagulogen has less than 2% by weight coagulogen relative to the total protein in the LAL. In some embodiments, the clarified LAL substantially free of coagulogen has less than 0.5% by weight coagulogen relative to the total protein in the LAL.

In some embodiments, the clarified LAL that is substantially free of coagulogen has a coagulogen concentration of less than 5 μ g/μ L. In some embodiments, the clarified LAL that is substantially free of coagulogen has a coagulogen concentration of less than 3 μ g/μ L. In some embodiments, the clarified LAL that is substantially free of coagulogen has a coagulogen concentration of less than 2 μ g/μ L. In some embodiments, a single cuvette spectroscopy, multiple cuvette spectroscopy or a microplate reader is used for the chromogenic assay.

In some embodiments, the method further comprises comparing the chromogenic effect to a standard to determine the amount of endotoxin in the sample.

In some embodiments, the present disclosure relates to a method of detecting endotoxin in a biological sample using a chromogenic assay, the method comprising: (a) Contacting the biological sample with an aqueous reagent comprising clarified Limulus Amebocyte Lysate (LAL) and Ac-Ile-Glu-Ala-Arg-pNA; (b) Measuring a change in absorbance at 405nm caused by enzymatic cleavage of pNA from Ac-Ile-Glu-Ala-Arg-pNA in the presence of endotoxin in the sample; wherein the LAL is substantially free of coagulogen.

In some embodiments, the methods of the present disclosure have increased sensitivity. In some embodiments, the method has a sensitivity of < 0.001EU/mL endotoxin.

In some embodiments, the methods of the present disclosure relate to a kit comprising: (a) A clarified Limulus Amebocyte Lysate (LAL), wherein the LAL is substantially free of coagulogen; (b) a chromogenic substrate; and (c) instructions for detecting endotoxin using LAL and a chromogenic substrate.

In some embodiments, the clarified LAL is lyophilized. In some embodiments, the clarified LAL is in an aqueous solution. In some embodiments, the LAL and the chromogenic substrate are in a single container. In some embodiments, the kit further comprises a sterile container containing the clarified LAL. In some embodiments, the sterile container is a sterile vial. In some embodiments, the kit further comprises a control standard endotoxin.

In some embodiments, the present disclosure relates to a method of preparing a clarified Limulus Amoebocyte Lysate (LAL) substantially free of coagulogen, the method comprising: centrifuging a solution from lysed amoeba cells of Limulus polyphemus (Limulus polyphemus) at 1000 to 3000rpm for 2 to 15 minutes at 2 to 10 ℃ to produce a supernatant; combining the supernatant from (a) with a buffer; filtering the pool from (b) using a 20kDa to 50kDa filter to produce a retentate; centrifuging the retentate from (c) at 3000 to 7000rpm for 2 to 10 minutes at 2 to 10 ℃ to produce a supernatant, wherein the supernatant comprises clarified LAL, which is substantially free of coagulogen.

In some embodiments, the filter is Tangential Flow Filtration (TFF). In some embodiments, the TFF filter is a modified polyethersulfone (mPES) membrane filter. In some embodiments, TFF is performed at a flow rate of 350 mL/min to 500 mL/min. In some embodiments, the buffer is a Tris buffer or a MES buffer. In some embodiments, the pH of the buffer is about 7.0 to 8.0.

In some embodiments, the present disclosure relates to a method of preparing a clarified Limulus Amoebocyte Lysate (LAL) substantially free of coagulogen, the method comprising:

(a) Obtaining a solution from lysed amoeba-like cells of horseshoe crab (Limulus polyphemus);

(b) Combining the solution from (a) with a buffer;

(c) Performing continuous Tangential Flow Filtration (TFF) on the pool from (b) using a 20kDa to 50kDa membrane filter to produce a retentate; and

(d) Centrifuging the retentate from (c) at greater than 20,000Xg for more than 25 minutes to produce a supernatant,

wherein the supernatant is clarified LAL, which is substantially free of coagulogen. In some embodiments, the solution in (a) comprises a supernatant obtained by centrifugation of lysed amoeba cells from horseshoe crab (Limulus polyphemus) at 1000 to 3000rpm for 2 to 15 minutes at 2 to 10 ℃. In some embodiments, the centrifugation in (a) comprises centrifugation at 2000 rpm. In some embodiments, the centrifugation in (a) comprises centrifugation for 8 minutes.

In some embodiments, centrifugation comprises centrifugation at 2 to 10 ℃, e.g., 4 ℃. In some embodiments, the continuous TFF of (c) comprises at least 4 diafiltration volumes. In some embodiments, the continuous TFF of (c) comprises at least 6 diafiltration volumes. In some embodiments, the centrifuging of (d) comprises centrifuging at 40,000xg. In some embodiments, the centrifuging of (d) comprises centrifuging for 30 minutes. In some embodiments, the buffer is a Tris buffer or a MES buffer. In some embodiments, the pH of the buffer is about 7.0 to 8.0.

Drawings

The foregoing and other features and aspects of the present technology may be better understood based on the following description of the embodiments and as illustrated in the accompanying drawings. The accompanying drawings, which are incorporated herein and form a part of the specification, further serve to explain the principles of the present technology.

FIG. 1: the response curves for the formulations containing clarified LAL ("LAL-co") substantially free of coagulogen (samples 1-6) and the formulations containing unclarified LAL-co (samples 7-12) were in the form of 50/50/50 formulations, or in the form of 50/80/20 formulations. See example 1.

FIG. 2: visual comparison of LAL substantially free of coagulogen ("LAL-co") with an embodiment of clarified LAL-co prepared by centrifuging LAL-co at 5000rpm for 5 minutes at 4 ℃.

FIG. 3: schematic of an embodiment of a method of making clarified LAL substantially free of coagulogen. A plurality of batches of lysed amebocytes from american horseshoe crabs (Limulus polyphemus) are pooled together ("LAL daywood") and centrifuged, and the resulting supernatant is filtered using tangential flow filtration to produce a retentate containing LAL substantially free of coagulogen, which is then centrifuged to produce a supernatant, clarifying the LAL substantially free of coagulogen.

FIG. 4 is a schematic view of: one embodiment of a method for detecting endotoxin using a chromogenic assay exhibits improved assay performance, such as a smooth reaction curve and large separation from a 0EU/mL control.

FIG. 5: the optical density of clarified, substantially coagulogen-free LAL in a formulation containing 40% (v/v) clarified, coagulogen-free LAL and 20% chromogenic substrate (50/80/20 formulation) was varied over time for 1 year, 2 years and 3 years LAL sources.

FIG. 6: a reaction parameter table for clarified, substantially coagulogen-free LAL in a formulation containing 40% (v/v) clarified coagulogen-free LAL and 20% chromogenic substrate (50/80/20 formulation) for 1 year, 2 years and 3 years LAL sources.

FIG. 7 is a schematic view of: the bar graph shows the time (seconds) for the 50/80/20 formulation to reach 50mOD,30mOD and 20mOD in the chromogenic assay of the invention versus the age of the clarified LAL-co source (1 year, 2 years or 3 years) using either the 0.005EU/mL standard (right panel) or the blank standard (left panel).

FIG. 8: clear LAL substantially free of coagulogen in a formulation containing 40% (v/v) clear coagulogen-free LAL and 20% chromogenic substrate (50/80/20 formulation) had a reaction time to 0.005EU/mL standard for 1 year, 2 years and 3 years LAL sources. Block diagram of reaction time of a preparation containing 40% (v/v) LAL and 20% chromogenic substrate (50/80/20 preparation) for LAL derived from LAL at 1 year, 2 years and 3 years for a standard of 0.005EU/mL.

FIG. 9: in a formulation containing 40% (v/v) of clarified coagulogen-free LAL and 20% of chromogenic substrate (50/80/20 formulation), the optical density of clarified LAL, which is substantially free of coagulogen, changes with time. In a formulation containing 40% (v/v) LAL and 20% chromogenic substrate (50/80/20 formulation), the optical density of the LAL varies with time. The table compares the reaction time for the 0.005EU/mL standard and the interval between the reaction times for the 0.005EU/mL and 0EU/mL blank.

FIG. 10: comparison of reaction times for 0EU/mL blank or 0.005EU/mL retentate after continuous tangential flow filtration of each Diafiltration Volume (DV) using a 30kDa filter. The target reaction time for the 0.005EU/mL retentate was 1200 seconds, indicated by the dashed line. The numbers below each set of bars represent the absolute difference ("interval (seconds)") and the percent difference ("interval") between the reaction time of the blank and the reaction time of 0.005EU/mL. A positive difference indicates that the blank reaction is slower than 0.005EU/mL. A negative difference indicates that the blank reaction is faster than 0.005EU/mL.

FIG. 11: SDS-PAGE gels of retentate from continuous tangential flow filtration after each diafiltration volume. The band of about 20kDa represents coagulogen.

FIG. 12: quantification of the protein band at 20kDa in the SDS-PAGE gel depicted in FIG. 11. The (left) table shows the total protein concentration (μ g/. Mu.L) of each retentate, as well as the percentage of the band of 20kDa in the whole lane of the gel, calculated for each retentate obtained after the indicated number of diafiltration volumes.

FIG. 13: estimated coagulogen concentration (μ g/μ L) after continuous tangential flow filtration of each diafiltration volume.

FIG. 14 is a schematic view of: table showing known proteins and peptides present in LAL. Highlighted proteins and peptides have molecular weights of less than 30kDa and can be removed with a 30kDa molecular weight cut-off filter. Reproduced from Iwanga and Kawabata, frontiers in Bioscience 3: d973-984 (1998).

