Patent Application
20050244299
The invention relates to automated measurement of endotoxin levels.
Bacterial endotoxin is a potentially widespread contaminant of a variety of materials, such as water, food, pharmaceutical products, and parenteral preparations. Bacterial endotoxins (lipopolysaccharides) are released from the outer cell membranes of Gram-negative bacteria during early stages of growth, phagocytic digestion, or autolysis of bacterial cells. Lipopolysaccharides are water-soluble stable molecules that have both hydrophobic and hydrophilic regions. The latter are composed of repeating oligosaccharide side chains attached to a polysaccharide core.
There is considerable variation in the details of the structure of endotoxins derived from different bacteria. While the polysaccharide moiety is responsible for the immunogenic properties of endotoxins, their toxicity is elicited by the hydrophobic part (called ‘lipid A,’ which is virtually invariant in composition across different bacterial species). Even in small doses, the introduction of endotoxins into the circulatory system of either humans or animals is capable of causing a wide spectrum of nonspecific pathophysiological changes, e.g., fever, increased erythrocyte counts, disseminated intravascular coagulation, hypotension, shock, cell death, etc. In large doses, it causes death in most mammals. Early-life exposure to endotoxins exerts long-term effects on endocrine and central nervous system development and increases predisposition to inflammatory diseases. Shanks et al., Proc. Natl. Acad. Sci. 97, 5645-50, 2000; see also Pearson III, in P
Given current concerns regarding bioterrorism, it is useful to note that inhalation of high concentration of endotoxins causes dry cough and shortness of breath, accompanied by a decrease in lung function and fever. Rylander, in O
It is thus essential to ensure that the endotoxin contents of parenterally administered drugs or other fluids remain below permissible levels (in the US, this is set by the US Food and Drug Administration). Sterile water for injection or irrigation, for example, has a maximum permissible limit of 0.25 Endotoxin Units (EU)/mL (for endotoxin derived from E. coli, 1 EU is approximately 75-200 pg). See the URL address: http file type, www host server, domain name “fda.gov,” file type “ora/inspect_ref/itg/itg40.html”; United States Pharmacopeia, USP 24-NF 19, Suppl. 2, 2761-62; Jul. 1, 2000.
Measurement of Endotoxins
The rabbit pyrogen test (fever induction in a rabbit) was introduced in the U.S. Pharmacopoeia in 1942 for the general testing of pyrogens, which include bacterial endotoxins. The test is slow and qualitative and has largely been replaced by some form of the Limulus amebocyte lysate (LAL) test. In 1964, Levin and Bang discovered that bacterial endotoxins can greatly accelerate the rate of clotting of blood from the horseshoe crab Limulus polyphemus. Levin & Bang, Bull. Johns Hopkins Hosp. 115, 265-74, 1964; see also the URL address: http file type, www host server, domain name “dnr.state.md.us,” file type “fisheries/education/horseshoe/horseshoefacts.html.” By 1987, the US Food and Drug Administration (FDA) published guidelines for the validation of the LAL test as an alternative to the USP Rabbit Pyrogen Test The superiority of the LAL based assay over the rabbit test has been known for some time. See Levin, in E
LAL contains several protease enzymes responsible for endotoxin induced gel/clot formation. Through a series of cascade reactions, the primary protein component sensitive to endotoxins activates the proclotting enzyme to form the clotting enzyme. Berzofsky & McCullough in I
Presently there are three major versions of LAL tests: the gel-clot assay (Levin & Bang, 1964; Levin, 1982; U.S. Pat. No. 5,310,657), the turbidimetric assay (Levin et al., J Lab. Clin. Med. 75, 903-11, 1970; Cooper et al., J Lab. Clin. Med. 78, 138-48, 1971; Pearson & Weary, J. Lab. Clin. Med. 78, 65-77, 1971); and the colorimetric assay (Teller & Kelly, in B
Turbidimetric assays measure turbidity due to gel formation; apparent turbidity is somewhat affected by the size and the number of particles, etc. but this problem can be largely overcome. Ohki et al., FEBS Lett. 120, 217-20, 1980. Turbidity measurement is generally unaffected by color present in the sample. A quartz oscillator has been used to measure the viscosity change that occurs during gelation; this technique allows turbid samples to be analyzed. Novitsky et al., in D
In a colorimetric assay, a synthetic chromogenic peptide is hydrolyzed by the clotting enzyme to release the terminal colored chromogenic moiety. It provides better quantitation and is less laborious than clotting based methods. It is also more sensitive because the amount of enzyme needed for the hydrolysis of the chromogenic substrate is less than the amount needed to form a clot. Friberger et al., in E
Turbidimetric and colorimetric assays can be practiced in two modes. In the endpoint mode, turbidity or color is measured after a fixed incubation period. In the kinetic assay mode, which offers greater dynamic range,.the turbidity or color development is measured continuously as a function of time. In the end point assay mode, a colorimetric reaction can be stopped by adding acid or a surfactant. solution (e.g., SDS), and the absorbance can be measured at any time thereafter. In a turbidimetric assay this is not possible; addition of acid also destroys the turbidity.
Automation
A degree of automation of the turbidirmetric end point assay has been achieved with a commercially available system (Muramatsu et al., Anal Chim. Acta 215, 91-98, 1988; Homma et al., Anal Biochem. 204, 398-404, 1992); however, poor correlation with other methods and generally higher results have been observed (Tsuji & Martin, 1978).
For some time now, the chromogenic LAL test is the most widely used. Jorgensen & Alexander, Appl. Environ. Microbiol. 41, 1316-20, 1981; Novitsky et al., Parenteral. Sci. Technol. 36, 11-16, 1982.
A robotic automated system has been developed for the chromogenic test. Tsuji & Martin, 1978. This early system and its subsequent commercial counterparts has impressive capabilities but the overall cost is very high. See Bussey & Tsuji, J. Parenter. Sci. Technol. 38, 228-33, 1984; Martin et al., J. Parenter. Sci. Technol. 40, 61-66, 1986. In fact, the cost is prohibitive for deployment at each point of use, as is necessary, for example, in sterile water testing applications. Rather, most users utilize microplate reader based instrumentation where 96-well plates are manually loaded with samples, standards, and reagents. See the URL address: http file type, www host server, domain name “Cambrex.com,” file name “biosciences/lal/b-EndotoxinDPS-instrument.htm#1.”
It is known in the art to use flow injection analysis or sequential injection analysis when attempting to detect the presence of a species. Conventional sequential injection analysis involves the use of a system comprising, typically, a rotary, multi-position selection valve around which multiple liquid solutions including samples and reagents are arranged. A bi-directional pump is used to draw up volumes of these samples and reagents through respective ports of the selection valve and into a holding coil where the samples and reagents are stacked and then delivered to a detector for analysis. This process causes mixing of the sample and reagent segments leading to chemistry that forms a detectable species before reaching the detector. The detector is typically attached to one port of the rotary valve via which the stacked segments can be made to flow by the pump. Stacking is the process of providing a plurality of aliquots, slugs or segments of fluids in a single conduit, either discrete and apart one slug or aliquot from another or adjacent to one another. Conventional systems can involve the use of a single pump (syringe or peristaltic) and a single rotary selection valve. Conventional multi-position selection valves permit random access of the ports that are connected to the samples, the reagents and the detector. Conventional selection valves that are usable in sequential injection analysis systems are can have between six and twenty-eight ports. Commonly, the section valves have between eight and ten ports. An electronic actuator that, in some instances, moves through the ports in both clockwise and counter-clockwise directions controls the operation of the selection valve. Typically, only one port is accessed at any time. When compared to flow injection analysis, sequential injection analysis systems have the advantage of being able to access an increased number of solutions with just one pump. However, these types of sequential injection analysis systems have not been used to determine the presence of the endotoxins due, at least in part, to the difficulties in cleaning the system between different test samples.
There is, therefore, a need in the art for an affordable, sensitive, and fully automated (“on-line”) endotoxin determination system that can be used for point of use endotoxin determinations.