FIG. 15 is a schematic view of: comparison of the retentate from continuous tangential flow filtration using a 30kDa filter (left) and a 10kDa filter (right) is depicted by SDS-PAGE gels. The table below each gel image represents the percentage of coagulogen removal estimated by the intensity of the bands in the gel.

FIG. 16: comparison of assay reaction times using unfiltered LAL,30kDa retentate and 10kDa retentate prepared from the original formulation and assay form (left) and the modified formulation and assay form (right). The line shows the difference in reaction time for the blank (open) compared to 0.005EU/mL (solid).

FIG. 17: comparison of the effects of centrifugation speed on the difference in reaction time between blank and 0.005EU/mL. The numbers above each bar indicate the reaction time.

FIG. 18A: comparison of the optical density of the unclarified 30kDa retentate with the clarified 30kDa retentate by centrifugation at 40,000Xg, 30,000Xg and 20,000Xg, determined by absorbance at 300-500 nm. FIG. 18B: the difference in reaction time between blank and 0.005EU/mL ("% interval") was correlated with centrifugation rate, as shown in FIG. 17.

FIG. 19: comparison of reaction time of 0.005EU/mL or blank of retentate frozen and then thawed before assay or not frozen retentate (left). The target reaction time for the 0.005EU/mL retentate was 1200 seconds, indicated by the dashed line. The numbers below each set of bars represent the absolute difference ("interval(s)") and the percent difference ("% interval") between the blank of the retentate and the reaction time of 0.005EU/mL. A positive difference indicates that the blank reaction is slower than 0.005EU/mL. A negative difference indicates that the blank reaction is faster than 0.005EU/mL.

Detailed Description

It should be understood that the embodiments shown and described herein are examples and are not intended to limit the scope of the present application in any way.

The published patents, patent applications, websites, company names and scientific literature referred to herein are incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Also, any conflict between a definition of a word or phrase, as understood in the art, and a definition of the word or phrase as specifically taught in the present specification shall be resolved in favor of the latter.

As used in this specification, the singular forms "a", "an" and "the" include specifically also the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term "about" as used herein means approximately, within a certain range, approximately, or around. When the term "about" is used in conjunction with a numerical range, it modifies that numerical range by extending the boundaries above and below the numerical values set forth. Generally, as used herein, the term "about" modifies an enumerated value by an amount that varies by 20%, either upwardly or downwardly.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Reference is made herein to various methods and materials known to those skilled in the art. The disclosures of any documents cited herein are incorporated by reference in their entirety.

In some embodiments, the invention relates to a method of detecting endotoxin. The term "endotoxin" generally refers to the lipopolysaccharide complex associated with the outer membrane of gram-negative bacteria. The term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin. Endotoxin refers to cell-associated lipopolysaccharide, while exotoxin refers to a toxin secreted by bacteria and is predominantly polypeptide in nature.

The biological activity of endotoxins is associated with Lipopolysaccharide (LPS). Lipopolysaccharides are part of the outer membrane of the cell wall of gram-negative bacteria. Lipopolysaccharides are always associated with gram-negative bacteria, whether or not the organism is pathogenic. Toxicity is associated with the lipid component (lipid a) and immunogenicity is associated with the polysaccharide component. The cell wall antigen (O antigen) of gram-negative bacteria is the polysaccharide component of LPS. In addition, LPS can elicit a variety of inflammatory responses in animals.

Gram-negative bacteria in animals release trace amounts of endotoxins during growth. This may result in stimulation of natural immunity. Young cultures grown in the laboratory are known to release small amounts of endotoxin in soluble form. However, in most cases, endotoxins remain associated with the cell wall until the organism disintegrates. The disintegration of bacterial organisms can result from autolysis, complement and lysozyme mediated external lysis and phagocytic digestion of bacterial cells. Bacterial endotoxins are abundant in the human intestinal tract. Elevated endotoxin concentrations are associated with a number of conditions, including some metabolic syndrome disorders. Metabolic syndrome disorders include, for example, atherosclerosis, insulin resistance, diabetes and obesity. Elevated endotoxin levels have also been associated with fatty liver and crohn's disease. Endotoxin may also leak from the gastrointestinal tract when present at high levels. Endotoxin is a potent inflammatory antigen and leakage of endotoxin can lead to systemic inflammatory responses.

Endotoxins act less efficiently and specifically than classical bacterial exotoxins because they do not exert an enzymatic action. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxins), but it has been reported that some strong oxidants such as superoxide, peroxide and hypochlorite can neutralize them. Since these are strong oxidizers, they are not particularly suitable for use in therapeutic compositions to neutralize endotoxin.

The endotoxin of the present invention may be derived from gram-negative bacteria. Exemplary gram-negative bacteria include, but are not limited to: escherichia (Escherichia spp.), shigella (Shigella spp.), salmonella (Salmonella spp.), campylobacter (Campylobacter spp.), neisseria (Neisseria spp.), haemophilus (Haemophilus spp.), aeromonas hydrophila (Aeromonas spp.), francisella (Francisella spp.), yersinia (Yersinia spp.), klebsiella (Klebsiella spp.), bordetella (boetrella spp.), legionella (Legionella spp.), corynebacterium (Citrobacter spp.), chlamydia (Chlamydia spp.), pseudomonas (Brucella spp.), pseudomonas (Helicobacter spp.), klebsiella spp.), chlamydia spp.). Gram-negative bacteria may also be those of the families Enterobacteriaceae (Enterobacteriaceae), pseudomonas (Pseudomonadaceae), neisseriaceae (Neisseria), veillonellaceae (Veillonellaceae), bacteroidaceae (Bacteroidaceae), vibrionaceae (Vibrionaceae), pasteurellaceae (Pasteurellaceae) and Clostridium (Fusobacteriaceae). In some embodiments, the endotoxin is from salmonella or escherichia coli.

As used herein, the term "endotoxin activity" refers to the fraction of gram-negative bacteria that can cause toxicity, including pyrogenicity and septic shock. The toxic effects attributed to endotoxins have been found to be associated with the glycosylated lipid a moiety of lipopolysaccharide molecules present in or derived from the outer membrane of gram-negative bacteria.

The term "lipopolysaccharide" (LPS) refers to a macromolecule consisting of lipids and polysaccharides (glycophospholipids) linked by covalent bonds. LPS consists of three parts: 1) O antigen, 2) core oligosaccharide, and 3) lipid A. The O antigen is a repeating glycan polymer linked to the core oligosaccharide and contains the outermost domain of the LPS molecule. The core oligosaccharide is directly linked to lipid a and typically contains sugars such as heptose and 3-deoxy-D-mannooctulonic acid (also known as KDO, keto-deoxinstolic acid). Lipid a is a phosphorylated glucosamine disaccharide linked to various fatty acids. The fatty acid anchors LPS in the bacterial membrane, and the remainder protrudes from the cell surface. If LPS is mutated or removed, bacterial death may result.

Endotoxin activity is present in the lipid a domain portion of LPS. When bacterial cells are lysed by the immune system, the membrane fragments containing lipid a are released into the circulation, causing fever (pyrogenicity), diarrhea and potentially fatal shock (known as endotoxic or septic shock). The toxicity of LPS is expressed by lipid a through interaction with B cells and macrophages of the mammalian immune system, a process that results in the secretion of pro-inflammatory cytokines, primarily Tumor Necrosis Factor (TNF), which may have fatal consequences for the host. Lipid a also activates human T lymphocytes (Th-1) in vitro and mouse CD4+ and CD8+ T cells in vivo, a property that allows the host immune system to produce a recall IgG antibody response specific to the variable size carbohydrate chain of LPS. On these grounds, LPS has recently been considered as a T cell-dependent antigen "in vivo". Thus, in some embodiments, the methods of the invention involve detecting lipid a.

In some embodiments, endotoxin is detected using a chromogenic assay. As used herein, a chromogenic assay measures or detects the change in absorbance in the presence of an endotoxin in a chromogenic substrate (i.e., a chromogen). In some embodiments, the change in absorbance in the chromogenic substrate is caused by an enzymatic activity. In some embodiments, the term "chromogenic substrate" refers to a substrate before and after enzymatic activity. For example, if the chromogenic substrate is a peptide-chromophore that is cleaved by an enzyme to produce a peptide and chromophore, the term "chromogenic substrate" refers to the peptide-chromophore, the cleaved peptide and the released chromophore. In some embodiments, synthetic chromogens may be used. In some embodiments, naturally occurring chromogens may be used. In some embodiments, the chromogenic substrate is a synthetic peptide. In some embodiments, the substrates are very sensitive, i.e., they can detect very low enzyme activity.

The ability of reagents comprising chromogenic substrates to detect low enzyme concentrations makes them useful, for example, in research or quality control procedures to look for the presence of certain enzyme activities associated with endotoxins. Sometimes, there is a lack of correspondence between natural (i.e., the natural substrate of the enzyme) and synthetic chromogenic substrates in a reaction to an enzyme preparation. In some embodiments, the chromogenic substrate is less selective, i.e., less reactive, with the enzyme of interest than the natural substrate.

The term "chromogenic substrate" refers to a substrate, such as a compound or polypeptide, in an assay that changes its absorption spectrum (e.g., a color change) in the presence of an endotoxin. Chromogenic substrates refer to substrates that (i) absorb and/or (ii) do not absorb at a particular wavelength. Thus, for example, in accordance with the present disclosure, a chromogenic substrate may initially not absorb at a particular wavelength (e.g., not absorb at visible wavelengths) and then begin to absorb at a particular wavelength (e.g., at visible wavelengths) in the presence of an endotoxin. Alternatively, for example, the chromogenic substrate initially absorbs at a specific wavelength (e.g., absorbs at visible wavelengths) and then does not absorb at a specific wavelength (e.g., does not absorb at visible wavelengths) in the presence of the endotoxin. In some embodiments, the chromogenic substrate may absorb at a given wavelength in the absence of endotoxin, and then at a different wavelength in the presence of endotoxin. Changes in the absorbance characteristics, i.e. the color effect, at one or more specific wavelengths can be correlated with the presence of endotoxin.

In some embodiments, the chromogenic substrate is a chromogenic peptide substrate. In some embodiments, the chromogenic peptide substrate is initially colorless. In some embodiments, the chromogenic peptide substrate initially has a color, such as in the visible spectrum (about 390-700 nm). In some embodiments, when the chromogenic peptide substrate is cleaved by an enzyme, a color change may occur, such as release of a chromophore, resulting in the resultingThe color of the product changed. In some embodiments, cleavage changes the optical properties of the product, which is different from the optical properties of the uncleaved substrate and can be measured spectrophotometrically. Non-limiting examples of chromogenic groups that can be coupled to a peptide substrate are: para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid (ANBA), 7-amino-4-methoxycoumarin (ANC), quinone amide (QUA), dimethyl 5-aminoisophthalate (DPA) and derivatives thereof. Fluorogenic substrates include, but are not limited to, Z-Gly-Pro-Arg-AMC [ Z = benzyloxycarbonyl; AMC = 7-amino-4-methylcoumarin]Homovanillic acid, 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines, reduced benzothiazines,resorufin β -D-galactopyranoside, fluorescein Digalactoside (FDG), fluorescein digluconate and structural variants thereof (U.S. Pat. nos. 5,208,148, 5,242,805, 5,362,628, 5,576,424, and 5,773,236, incorporated by reference), 4-methylumbelliferyl β -D-galactopyranoside, carboxyumbelliferyl β -D-galactopyranoside, and fluorinated coumarin β -D-galactopyranoside (U.S. Pat. nos. 5,830,912, incorporated by reference).

A non-limiting chromogenic assay is an enzyme activity assay based on the factor C/factor B cascade. Factor C is the first component in the cascade, a protease zymogen activated by endotoxin binding. In some embodiments, the chromogenic assay uses a recombinant form of factor C (rFC). In this pathway, factor B is activated by factor C. Factor B activates proclotting enzyme to clotting enzyme. In some embodiments, the pro-clotting enzyme affects a chromogenic change in a chromogenic substrate. In some embodiments, the chromogenic assay is a LAL assay, such as an end-point chromogenic LAL assay from lomsa (Lonza).

In some embodiments, the chromogenic assay is a LAL assay, wherein the initial portion of the LAL endotoxin reaction activates the proclotting enzyme, which in turn enzymatically cleaves para-nitroaniline (pNA) from a synthetic substrate to yield a yellow color.

Zymogen + endotoxin → enzyme

Substrate + H ₂ O + enzyme → peptide + pNA (yellow)

In some embodiments, gram-negative bacterial endotoxins can indirectly catalyze the activation of zymogens in LAL. The initial activation rate can be determined by the concentration of endotoxin present.

In some embodiments, the chromogenic substrate is altered as a result of an enzymatic reaction. In some embodiments, the enzymatic reaction results in cleavage of a peptide bond, thereby cleaving the chromophore substituent (e.g., p-NA) from the polypeptide. For example, the activated enzyme can catalyze the release of pNA from a colorless peptide substrate (e.g., ac-Ile-Glu-Ala-Arg-pNA). In some embodiments, the peptide substrate is p-nitroaniline covalently bonded to more than three amino acids. In some embodiments, the chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the chromogenic assay measures free pNA. In some embodiments, the chromogenic assay optically measures free pNA at an absorbance of 380nm to 410nm, for example 400nm to 410nm, or 405 nm. Methods for measuring absorbance are well known to those skilled in the art. In some embodiments, a single cuvette spectroscopy, multiple cuvette spectroscopy or microplate reader is used to perform a chromogenic assay to measure absorbance.

After termination of the reaction with the termination reagent, free pNA can be measured optically at 380nm to 410nm, e.g. 405 nm. The concentration of endotoxin in the sample was calculated from a standard curve of absorbance values of solutions containing known amounts of endotoxin standards.

One standard chromogenic assay for detecting endotoxin comprises contacting a sample with a reagent, wherein the reagent comprises Limulus Amebocyte Lysate (LAL). In some embodiments, the reagent is a liquid, such as an aqueous liquid. Alternatively, the reagents may be lyophilized and then reconstituted in an aqueous liquid, such as sterile water or buffer solution, prior to contact with the sample. In some embodiments, the reagent is a liquid. In some embodiments, the reagent is an aqueous liquid. In some embodiments, the reagents are lyophilized and then reconstituted in an aqueous liquid prior to contact with the sample. In some embodiments, the LAL is lyophilized and then reconstituted in an aqueous liquid prior to contact with the sample. In some embodiments, the chromogenic substrate is lyophilized and then reconstituted in an aqueous liquid prior to contact with the sample. In some embodiments, lyophilization allows for longer and/or more robust storage of reagents, LAL, and/or chromogenic substrates in a chromogenic assay. For example, in some embodiments, the lyophilized reagent, LAL, and/or chromogenic substrate allow for an increase in stabilization time of greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 100% relative to the non-lyophilized reagent, LAL, and/or chromogenic substrate in the chromogenic assay. "stability" as used herein refers to an assay for its intended purpose, i.e., for detecting endotoxin at the same rate and sensitivity. For example, if a non-lyophilized reagent is stable for 3 weeks, the stabilization time for a lyophilized reagent that is stable for 6 weeks will "increase 100%".

In some embodiments, the LAL is frozen and then thawed prior to contact with the sample. In some embodiments, lyophilizing the LAL allows for longer and/or more robust storage of the LAL. In some embodiments, the LAL that has been frozen and then thawed has the same or substantially the same assay performance as the LAL that has not been frozen. Herein, assay performance that is "the same or substantially the same" means that the reaction time reduction is similar when comparing the blank reaction with the reaction using LAL. In some embodiments, the difference in reduction in reaction time between the LAL that is not frozen and the LAL that has been frozen and then thawed is less than 20%. In some embodiments, the difference in reduction in reaction time is less than 10%. In some embodiments, the difference in reduction of reaction time is less than 5%. In some embodiments, the difference in reduction of reaction time is less than 1%. In some embodiments, the LAL is frozen and stored at about-20 ℃. In some embodiments, the LAL is frozen and stored at about-30 ℃, about-40 ℃, about-50 ℃, about-60 ℃, about-70 ℃, or about-80 ℃. In some embodiments, the LAL is flash-frozen. In some embodiments, LAL is flash frozen using dry ice or liquid nitrogen.

In some embodiments, the LAL is frozen for more than one week, greater than 1 month, greater than 3 months, greater than 6 months, greater than 9 months, greater than 12 months, greater than 15 months, greater than 18 months, or greater than 24 months, after which the LAL that has been frozen is thawed and has the same or substantially the same assay performance as the LAL that has not been frozen. In some embodiments, the LAL is frozen for one to five years, one to four years, three to three years, or 6 to two years, after which the frozen LAL is thawed and has the same or substantially the same assay performance as the LAL that has not been frozen.

The present disclosure provides improved methods for detecting endotoxin in a sample. The term "sample" may include any substance, compound, tool or instrument. However, for practical purposes, a sample may include a substance, compound, tool or instrument that comes into contact with a biological organism, including, for example, a mammal, a human, a domesticated animal or a zoo animal. The term "sample" may refer to any medical device, pharmaceutical and biotechnological product where the source of endotoxin (received from the raw material to the end of the manufacturing process) may render the sample unsuitable for contact with cerebrospinal fluid or the cardiovascular system. In some embodiments, the term sample refers to a medical device that is in contact with cerebrospinal fluid or cardiovascular system in vivo, such as in contact with a human. In some embodiments, the term sample refers to a biological sample. In some embodiments, the sample is selected from: parenteral dosage forms, vaccines, antibiotics, therapeutic proteins, therapeutic nucleic acids, therapeutic antibodies, and biological products.

The term "Limulus amoebocyte lysate" (LAL) refers to an aqueous extract of blood cells (amoebocyte cells) from horseshoe crab (horseshoe crab), horseshoe crab (Limulus polyphemus). An aqueous extract of blood cells from horseshoe crab (horseshoe crab) contains coagulogens, which are gel-forming proteins of hemolymph and block the spread of invaders by fixing them. See, e.g., iwanga S et al, j. Biochem.98:305-318 (1985) and Iwanaga S et al, J.biochem.95 (6): 1793-1801 (1984).

The coagulation cascade of the horseshoe crab is involved in hemostasis and host defense. This cascade results in the conversion of coagulogen (a soluble protein) to an insoluble coagulin gel. The clotting enzyme cleaves fragment peptide C from coagulogen, causing aggregation of the monomers.

The term "coagulogen" refers to the polypeptide chain found in Iwanaga (1984) and Iwanaga (1985), which is a single 175 residue polypeptide chain that is cleaved after Arg-18 and Arg-46 by the clotting enzymes contained in blood cells and activated by bacterial endotoxin (lipopolysaccharide). Cleavage releases the two chains a and B of coagulin, which are linked to peptide C by two disulfide bonds. Gel formation results from the interconnection of coagulin molecules. Secondary structure prediction indicates that the C peptide forms a d-helix, which is released during the proteolytic conversion of coagulogen to coagulin gel. The 16 cysteines found in the molecule and the beta-sheet structure appear to produce compact proteins that are stable to acid and heat.

While coagulogen is important for gel formation (e.g., coagulation assays), the present disclosure finds it unnecessary in chromogenic assays. The present disclosure finds that chromogenic assays comprising clarified LAL substantially free of coagulogen achieve increased speed, sensitivity, and spacing levels relative to chromogenic assays comprising LAL having a naturally occurring amount of coagulogen and not clarified. Thus, in some embodiments, the invention relates to a chromogenic assay comprising clarified LAL substantially free of coagulogen.