One embodiment of the invention provides a system for detecting the presence of an endotoxin in a fluid. The system comprises a fluid delivery pump for introducing and moving fluids within said system; a fluid selection valve having a plurality of ports, each of said ports adapted to receive at least one fluid therethrough in response to the operation of said fluid delivery pump; a fluid holding member in fluid communication with said fluid selection valve and said fluid delivery pump, wherein selected fluids received by said fluid selection valve are stacked in a predetermined order within said fluid holding member, a solid state detector in fluid communication with said fluid selection valve for receiving the fluids that enter said selection valve from the fluid holding member; a multiport fluid valve in fluid communication with said fluid delivery pump and said fluid holding member; and a solenoid valve for connecting to a source of pressurized air, said solenoid valve being coupled to one of said ports of said multiport valve, said solenoid valve introducing air into said system after the
Turbidimetric assays measure turbidity due to gel formation; apparent turbidity is somewhat affected by the size and the number of particles, etc. but this problem can be largely overcome. Ohki et aL, FEBS Lett. 120, 217-20, 1980. Turbidity measurement is generally unaffected by color present in the sample. A quartz oscillator has been used to measure the viscosity change that occurs during gelation; this technique allows turbid samples to be analyzed. Novitsky et al., in D
In a colorimetric assay, a synthetic chromogenic peptide is hydrolyzed by the clotting enzyme to release the terminal colored chromogenic moiety. It provides better quantitation and is less laborious than clotting based methods. It is also more sensitive because the amount of enzyme needed for the hydrolysis of the chromogenic substrate is less than the amount needed to form a clot. Friberger et aL, in E
Turbidimetric and colorimetric assays can be practiced in two modes. In the endpoint mode, turbidity or color is measured after a fixed incubation period. In the kinetic assay mode, which offers greater dynamic range,.the turbidity or color development is measured continuously as a function of time. In the end point assay mode, a colorimetric reaction can be stopped by adding acid or a surfactant. solution (e.g., SDS), and the absorbance can be measured at any time thereafter. In a turbidimetric assay this is not possible; addition of acid also destroys the turbidity.
Automation
A degree of automation of the turbidimetric end point assay has been achieved with a commercially available system (Muramatsu et al., Anal Chim. Acta 215, 91-98, 1988; Homma et al., Anal. Biochem. 204, 398-404, 1992); however, poor correlation with other methods and generally higher results have been observed (Tsuji & Martin, 1978).
For some time now, the chromogenic LAL test is the most widely used. Jorgensen & Alexander, Appl. Environ. Microbial 41, 1316-20, 1981; Novitsky et al., Parenteral. Sci. Technol. 36, 11-16, 1982.
A robotic automated system has been developed for the chromogenic test. Tsuji & Martin, 1978. This early system and its subsequent commercial counterparts has impressive capabilities but the overall cost is very high. See Bussey & Tsuji, J. Parenter. Sci. Technol. 38, 228-33, 1984; Martin et al., J. Parenter. Sci. Technol 40, 61-66, 1986. In fact, the cost is prohibitive for deployment at each point of use, as is necessary, for example, in sterile water testing applications. Rather, most users utilize microplate reader based instrumentation where 96-well plates are manually loaded with samples, standards, and reagents. See the URL address: http file type, www host server, domain name “Cambrex.com,” file name “biosciences/lal/b-EndotoxinDPS-instrument.htm#1.”
It is known in the art to use flow injection analysis or sequential injection analysis when attempting to detect the presence of a species. Conventional sequential injection analysis involves the use of a system comprising, typically, a rotary, multi-position selection valve around which multiple liquid solutions including samples and reagents are arranged. A bi-directional pump is used to draw up volumes of these samples and reagents through respective ports of the selection valve and into a holding coil where the samples and reagents are stacked and then delivered to a detector for analysis. This process causes mixing of the sample and reagent segments leading to chemistry that forms a detectable species before reaching the detector. The detector is typically attached to one port of the rotary valve via which the stacked segments can be made to flow by the pump. Stacking is the process of providing a plurality of aliquots, slugs or segments of fluids in a single conduit, either discrete and apart one slug or aliquot from another or adjacent to one another. Conventional systems can involve the use of a single pump (syringe or peristaltic) and a single rotary selection valve. Conventional multi-position selection valves permit random access of the ports that are connected to the samples, the reagents and the detector. Conventional selection valves that are usable in sequential injection analysis systems are can have between six and twenty-eight ports. Commonly, the section valves have between eight and ten ports. An electronic actuator that, in some instances, moves through the ports in both clockwise and counter-clockwise directions controls the operation of the selection valve. Typically, only one port is accessed at any time. When compared to flow injection analysis, sequential injection analysis systems have the advantage of being able to access an increased number of solutions with just one pump. However, these types of sequential injection analysis systems have not been used to determine the presence of the endotoxins due, at least in part, to the difficulties in cleaning the system between different test samples.