In some embodiments, the clarified LAL is substantially free of coagulogen. For convenience, in some embodiments herein, LAL substantially free of coagulogen is also referred to as "LAL-co". Upon reading this disclosure, one skilled in the art will appreciate that a reduction in various amounts of coagulogen will result in an increase in the speed, sensitivity, and/or spacing levels in a chromogenic assay (e.g., a LAL assay). In some embodiments, the term "substantially free" refers to LAL having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, or less than 0.5% by weight of coagulogen relative to the total protein in the LAL as measured by protein-stained SDS-PAGE and confirmed by Western blot. In some embodiments, the term "substantially free" refers to LAL having less than 10% by weight or less than 5% by weight coagulogen relative to total protein in the LAL as measured by SDS-PAGE and confirmed by Western blot. In some embodiments, the term "substantially free" refers to LAL having a coagulogen concentration of less than about 20 μ g/μ L, less than about 15 μ g/μ L, less than about 10 μ g/μ L, less than about 5 μ g/μ L, less than about 4 μ g/μ L, less than about 3 μ g/μ L, less than about 2 μ g/μ L, or less than about 1 μ g/μ L. In some embodiments, the term "substantially free" refers to LAL having a coagulogen concentration of 20 μ g/μ L to 0.001 μ g/μ L,15 μ g/μ L to 0.01 μ g/μ L,10 μ g/μ L to 0.1 μ g/μ L,5 μ g/μ L to 0.5 μ g/μ L,4 μ g/μ L to 0.5 μ g/μ L,3 μ g/μ L to 0.5 μ g/μ L,2 μ g/μ L to 0.5 μ g/μ L, or less than 1 μ g/μ L. In some embodiments, the term "substantially free" refers to LAL having a coagulogen concentration of 10 μ g/μ L to 1 μ g/μ L,5 μ g/μ L to 1 μ g/μ L,4 μ g/μ L to 1 μ g/μ L,3 μ g/μ L to 1 μ g/μ L,2 μ g/μ L to 1 μ g/μ L, or less than 1 μ g/μ L. The concentration of coagulogen may be determined, for example, using absorbance spectroscopy, quantification of SDS-PAGE gel bands or Western blot bands, or any other method known to measure coagulogen concentration. In some embodiments, the measured concentration of coagulogen in the "LAL substantially free of coagulogen" cannot be accurately determined because it is within the error range of the minimum detection amount using conventional detection methods.

In some embodiments, the term LAL substantially free of coagulogen refers to LAL from which at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% by weight of coagulogen has been removed relative to the amount of coagulogen in LAL from which no coagulogen has been removed.

It will be appreciated by those skilled in the art that different methods may be used to remove coagulogen from LAL. Each of these methods may vary in efficiency, purification rate, cost and effort, but are within the knowledge of one skilled in the art. The present disclosure includes methods of making clarified LAL substantially free of coagulogen using tangential flow filtration. Tangential Flow Filtration (TFF) refers to cross-flow filtration, in which the majority of the feed stream passes tangentially across the surface of the filter, rather than entering the filter. By using TFF, the retentate containing most of the LAL protein (which may contaminate the filter) is essentially washed away during filtration and the coagulogen is filtered into the permeate. In some embodiments, TFF is a continuous process, i.e., continuous tangential flow filtration or continuous TFF, as opposed to batch dead-end filtration. In some embodiments, continuous TFF involves adding a diafiltration solution, i.e., water or buffer, to the sample at the same rate that the permeate is produced, so that the sample volume remains constant while components of the freely permeable filter are washed away. In some embodiments, diafiltration is a type of tangential flow filtration. Diafiltration refers to a fractionation process that washes smaller molecules through a membrane or filter and leaves larger molecules in the retentate without ultimately changing the volume. The diafiltration volume or DV is the sample volume before addition of the diafiltration solution. In embodiments, using more diafiltration volume in tangential flow filtration results in more permeate removal.

The term "clarified limulus amoeboid cell lysate" (or "clarified LAL") substantially free of coagulogen refers to LAL substantially free of coagulogen as discussed above that has been further treated to remove components that give the LAL a cloudy appearance. In embodiments, clarified LAL is produced by centrifuging LAL substantially free of coagulogen. In some embodiments, the term "clarified LAL" refers to a LAL that has been centrifuged at greater than 1800g (i.e., 1800x gravity), greater than 2200g, greater than 2600g, greater than 3000g, greater than 3400g, greater than 3800g, greater than 4200g, greater than 4600g, greater than 5000g, greater than 5400g, greater than 5800g, greater than 6000g, greater than 6100g, or greater than 6200g for a time sufficient to significantly clarify the LAL without disruption of the enzyme. In some embodiments, the term "clarified LAL" refers to an LAL that has been centrifuged at 1800 to 8000g,2200g to 7600g,2600g to 7200g,3000g to 7200g,3400g to 7200g,3800g to 7200g,4200g to 7200g,4600g to 7200g,5000g to 7200g,5400g to 7200g,5800g to 7200g, or 6100g to 7200g for a time sufficient to significantly clarify the LAL without destroying the enzyme.

In some embodiments, the term "clarified limulus amoebocyte lysate" (or "clarified LAL") that is substantially free of coagulogen refers to a LAL substantially free of coagulogen discussed above that has been further processed to remove components that produce a cloudy appearance by centrifugation of the LAL substantially free of coagulogen at greater than 20,000x g, greater than 22,000x g, greater than 24,000x g, greater than 25,000x g, greater than 26,000x g, greater than 28,000x g, greater than 30,000x g, greater than 35,000x g, greater than 40,000x g, greater than 45,000x g, or greater than 50,000x g. In some embodiments, LAL substantially free of coagulogen is centrifuged at greater than 20,000 to 50,000x g,20,000 to 40,000x g,25,000 to 50,000x g,25,000 to 40,000x g, or 30,000 to 40,000x g. In some embodiments, the LAL substantially free of coagulogen is centrifuged for greater than 20 minutes, greater than 30 minutes, greater than 40 minutes, or greater than 60 minutes. In some embodiments, the LAL substantially free of coagulogen is centrifuged for 20 to 120 minutes, 20 to 90 minutes, 20 to 60 minutes, 20 to 40 minutes, or about 30 minutes.

In some embodiments, the term "clarified LAL" refers to LAL that has been centrifuged for greater than 3 minutes, greater than 4 minutes, greater than 5 minutes, greater than 6 minutes, greater than 7 minutes, greater than 8 minutes, greater than 9 minutes, or greater than 10 minutes. In some embodiments, the term "clarified LAL" refers to LAL that has been centrifuged for 3 to 30 minutes, 4 to 25 minutes, 4 to 20 minutes, 5 to 15 minutes, or 5 to 10 minutes. One skilled in the art will appreciate that a lower centrifugation speed may require a longer centrifugation time, and that the time and/or speed will be adjusted accordingly to reduce visual clouding of the LAL. In some embodiments, the term "clarified LAL" refers to LAL substantially free of coagulogen that is centrifuged at about 5000g to about 7000g for about 3 minutes to about 10 minutes, or at about 6120g for 5 minutes. In embodiments, clarified LAL substantially free of coagulogen is prepared by centrifuging a solution of lysed amoebocytes from a horseshoe crab (Limulus polyphemus) at 2,000rpm (980 g) for 8 minutes at 4 ℃. After centrifugation, clear LAL was found in the supernatant. In some embodiments, the resulting supernatant is then combined with a buffer; the resulting combination of supernatant and buffer was then subjected to tangential flow filtration using a 30kDa membrane filter to produce a retentate; the retentate was centrifuged at 5,000rpm (6120 g) for 5 minutes at 4 ℃ to produce a supernatant, wherein the supernatant was clarified LAL substantially free of coagulogen. In an embodiment, the solution of lysed amoebocyte cells from a horseshoe crab (Limulus polyphemus) is a collection of lysed amoebocyte cells from a plurality of horseshoe crabs (Limulus polyphemus).

In some embodiments, the clarified LAL substantially free of coagulogen is prepared by obtaining a solution of lysed amoeboid cells from horseshoe crab (Limulus polyphemus). In some embodiments, the solution is then combined with a buffer; the resulting solution and buffer pool was then subjected to continuous Tangential Flow Filtration (TFF) using a 20kDa to 50kDa membrane filter to produce a retentate; the retentate was centrifuged at greater than 20,000Xg for more than 25 minutes at 4 ℃ to produce a supernatant, wherein the supernatant was clarified LAL substantially free of coagulogen. In an embodiment, the solution of lysed amoebocyte cells from a horseshoe crab (Limulus polyphemus) is a collection of lysed amoebocyte cells from a plurality of horseshoe crabs (Limulus polyphemus). In some embodiments, the continuous TFF comprises at least 4 Diafiltration Volumes (DV). In some embodiments, the continuous TFF comprises at least 5 diafiltration volumes. In some embodiments, the continuous TFF comprises at least 6 diafiltration volumes. In some embodiments, the continuous TFF comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 diafiltration volumes.

In some embodiments, the clarified LAL substantially free of coagulogen according to the invention is produced by a method utilizing any combination of the technical features described herein. Thus, one skilled in the art can use any of the listed filters, filter sizes, filter flow rates, buffers, centrifugation speeds, centrifugation temperatures, centrifugation times, etc., sufficient to render the LAL substantially free of coagulogen. For example, in some embodiments, LAL (i) is centrifuged at greater than 20,000xg, greater than 22,000xg, greater than 24,000xg, greater than 25,000xg, greater than 26,000xg, greater than 28,000xg, greater than 30,000xg, greater than 35,000xg, greater than 40,000xg, greater than 45,000xg, or greater than 50,000xg, (ii) centrifuged at a temperature of 2 ℃ to 10 ℃,2 ℃ to 8 ℃, or 4 ℃, (iii) centrifuged for 20 to 120 minutes, 20 to 90 minutes, 20 to 60 minutes, 20 to 40 minutes, or about 30 minutes, (iv) centrifuged at a flow rate of greater than 500 mL/minute, such as 500 mL/minute to 2000 mL/minute, 800 mL/minute to 1500 mL/minute, or 1000 mL/minute to 1200 mL/minute, (v) TFF is performed using a 50kDa filter, 45kDa filter, 40kDa, 35kDa filter, 30kDa filter, 25 DV filter, or 20 DV filter, (v) is performed using at least 4, at least 5, at least 8, at least 7, at least 8, and so forth.

In some embodiments, the chromogenic assay determines the presence or absence of endotoxin in a sample. In other embodiments, the chromogenic assay may quantify the amount of endotoxin in the sample. In some embodiments, the method further comprises comparing the chromogenic effect of endotoxin in the sample to a known endotoxin standard to determine the amount of endotoxin in the sample.

In some embodiments, the present disclosure relates to a method of detecting endotoxin in a biological sample using a chromogenic assay, the method comprising: (a) Contacting the biological sample with an aqueous reagent comprising clarified Limulus Amebocyte Lysate (LAL) and Ac-Ile-Glu-Ala-Arg-pNA; (b) Measuring the change in absorbance at 405nm caused by enzymatic cleavage of pNA from Ac-Ile-Glu-Ala-Arg-pNA in the presence of endotoxin in the sample; wherein the LAL is substantially free of coagulogen.

In embodiments, the invention provides a method of detecting endotoxin in a sample using a chromogenic assay, the method comprising (a) combining a first solution comprising 30% to 60% (v/v) clarified LAL substantially free of coagulogen with a second solution comprising a chromogenic substrate to produce a third solution, wherein 75% -85% (v/v) of the third solution is the first solution and 15% -25% (v/v) of the third solution is the second solution; (b) combining the third solution with a sample containing endotoxin; and (c) measuring the change in absorbance of the chromogenic substrate. In embodiments, the invention provides a method of detecting endotoxin in a sample using a chromogenic assay, the method comprising (a) combining a first solution comprising 50% (v/v) clarified LAL substantially free of coagulogen with a second solution comprising a chromogenic substrate to produce a third solution, wherein 80% (v/v) of the third solution is the first solution and 20% (v/v) of the third solution is the second solution; (b) combining the third solution with a sample containing endotoxin; and (c) measuring the change in absorbance of the chromogenic substrate. In embodiments, the first solution further comprises a buffer and a detergent. In embodiments, the invention provides a method of detecting endotoxin in a sample using a chromogenic assay, the method comprising (a) contacting a solution comprising 35 to 45% (v/v) clarified LAL substantially free of coagulogen and 15% to 25% (v/v) chromogenic substrate with a biological sample, and (b) measuring the change in absorbance of the chromogenic substrate. In embodiments, the invention provides a method of detecting endotoxin in a sample using a chromogenic assay, the method comprising (a) contacting a solution comprising 40% (v/v) clarified LAL substantially free of coagulogen and 20% (v/v) chromogenic substrate with a biological sample, and (b) measuring the change in absorbance of the chromogenic substrate. In embodiments, the solution comprising clarified LAL substantially free of coagulogen further comprises a buffer and a detergent.

In embodiments, the chromogenic assay surprisingly has increased sensitivity by removing coagulogen from LAL and clarifying the LAL without coagulogen. In some embodiments, the method has a sensitivity of 0.0001EU/mL to 1.0EU/mL endotoxin. In embodiments, the method has a sensitivity of 0.0005EU/mL to 1.0EU/mL endotoxin, 0.008EU/mL to 1.0EU/mL endotoxin, 0.001EU/mL to 1.0EU/mL endotoxin, 0.005EU/mL to 1.0EU/mL endotoxin, 0.01EU/mL to 1.0EU/mL endotoxin, 0.02EU/mL to 1.0EU/mL endotoxin, 0.03EU/mL to 1.0EU/mL endotoxin, or 0.05EU/mL to 1.0EU/mL endotoxin. In embodiments, less than 0.05EU/mL, less than 0.03EU/mL, less than 0.01EU/mL, less than 0.008EU/mL, less than 0.006EU/mL, less than 0.005EU/mL, less than 0.004EU/mL, less than 0.003EU/mL, less than 0.002EU/mL, or less than 0.001EU/mL.

In some embodiments, clarified LAL-co produces a smoother response curve relative to unclarified LAL-co. In some embodiments, the reaction curve between separate batches is more consistent because the reaction curve is smoother. In some embodiments, the response curve of clarified LAL-co fits better to the curve relative to unclarified LAL-co.

In some embodiments, clarified LAL-co produces a smoother response curve relative to unclarified LAL-co. In some embodiments, the reaction profile between separate batches is more consistent because the reaction profile is smoother. In some embodiments, the response curve of clarified LAL-co is better fit to the curve relative to unclarified LAL-co.

In some embodiments, clarified LAL-co reaches a specified optical density more quickly than non-clarified LAL-co. In some embodiments, clarified LAL-co reaches 30mOD faster than non-clarified LAL-co. In some embodiments, the clarified LAL-co achieves 30mOD 20%,30%,40%,50%,60%,70% or 80% faster than the unclarified LAL-co. In some embodiments, clarified LAL-co reaches 50mOD faster than non-clarified LAL-co. In some embodiments, the clarified LAL-co achieves 50mOD faster than the unclarified LAL-co by 20%,30%,40%,50%,60%,70% or 80%.

In some embodiments, clarified LAL-co has a greater interval (time) between the 0EU/mL standard (blank standard) and the 0.005EU/mL standard relative to unclarified LAL-co. In some embodiments, clarified LAL-co has a 20%,30%,40%,50%,60%,70%, or 80% greater separation (time) between the 0EU/mL standard (blank standard) and the 0.005EU/mL standard relative to the unclarified LAL-co. In some embodiments, clarified LAL-co has a greater separation (time) between the 0EU/mL standard (blank standard) and the 0.0005EU/mL standard relative to unclarified LAL-co. In some embodiments, clarified LAL-co has a 20%,30%,40%,50%,60%,70%, or 80% greater separation (time) between the 0EU/mL standard (blank standard) and the 0.0005EU/mL standard relative to the unclarified LAL-co. The invention further provides a composition comprising (a) clarified LAL substantially free of coagulogen ("LAL-co") and (b) a buffer. In embodiments, such a composition comprisesComprises 30% to 60% (v/v) clarified LAL-co, and in embodiments comprises 40% to 60% (v/v), and in embodiments, 50% (v/v) clarified LAL-co. In an embodiment, the present invention provides a composition comprising (a) clarified LAL substantially free of coagulogen, (b) a buffer, and (c) a detergent. In embodiments, such compositions comprise 30% to 60% clarified LAL substantially free of coagulogen, and in embodiments 40% to 60% (v/v), and in embodiments, 50% clarified LAL-co. In an embodiment, a composition of the invention comprising clarified LAL substantially free of coagulogen comprises a Tris buffer at pH 7.4-7.5. In embodiments, a composition of the invention comprising clarified LAL substantially free of coagulogen comprises Tris buffer, sodium chloride, trehalose, and magnesium chloride. In embodiments, the compositions of the invention comprise clarified LAL substantially free of coagulogen, about 25mM to about 50mM Tris buffer, about 80mM sodium chloride, about 70mM trehalose, and about 10mM magnesium chloride. In embodiments, the compositions of the invention comprise clarified LAL substantially free of coagulogen, about 50% LAL-co, about 50mM Tris buffer, about 75mM trehalose, about 77mM sodium chloride and about 10mM magnesium chloride, and a pH of 7.4-7.5. In an embodiment, the invention provides a composition comprising clarified LAL substantially free of coagulogen, a buffer, and a zwitterionic detergent that retains its zwitterionic character over a wide pH range. In embodiments, the buffer is of formula C ₁₉ H ₄₁ NO ₃ N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid salt of S: (3-14 detergent). In embodiments, in a composition comprising clarified LAL substantially free of coagulogen, the detergent is present at about 0.2mM to 0.5mM, about 0.3mM to 0.4mM, or about 0.44 mM. In embodiments, the compositions of the invention comprise clarified LAL substantially free of coagulogen, about 25mM to about 50mM Tris buffer, about 20mM to about 90mM trehalose, about 80mM chlorideSodium and about 10mM magnesium chloride. In an embodiment, the composition of the invention comprises about 50% LAL-co, about 50mM Tris buffer, about 75mM trehalose, about 77mM sodium chloride, about 10mM magnesium chloride and about 0.44mM of a compound of formula C ₁₉ H ₄₁ NO ₃ N-tetradecyl-N, N-dimethyl-3-amino-1-propanesulfonic acid salt of S: (3-14 detergent) at a pH of about 7.4-7.5.

The present invention also relates to a composition comprising clarified LAL, wherein the composition is substantially free of coagulogen, wherein the composition is prepared by a process comprising the steps of: centrifuging a solution of lysed amoebocytes from Limulus polyphemus (Limulus polyphemus) at 2000rpm for 8 minutes at 4 ℃ to produce a supernatant; combining the supernatant from (a) with a buffer; tangential flow filtration of the pool from (b) using a 30kDa membrane filter to produce a retentate; and centrifuging the retentate from (c) at 5000rpm (e.g., 6120 g) for 5 minutes at 4 ℃ to produce a supernatant, wherein the supernatant is clarified LAL, which is substantially free of coagulogen.