There is, therefore, a need in the art for an affordable, sensitive, and fully automated (“on-line”) endotoxin determination system that can be used for point of use endotoxin determinations.
One embodiment of the invention provides a system for detecting the presence of an endotoxin in a fluid. The system comprises a fluid delivery pump for introducing and moving fluids within said system; a fluid selection valve having a plurality of ports, each of said ports adapted to receive at least one fluid therethrough in response to the operation of said fluid delivery pump; a fluid holding member in fluid communication with said fluid selection valve and said fluid delivery pump, wherein selected fluids received by said fluid selection valve are stacked in a predetermined order within said fluid holding member, a solid state detector in fluid communication with said fluid selection valve for receiving the fluids that enter said selection valve from the fluid holding member, a multiport fluid valve in fluid communication with said fluid delivery pump and said fluid holding member; and a solenoid valve for connecting to a source of pressurized air, said solenoid valve being coupled to one of said ports of said multiport valve, said soleneid valve introducing air into said system after the fluids from the fluid holding member have been received by said fluid selection valve and said detector.
Another embodiment of the invention provides a system for detecting the presence of an endotoxin in a fluid sample, said system comprising a fluid delivery pump for introducing and moving fluids within said system; a fluid selection valve having a plurality of ports, each of said ports adapted to receive at least one fluid therethrough in response to the operation of said fluid delivery pump; a fluid holding member in fluid communication with said fluid selection valve and said fluid delivery pump, wherein selected fluids received by said fluid selection valve are stacked in a predetermined order within said fluid holding member; a detector in fluid communication with said fluid selection valve for receiving the stacked fluids from the fluid holding member, said detector comprising multiple detector cells each comprising a tube extending within a detector block, a light source, a first fiber optic extending between said light source and said tube, a second fiber optic extending between said tube and a signal photodiode for delivering light from said tube containing the fluid sample, a third fiber optic extending between said light source and a reference photodiode, said third fiber optic for delivering light from the light source to the reference photodiode, and a system for comparing an output of the signal diode with an output from said reference diode to determine if an endotoxin is present within said tube.
Yet another embodiment of the invention provides a method of detecting the presence of an endotoxin in a test fluid sample. The method comprises steps of: (a) introducing gas into a fluid holding member to form a first gas buffer, (b) introducing an LAL reagent fluid, a chromogenic substrate fluid, and a test fluid sample into a fluid holding member to form a stacked fluid slug adjacent to the first gas buffer; (c) introducing gas into the fluid holding member to form a second gas buffer adjacent to the stacked fluid slug; (d) mixing said stacked fluid slug to form a mixed fluid; (e) introducing said mixed fluid into a portion of a detector cell; (f) introducing a first light emitted from a light source into said portion of said detector cell including said mixed fluids; (g) measuring the light emitted from said light source; (h) measuring a second light from within said portion of said detector cell; and (i) comparing the measured first and second lights to determine if an endotoxin has changed the light from the light source introduced into said portion of said detector cell.
Still another embodiment of the invention provides a method of washing an endotoxin detection system, comprising the steps of (a) introducing a basic solution into an endotoxin detection system to remove endotoxin; (b) rinsing the basic solution from the endotoxin detection system with an aqueous solution comprising about 50% ethanol; and (c) rinsing the aqueous solution from the endotoxin detection system with endotoxin-free water.
A further embodiment of the invention provides a method of washing an endotoxin detection system, comprising the steps of (a) rinsing an endotoxin detection system with deionized water, (b) displacing the deionized water with about 0.05% triethylamine (TEA) to remove endotoxin; and (c) displacing the TEA with endotoxin-free water.