The invention also relates to a composition comprising (1) clarified LAL substantially free of coagulogen, a buffer and a detergent, and (2) a chromogenic substrate. In some embodiments, the chromogenic substrate in the composition comprises pNA. In some embodiments, the chromogenic substrate in the composition is Ac-Ile-Glu-Ala-Arg-pNA. In some embodiments, the composition comprises 30% to 50% by volume of the clarified LAL substantially free of coagulogen formulation, and 10% to 30% by volume of the chromogenic substrate. In some embodiments, the composition comprises from about 35% to about 45% of clear LAL substantially free of coagulogen, and from 15% to 25% of a chromogenic substrate. In some embodiments, the composition comprises 40% by weight LAL substantially free of coagulogen preparation and 20% by weight chromogenic substrate. In some embodiments, the composition comprises about 40% by weight LAL substantially free of coagulogen preparation and 20% by weight Ac-lle-Glu-Ala-Arg-pNA. In some embodiments, a composition as described herein is in a single container, e.g., a single vial. In some embodiments, the compositions described herein are lyophilized. For example, the present disclosure specifically describes a lyophilized composition comprising 40% by weight LAL substantially free of a coagulogen preparation and 30% by weight Ac-Ile-Glu-Ala-Arg-pNA.

In some embodiments, the invention relates to a chromogenic assay kit. The kit may include one or more components typically associated with a LAL chromogenic assay, including reagents comprising clarified LAL and a chromogenic substrate. In some embodiments, the methods of the present disclosure relate to a kit comprising: (a) A clarified Limulus Amebocyte Lysate (LAL), wherein the LAL is substantially free of coagulogen; (b) a chromogenic substrate; and (c) instructions for detecting endotoxin using LAL and a chromogenic substrate. In some embodiments, the kit comprises various reagents, each having a clarified LAL with a different amount of coagulogen removed therefrom.

The kit may comprise one or more containers. In some embodiments, the clarified LAL and the chromogenic substrate are in a single container. In some embodiments, the clarified LAL and chromogenic substrate are in two different containers. In some embodiments, the kit comprises a sterile container containing the clarified LAL. In some embodiments, the kit comprises a reconstitution buffer that can reconstitute the clarified LAL and/or chromogenic substrate for use in the assay. In some embodiments, the sterile container is a sterile vial. In some embodiments, the kit further comprises a control standard endotoxin, which can be used as a positive endotoxin control, or can be used to quantify the amount of endotoxin in the standard. In some embodiments, the kit comprises one or more concentrations of one or more control standard endotoxins.

It will be appreciated by those skilled in the art that different methods may be used to remove coagulogen from LAL. Each of these methods may vary in efficiency, purification rate, cost and effort, but are within the knowledge of one skilled in the art. The present disclosure includes methods of making clarified LAL substantially free of coagulogen using tangential flow filtration. Tangential Flow Filtration (TFF) refers to cross-flow filtration, wherein the majority of the feed stream passes tangentially across the surface of the filter, rather than entering the filter. By using TFF, the retentate, which contains most of the LAL protein (which can contaminate the filter), is essentially washed away during filtration and coagulogen is filtered into the permeate. In some embodiments, TFF is a continuous process, as opposed to batch dead-end filtration.

In some embodiments, the present disclosure relates to a method of preparing clarified LAL substantially free of coagulogen, the method comprising centrifuging a solution of lysed amoebocytes from a horseshoe crab (Limulus polyphemus) at 1000 to 3000rpm for 2 to 15 minutes at 2 to 10 ℃ to produce a first supernatant ("first centrifugation"); combining the supernatant with the buffer; filtering the pool using a 20kDa to 50kDa filter, resulting in a retentate; the retentate is centrifuged at 3000 to 7000rpm at 2 to 10 ℃ for 2 to 10 minutes, resulting in a second supernatant ("second centrifugation"), wherein the second supernatant comprises clarified LAL substantially free of coagulogen. In embodiments, the filtration is subjecting the LAL to TFF. In some embodiments, the LAL is placed in a buffer prior to TFF. In some embodiments, the buffer is a Tris buffer or a MES buffer. In some embodiments, the pH of the buffer is from about 6.0 to about 9.0, or from about 7.0 to about 8.0. In an embodiment, the first centrifugation comprises centrifugation at 2000 rpm. In an embodiment, the first centrifugation comprises centrifugation for 8 minutes. In an embodiment, the first centrifugation comprises centrifugation at 4 ℃. In embodiments, the second centrifugation comprises centrifugation at 5000 rpm. In embodiments, the second centrifugation comprises centrifugation for 5 minutes. In embodiments, the second centrifugation comprises centrifugation at 4 ℃.

Various membranes can be used in TFF. Filters of different pore sizes can be used for TFF, depending on the size of the desired protein to be reduced in the resulting retentate. In the present disclosure, factor C, factor B, factor G and prothrombin are known to participate in the coagulation cascade of LAL, resulting in the conversion of coagulogen to insoluble coagulin gel. For the purposes of the disclosure provided herein, any TFF procedure (and accompanying filter pore size, pore type and buffer system) can be used that results in a reduction of coagulogen and retention of factor C, factor B, factor G and coagulogen. Thus, in some embodiments, the TFF procedure uses a 50kDa filter, a 45kDa filter, a 40kDa filter, a 35kDa filter, a 30kDa filter, a 25kDa filter, or a 20kDa filter. In some embodiments, a 40kDa to 25kDa filter is used. In some embodiments, the membrane is a 10 to 80kDa filter, or a 20 to 50kDa filter. In some embodiments, the filter is a 30kDa filter.

Membranes used in the methods disclosed herein may include, but are not limited to, modified polyethersulfone (mPES), polysulfone (PS), and Polyethersulfone (PES). In some embodiments, the method of making LAL substantially free of coagulogen is performed with TFF using a modified polyethersulfone (mPES) membrane filter. The flow rate of LAL through the membrane may be adjusted to optimize removal of coagulogen from the LAL. In some embodiments, TFF is performed at a flow rate of 200 mL/min to 800 mL/min, 300 mL/min to 600 mL/min, or 350 mL/min to 500 mL/min. In some embodiments, TFF is performed at a flow rate greater than 500 mL/min, such as 500 mL/min to 2000 mL/min, 800 mL/min to 1500 mL/min, or 1000 mL/min to 1200 mL/min. In some embodiments, TFF is performed at 1000 mL/min, 1100 mL/min, 1200 mL/min, 1300 mL/min, or 1400 mL/min. In some embodiments, TFF is performed at 1100 mL/min.

In embodiments, the present invention provides methods for producing clarified LAL substantially free of coagulogen by centrifuging LAL substantially free of coagulogen.

Examples

Example 1

Improved endotoxin detection with clarified LAL-co

Samples containing LAL (LAL-co) substantially free of coagulogen and clarified LAL-co were tested in 96-well plates for reaction rate and sensitivity of chromogenic endotoxin assay, as shown in FIG. 1. Rows A-F include increased amounts of endotoxin standards. The first solution containing 50% LAL-co or clarified LAL-co is combined with the second solution containing the chromogenic substrate to form a third and final solution, which is then contacted with the sample containing endotoxin. In columns 1-3 of FIG. 1, the third solution contains 50% clarified LAL-co first solution and 50% second solution (i.e., 25% of the final third solution contains clarified LAL-co) (50/50/50 solution). In columns 4-6 of FIG. 1, the third solution contains 80% clarified LAL-co first solution and 20% second solution (i.e., 40% of the final third solution contains clarified LAL-co) (50/80/20 solution). In columns 7-9 of FIG. 1, the third solution contains 50% of the total LAL-co solution, and 50% of the second solution (i.e., 25% of the final solution contains total LAL-co). In columns 10-12 of FIG. 1, the third solution contains 80% of the total LAL-co solution, and 20% of the second solution (i.e., 40% of the final solution contains total LAL-co).

Figure 1 illustrates that the use of clarified LAL-co and formulations with more LAL-co and less substrate surprisingly provides increased spacing and speed between blank intervals with a smooth response curve. This phenomenon is further illustrated in fig. 4. Three different 50/80/20 solutions were prepared and tested with 0.0005EU/mL and 0.005EU/mL standards. The mean 30mOD times for these clear LAL-co solutions were 31 min and 17 min, respectively. The reaction curve is smoother and more consistent. Furthermore, the time interval between 0EU/mL sample and 0.0005EU/mL sample (940 seconds) and 0EU/mL sample and 0.005EU/mL sample (1755 seconds) was greater. Thus, clarified LAL-co resulted in improved assay performance (e.g., increased reaction rate, greater differentiation from 0EU/mL control), lower amounts of substrate, and a smoother reaction curve resulted in more agreement.

Example 2

Preparation of clarified LAL-co

FIG. 3 is a schematic diagram illustrating a process for preparing clarified LAL-co. First, multiple batches of lysed amoebocytes from horseshoe crab (Limulus polyphemus) were pooled to form LAL daywood. LAL Daypool was centrifuged at 2000rpm for 8 min at 4 ℃. Carefully remove the supernatant and combine with buffer. The resulting solution was filtered by Tangential Flow Filtration (TFF) using a 30kDa mPES membrane filter. The retentate contained LAL-co. The retentate was then centrifuged at 5000rpm for 5 minutes at 4 ℃ (see fig. 2) and the supernatant was transferred to a storage vessel and stored at 4 ℃.

Example 3

Stability of LAL Daypool for forming clarified LAL substantially free of coagulogen

The stability of LAL Daypool for clear LAL-co formation was investigated using 0.0005EU/mL and 0.005EU/mL standards. LAL daywood was stored for 1, 2 and 3 years and used to form clarified LAL-co. At the end of the indicated time period, the clarified LAL-co sample was used to form a solution containing 50% clarified LAL-co, which was then mixed with the LAL chromogenic reagent at a ratio of 80: 20 and placed in a 96 well plate in a plate reader and the absorbance at 405nm was measured. The reaction was automatically monitored over time for the appearance of yellow color.