Another embodiment of the invention provides a method of maintaining stability of a chromogenic substrate and an LAL reagent in an endotoxin detection system. The method comprises steps of: (a) introducing gas into a fluid holding member to form a first gas buffer; (b) introducing an LAL reagent fluid, a chromogenic substrate fluid, and a test fluid sample into a fluid holding member to form a stacked fluid slug adjacent to the first gas buffer; (c) introducing gas into the fluid holding member to form a second gas buffer adjacent to the stacked fluid slug; and (d) mixing said stacked fluid slug to form a mixed fluid before delivering the sample mixture to a detection portion of the endotoxin detection system.
Even another embodiment of the invention provides a method for testing a fluid to determine whether said fluid contains endotoxin, said fluid being transported through a conduit. The method comprises (1) withdrawing through a first flow path a sample volume of said fluid from said conduit and flowing said sample volume of said fluid into a mixing zone; (2) mixing said sample volume of said fluid in said mixing zone with an amount of LAL reagent and an amount of chromogenic substrate sufficient to detect endotoxin in said sample volume of fluid; (3) flowing said mixture prepared in step (2) from said mixing zone to an endotoxin detector cell through a second flow path which is in flow communication with at least a portion of said first flow path, and (4) determining whether said sample volume of said fluid contains endotoxin.
The invention thus provides automated “on-line” flow analysis systems that can perform a Limulus amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins.
The invention provides automated endotoxin detection systems (ie., automated “on-line” flow analysis systems) that can perform a Limulus amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins. The systems can be used to test fluid samples from production lines to detect the presence of endotoxin during the preparation of, for example, water, food, drink, pharmaceutical products (including those for animal and human health), and parenteral preparations.
In automated systems of the invention, a test fluid sample is mixed with a chromogenic substrate and an LAL reagent to form an assay mixture at the point of use. Assay mixtures are then delivered to individual detector cells for the simultaneous collection of time-based absorbance data. This automated system determines endotoxin concentration with good accuracy and reproducibility in the range of 0.005-0.5 endotoxin units (EU)/mL (r2≧0.99). Based on three times the standard deviation of a blank and the slope of a calibration curve, systems of the invention can detect endotoxin concentrations of 0.003 EU/mL or lower. The variability of the assay method is less than 5% (n=10). Analysis time required for a 0.005 EU/mL standard typically is less than 100 minutes.
LAL Reagent and Chromogenic Substrate
“LAL reagent” as used herein refers both to amebocyte lysates obtained from horseshoe crabs (e.g., Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleudus tridentata, or Tachypleudus gigas) and to “synthetic” LAL reagents. Synthetic LAL reagents include, for example, purified horseshoe crab Factor C protein (naturally occurring or recombinant) and, optionally, a surfactant, as described in WO 03/002976. One such reagent, “PyroGene™,” is available from Cambrex Bio Science Walkersville, Inc. LAL reagents preferably are obtained from Cambrex Bio Science Walkersville, Inc. Lyophilized LAL reagent can be reconstituted with 1.4 mL of LAL reagent water (endotoxin-free water) and kept refrigerated until use.
Any chromogenic substrate that can be used to detect an active serine protease (thrombin, trypsin, etc.) (i.e., has the sequence “Arg-chromogenic substrate) can be used in the automated systems disclosed herein. Such substrates are well-known and are commercially available. For example, the buffered chromogenic substrate (p-nitroaniline terminated pentapeptide (Ac-Ile-Glu-Ala-Arg-pNA, S50-640) is suitable and can be reconstituted with LAL reagent water and stored under refrigeration until use. Fluorogenic substrates having the sequence “Arg-fluorogenic substrate” also can be used and are encompassed within the term “chromogenic substrate.”
E. coli 055:B5 lyophilized endotoxin obtained from Cambrex Bio Science Walkersville, Inc. can be used to generate standard curves. Typically, lyophilized endotoxin is reconstituted with endotoxin-free water (LAL reagent water, Cambrex Bio Science Walkersville, Inc.) and vortexed for at least five minutes to yield a concentration of 50 EU/mL. Refrigerated reconstituted endotoxin is stable for at least one month. For the preparation of working standards, the stock solution is warmed to room temperature, vortexed for 5 minutes, diluted with LAL reagent water, and vortexed again before use.
Generally, detection systems of the invention involve withdrawing a sample volume of a fluid from a conduit (e.g., a production line) through a first flow path (as shown in an embodiment in
In alternative embodiments, other known bi-directional fluid delivery pumps 20 can be used. For example, in an alternative embodiment the system 10 can include a conventional bi-directional peristaltic pump (not shown). These peristaltic pumps pull fluid from one side of an internal area and push it to an opposing side of the area using a set of rollers and compressible pump tubing.