In the presence of endotoxins, the lysate will cleave the chromogenic substrate, resulting in a yellowing of the solution. The time required for the change is inversely proportional to the amount of endotoxin present. The reactions and the time intervals for each sample are shown in fig. 5, fig. 6, fig. 7, fig. 8 and fig. 9. Fig. 5,6 and 7 are from the same samples and experiments. Fig. 8 and 9 are from the same example and experiment. The x-axis of FIG. 5 is time (seconds) and the y-axis is the change in mOD. The y-axis of FIG. 7 is time (in seconds). The data indicate that LAL Daypool used to form clarified LAL-co was stable for at least 3 years and maintained its increased reaction time, smooth reaction curve, and larger interval between blank standard (0 EU/mL) and endotoxin controls (0.005 EU/mL and 0.0005 EU/mL). The y-axis of FIG. 8 is 0.005EU/mL reaction time in seconds, and the x-axis represents the LAL source for different years. The left panel is data from clarified LAL-co and the right panel from untreated LAL. The data show that the differences in LAL performance over time can be overcome by treating LAL with TFF and centrifuging the resulting LAL-co. The y-axis of the graph in fig. 9 shows the change in mOD, and the x-axis is time in seconds. The table contains the reaction times obtained from the 0.005EU/mL standard plot and the interval between this time and the 0EU/mL blank. The data show that clarified LAL-co has faster standard reaction time and greater separation between blank and low standard compared to untreated LAL counterpart.

Example 4

Each Diafiltration Volume (DV) of continuous tangential flow filtration was investigated to determine which volume gave the best sensitivity (blank compared to 0.005EU/mL standard) while achieving a reaction time of 1200 seconds. See fig. 10. Greater than 4DV was found to remove coagulogen to achieve a reaction time of less than 1200 seconds. 5,6 and 7 DVs yielded the required reaction times while yielding the highest sensitivity and minimizing the diafiltration volume.

SDS-PAGE analysis of the retentate from each DV demonstrated that the band density of the 20kDa band (approximate MW of coagulogen) decreases when more diafiltration volumes are used to wash the LAL. See, for example, fig. 11. Using the band densities from SDS-PAGE, it was shown that coagulogen comprised about 37% of the total stained protein in the unfiltered LAL and that each diafiltration volume reduced the 20kDa protein band. The total protein concentration in each retentate collected after each diafiltration volume was determined by measuring absorbance at 280 nm. See, for example, fig. 12. The percentage of coagulogen in each retentate sample using data from SDS-PAGE was multiplied by the total concentration of protein and an estimate of the coagulogen concentration in each retentate was calculated. The concentration of each post-DV coagulogen is shown in FIG. 13.

Example 5

Many proteins are known to be present in unfiltered LAL compositions. A list of some known proteins is shown in figure 14. Iwanaga S, et al Frontiers in Bioscience 3.973:973-984 (1998) proteins with molecular weights of less than 30kDa are shaded. Studies were conducted to determine if removal of other small molecules helped to improve performance. The LAL composition is subjected to continuous tangential flow filtration using a 30kDa filter or a 10kDa filter. As expected, SDS-PAGE demonstrated that the use of a 30kDa filter removed coagulogen (approximately 20 kDa), but a 10kDa filter did not. See fig. 15. Continuous tangential flow filtration using a 30kDa filter reduced reaction time relative to unfiltered LAL and continuous tangential flow filtration using a 10kDa filter. Continuous tangential flow filtration using a 30kDa filter also improved the separation between blank and 0.005EU/mL sample relative to unfiltered LAL and continuous tangential flow filtration using a 10kDa filter.

Example 6

The effect of centrifugation speed on reaction speed and interval was evaluated. The LAL composition was prepared and then centrifuged at 10,000 Xg, 20,000Xg, 30,000Xg or 40,000Xg for 30 minutes. The retentate was then tested for reaction time and interval using a blank and 0.005EU/mL standard. The optical density of each retentate was also measured. The results are shown in FIGS. 17 and 18. The unclarified material has a higher optical density than the centrifuged retentate. A correlation between the interval and the centrifugation speed was also observed, where increasing the centrifugation speed resulted in a greater interval between the blank and the 0.005EU/mL standard. A kinetic chromogenic assay (Δ t (sec): 30, Δ mOD: 50) was performed at 405nM using an absorbance reader to assess the performance of the clarified retentate, indicating that centrifugation removed material that contributed to the cleavage of the substrate without relying on endotoxin, indicating an additional benefit of the clarification step.

Example 7

The effect on performance of freezing and thawing clarified LAL substantially free of coagulogen was investigated. The activity of the retentate produced by the method described herein (30 kDa continuous TFF, centrifuged at 30,000 × g) was measured before freezing, and then the retentate was frozen and thawed at room temperature. The results are shown in FIG. 19. The study showed comparable performance before and after freeze/thaw.

It will be apparent to those of ordinary skill in the relevant art that other suitable modifications and adaptations to the methods and applications described herein may be made without departing from the scope of any of the embodiments. The above examples are for illustrative purposes only and are not limiting.

It is to be understood that while certain embodiments have been illustrated and described herein, the claims are not to be limited to the specific forms or arrangements of parts so described and shown. In the specification, there have been disclosed illustrative embodiments and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is, therefore, to be understood that the embodiments may be practiced otherwise than as specifically described.

While various embodiments are described above, they are merely illustrative and examples of the present technology and are not intended to be limiting. It will be apparent to persons skilled in the relevant art that various modifications and variations can be made in the form and details of the present invention without departing from the spirit and scope of the technology. Thus, the breadth and scope of the present technology should not be limited by any of the above-described embodiments, but should be defined only in accordance with the following claims and their equivalents. It will also be understood that each feature of each embodiment of each reference described and cited herein can be used in combination with the features of any other embodiment. All patents and publications cited herein are incorporated by reference in their entirety.

Claims (80)

1. A composition comprising limulus amebocyte lysate LAL, wherein the LAL has been (i) filtered to remove at least 60% by weight of its coagulogen and then (ii) centrifuged at greater than 1800xg for more than 3 minutes, wherein the LAL comprises less than 5% by weight of coagulogen relative to the total protein in the LAL as measured by SDS-PAGE and protein staining.

2. The composition of claim 1, further comprising a buffer, wherein the composition comprises 30% to 60% by volume of LAL.

3. The composition of claim 2, wherein the buffer is Tris buffer.

4. The composition of claim 2 or 3, wherein the buffer is at a concentration of 25mM to 50mM.

5. The composition of claim 1, further comprising a detergent.

6. The composition of claim 5, wherein the detergent is a zwitterionic detergent.

7. The composition of claim 5 or 6, wherein the concentration of detergent is 0.002% to 0.02%.

8. The composition of claim 1, wherein the composition comprises 50% LAL.

9. The composition of claim 1, further comprising a chromogenic substrate, wherein said chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA.

10. The composition of claim 9, wherein the concentration of the chromogenic substrate is between 0.11mg/mL and 0.77mg/mL.

11. The composition of claim 1, wherein the composition is lyophilized.

12. The composition of claim 1, wherein the composition is in an aqueous solution.

13. A composition, comprising:

a. the limulus amoeboid cell lysate LAL of claim 1, and

b. a buffer solution is added to the reaction kettle,

c. a detergent, and

d. a chromogenic substrate, wherein said chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA,

wherein the composition comprises 30 to 50 vol% LAL and 10 to 30 vol% chromogenic substrate.

14. The composition of claim 13, wherein the composition comprises from 35% to 45% LAL and from 15% to 25% chromogenic substrate.

15. The composition of claim 13, wherein the composition comprises 40% LAL and 20% chromogenic substrate.

16. A composition comprising clarified LAL, wherein the LAL comprises less than 5% by weight coagulogen relative to total protein in the LAL as shown by SDS-PAGE and protein staining measurements, wherein the composition is prepared by a method comprising the steps of:

a. centrifuging the solution of lysed amoebocytes from Limulus polyphemus at 2000rpm for 8 minutes at 4 ℃ to produce a supernatant;

b. combining the supernatant from (a) with a buffer;

c. tangential flow filtration of the pool from (b) using a 30kDa membrane filter to produce a retentate; and is

d. Centrifuging the retentate from (c) at 5,000rpm for 5 minutes at 4 ℃ to produce a supernatant, wherein the supernatant comprises clarified LAL.

17. A composition comprising clarified LAL, wherein said LAL comprises less than 5% by weight of coagulogen relative to total protein in the LAL as shown by SDS-PAGE and protein staining measurements, wherein said composition is prepared by a method comprising the steps of:

a. obtaining a solution of lysed amoeboid cells from a horseshoe crab;

b. combining the solution from (a) with a buffer;

c. subjecting the pool from (b) to continuous tangential flow filtration using a 20kDa to 50kDa membrane filter to produce a retentate; and is provided with

d. Centrifuging the retentate from (c) at greater than 20,000x g for more than 25 minutes to produce a supernatant, wherein the supernatant comprises clarified LAL.

18. The method of claim 17, wherein the continuous tangential flow filtration of (c) comprises at least 4 diafiltration volumes.

19. The process of claim 17 or 18, wherein the continuous tangential flow filtration of (c) comprises at least 6 diafiltration volumes.

20. A method of detecting endotoxin in a sample using a chromogenic assay, the method comprising:

a. contacting the sample with a reagent comprising limulus amebocyte lysate LAL and a chromogenic substrate, wherein said chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA;

b. measuring the chromogenic effect caused by a change in the chromogenic substrate in the presence of endotoxin in the sample;

wherein the LAL has been (i) filtered to remove at least 60% by weight of its coagulogen, and then (ii) centrifuged at greater than 1800x g for more than 3 minutes, wherein the LAL comprises less than 5% by weight of coagulogen relative to total protein in the LAL as shown by SDS-PAGE and protein staining.

21. The method of claim 20, wherein the change in the chromogenic substrate occurs as a result of an enzymatic reaction.

22. The method of claim 21, wherein the enzymatic reaction is the cleavage of a chromophore from the chromogenic substrate.

23. The method of claim 20, wherein measuring the chromogenic effect comprises measuring absorbance at 380nm to 420 nm.

24. The method of claim 20, wherein measuring a chromogenic effect comprises measuring absorbance at 405 nm.

25. The method of claim 20, wherein the reagent is a liquid.

26. The method of claim 20, wherein the reagent is an aqueous liquid.

27. The method of claim 20, wherein the reagent is lyophilized and then reconstituted in an aqueous liquid prior to contact with the sample.

28. The method of claim 20, wherein the LAL is lyophilized and then reconstituted prior to contacting with the sample.

29. The method of claim 20, wherein the LAL is frozen and then thawed prior to contacting with the sample.

30. The method of claim 20, wherein the chromogenic substrate is lyophilized and then reconstituted prior to contact with said sample.

31. The method of claim 20, wherein the sample is a biological sample.

32. The method of claim 20, wherein the sample is selected from the group consisting of: parenteral dosage forms, vaccines, antibiotics, therapeutic proteins, therapeutic nucleic acids, therapeutic antibodies, and biological products.

33. The composition or method of any one of claims 1, 13, 16, 17, and 20, wherein the LAL comprises less than 2% by weight coagulogen relative to total protein in the LAL as measured by SDS-PAGE and protein staining.

34. The composition or method of any one of claims 1, 13, 16, 17, and 20, wherein the LAL comprises less than 0.5% by weight coagulogen relative to total protein in the LAL as measured by SDS-PAGE and protein staining.

35. The composition or method of any one of claims 1, 13, 16, 17, and 20, wherein the LAL comprises less than 5 μ g/μ L of coagulogen.

36. The composition or method of any one of claims 1, 13, 16, 17, and 20, wherein the LAL comprises less than 3 μ g/μ L of coagulogen.

37. The composition or method of any one of claims 1, 13, 16, 17, and 20, wherein the LAL comprises less than 2 μ g/μ L of coagulogen.

38. The method of claim 20, wherein the chromogenic assay is performed using single-cell spectroscopy, multi-cell spectroscopy, or a microplate reader.

39. The method of claim 20, further comprising: the chromogenic effect is compared to a standard to determine the amount of endotoxin in the sample.

40. A method of detecting endotoxin in a biological sample using a chromogenic assay, said method comprising:

a. contacting the biological sample with a reagent comprising clarified LAL and Ac-Ile-Glu-Ala-Arg-pNA; and

b. measuring a change in absorbance at 405nm resulting from enzymatic cleavage of pNA from Ac-Ile-Glu-Ala-Arg-pNA in the presence of endotoxin in the sample,

wherein the LAL has been (i) filtered to remove at least 60% by weight of its coagulogen, and then (ii) centrifuged at greater than 1800x g for more than 3 minutes, wherein the LAL comprises less than 5% by weight of coagulogen relative to total protein in the LAL as shown by SDS-PAGE and protein staining.

41. The method of claim 40, wherein the reagent further comprises a buffer and a detergent.

42. The method of claim 40 or 41, wherein prior to (a), a solution comprising a buffer, a detergent, and clarified LAL is mixed with a solution comprising Ac-Ile-Glu-Ala-Arg-pNA.

43. The method of claim 42, wherein the solution comprising buffer, detergent, and LAL comprises 45% -55% LAL.

44. The method of claim 43, wherein the solution comprising buffer, detergent, and LAL comprises 50% LAL.

45. The method of claim 40, wherein the agent comprises 15% -25% Ac-Ile-Glu-Ala-Arg-pNA.

46. The method of claim 45, wherein the agent comprises 20% Ac-Ile-Glu-Ala-Arg-pNA.

47. The method of claim 40, wherein the method has a sensitivity of 0.0001EU/mL to 1.0EU/mL endotoxin.

48. A kit, comprising:

a. a limulus amebocyte lysate LAL, wherein the LAL has been (i) filtered to remove at least 60% by weight of its coagulogen and then (ii) centrifuged for more than 3 minutes at greater than 1800x g, wherein the LAL comprises less than 5% by weight of coagulogen relative to the total protein in the LAL as shown by SDS-PAGE and protein staining;

b. a chromogenic substrate, wherein said chromogenic substrate is Ac-Ile-Glu-Ala-Arg-pNA; and

c. instructions for detecting endotoxin using LAL and chromogenic substrate.

49. The kit of claim 48, wherein the LAL is lyophilized.

50. The kit of claim 48, wherein the LAL is an aqueous solution.

51. The kit of any one of claims 48-50, wherein the LAL and the chromogenic substrate are in a single container.

52. The kit of any one of claims 48-50, further comprising a sterile container containing the LAL.

53. The kit of claim 52, wherein the sterile container is a sterile vial.

54. The kit of any one of claims 48-50, further comprising a control standard endotoxin.

55. A method for preparing clarified limulus amoebocyte lysate LAL comprising less than 5 wt.% coagulogen, relative to the total protein in LAL, as measured by SDS-PAGE and protein staining, comprising:

a. centrifuging a solution of lysed amoebocyte cells from a horseshoe crab at 1000 to 3000rpm for 2 to 15 minutes at 2-10 ℃ to produce a supernatant;

b. combining the supernatant from (a) with a buffer;

c. filtering the pool from (b) using a 20kDa to 50kDa filter to produce a retentate;

d. centrifuging the retentate from (c) at 3000 to 7000rpm for 2 to 10 minutes at 2 to 10 ℃ to produce a supernatant, wherein the supernatant comprises clarified LAL.

56. The method of claim 55, wherein the centrifuging in (a) comprises centrifuging at 2000 rpm.

57. The method of claim 55 or 56, wherein the centrifuging in (a) comprises centrifuging for 8 minutes.

58. The method of claim 55 or 56, wherein the centrifuging in (a) comprises centrifuging at 4 ℃.

59. The method of claim 55 or 56, wherein the filtering of (c) comprises using a 30kDa filter.

60. The method of claim 55 or 56, wherein centrifuging in (d) comprises centrifuging at 5000 rpm.

61. The method of claim 55 or 56, wherein the centrifuging in (d) comprises centrifuging for 5 minutes.

62. The method of claim 55 or 56, wherein the centrifuging in (d) comprises centrifuging at 4 ℃.

63. The method of claim 55 or 56, wherein the buffer is Tris buffer or MES buffer.

64. The method of claim 55 or 56, wherein the pH of the buffer is 7.0 to 8.0.

65. The method of claim 55 or 56, wherein the filtration is tangential flow filtration.

66. The method of claim 65, wherein the TFF is performed at a flow rate of 350 ml/min to 500 ml/min.

67. The method of claim 55 or 56, wherein the filter is a modified polyethersulfone membrane filter.

68. A method for preparing clarified limulus amoebocyte lysate LAL comprising less than 5 wt.% coagulogen, relative to the total protein in LAL, as measured by SDS-PAGE and protein staining, comprising:

a. obtaining a solution of lysed amoeboid cells from a horseshoe crab;

b. combining the solution from (a) with a buffer;

c. subjecting the pool from (b) to continuous tangential flow filtration using a 20kDa to 50kDa membrane filter to produce a retentate; and

d. centrifuging the retentate from (c) at greater than 20,000x g for more than 25 minutes to produce a supernatant, wherein the supernatant is clarified LAL.

69. A method of preparing clarified limulus amebocyte lysate LAL comprising less than 5 wt.% coagulogen relative to the total protein in LAL as measured by SDS-PAGE and protein staining, comprising:

a. obtaining a solution of lysed amoeboid cells from a horseshoe crab;

b. combining the solution from (a) with a buffer;

c. subjecting the pool from (b) to continuous tangential flow filtration using a 20kDa to 50kDa membrane filter to produce a retentate; and

d. centrifuging the retentate from (c) at greater than 20,000x g to produce a supernatant, wherein the supernatant is clarified LAL.

70. The method of claim 68 or 69, wherein the solution in (a) comprises a supernatant obtained by centrifugation of lysed amoebocytes from Limulus tridentatus at 1000 to 3000rpm for 2 to 15 minutes at 2 to 10 ℃.

71. The method of claim 70, wherein centrifuging in (a) comprises centrifuging at 2000 rpm.

72. The method of claim 70, wherein centrifuging in (a) comprises centrifuging for 8 minutes.

73. The method of claim 68 or 69, wherein centrifuging in (d) comprises centrifuging at 2 to 10 ℃.

74. The process of claim 68 or 69, wherein the continuous tangential flow filtration of (c) comprises at least 4 diafiltration volumes.

75. The process of claim 68 or 69, wherein the continuous tangential flow filtration of (c) comprises at least 6 diafiltration volumes.

76. The method of claim 68 or 69, wherein the centrifuging of (d) comprises centrifuging at 40,000x g.

77. The method of claim 68 or 69, wherein the centrifuging of (d) comprises centrifuging for 30 minutes.

78. The method of claim 69, wherein the centrifuging of (d) comprises centrifuging for at least 20 minutes.

79. The method of claim 68 or 69, wherein the buffer is Tris buffer or MES buffer.

80. The method of claim 68 or 69, wherein the pH of the buffer is between 7.0 and 8.0.