A connection adapter or union 40 can be positioned between the bi-directional fluid delivery pump 20 and the three-port valve 30 for coupling the bi-directional fluid delivery pump 20 to the three-port valve 30. As shown in
The interior of the connection adapter 40 is substantially open so that fluid entering through either an upstream or downstream end of the connection adapter 40 can pass through and exit the connection adapter 40 at its opposite end. The connection adapter 40 includes a first port 42 that is connected to, and in fluid communication with, an outlet/inlet port 27 of the syringe housing 23. As illustrated in
As illustrated in
A second port 34 of the valve 30 is connected to an on/off solenoid valve 60 as shown in
The on/off solenoid valve 60 is in fluid communication with the second port 34 and a filtered compressed air source 63. In an embodiment of the present invention, the solenoid valve 60 includes a conventional solenoid valve. Such a solenoid valve can be obtained from Bio-Chem Valve Corp. of Hanover N.J. under part type 075T2. The solenoid valve 60 can be regulated for a predetermined pressure. In a preferred embodiment, the solenoid valve 60 can be set at 15 psi. The operation of the solenoid valve 60 can be controlled by a computer running WinPump™ software via a programmable digital output and a low current relay. In an alternative embodiment, a 3-way solenoid valve can be in positioned in communication with the pump 20. A filter 64 can be included within the compressed air source 63 or downstream of the air source 63 for filtering any impurities from the air exiting the compressed air source 63. In an embodiment, the filter 64 can include an Acrodisce® glass fiber filter from Pall-Gelman.
A third port 36 of the three-port valve 30 is located downstream of the first two ports 32, 34 as the piston 23 of the syringe pump 20 forces fluid (including air) toward the selection valve 70. The third port 36 receives an end of the holding coil 50 so that the interior of the three-port valve 30 and the holding coil are in fluid communication when the third port 36 is open. A bi-directional valve 37 that permits fluid to enter and exit the three-port valve 30 can be positioned at the third port 36. Like the other bi-directional valves 33, 35, bi-directional valve 37 can include any known valve that allows fluid to enter and exit the three-port valve 30 at the third port 36.
The system 10 also includes the holding coil 50 in which liquid segments (i.e., an LAL reagent, a chromogenic substrate, and a test fluid sample) are “stacked” to form a stacked fluid slug. The holding coil 50 extends between the three-port connector valve 30 and a rotary selection valve 70. As shown in
The term “coil” is used herein to describe the holding coil 50 because in normal practice a coiled tubing of length, for example, of between about 1 meter and about 5 meters with an internal diameter of about 0.5 mm to about 0.8 mm is used in a tightly coiled or knotted orientation so as to decrease longitudinal dispersion. However, the holding coil 50 is not limited to a coiled length of tubing. Use of the term “coil” is common practice in flow analysis and should not be taken to preclude use of other shaped tubing including straight tubing and knotted tubing, tubing containing beads or other dispersion modifying aids, reactive materials such as particles of solid phase catalysts, or even mixing chambers. In an embodiment, the temperature of the holding coil 50 can be controlled either by means of control computer (not shown) or an auxiliary system (not shown). The contents of the holding coil 50 may also be subjected to external excitation such as ultraviolet light, ultrasound, heat, radiation or microwave energy, with the source of these exciting phenomena being either controlled by control computer (not shown) or by auxiliary systems (not shown).
The individual, isolated ports 81-88 of the selection valve are distributed around a common access port 72 that receives the mixed liquids from the holding coil. The common access port 72 can establish a fluid flow path with each of the isolated ports 81-88. The selection valve 70 includes a selector mechanism whereby at least one of the ports 81-88 has access to the common access port 72 so that fluid flow is established between the ports 81-88 and the holding coil 50. As illustrated in
As illustrated in
The detector 100 is schematically illustrated in
Although the following is related to the single detector cell 105 illustrated in
The tube 120 is connected to one of the outlet ports 81-84 of the valve 70 via coupled conduit 132 so that the detector cell 105 and the holding coil 50 can be in fluid communication when the common access port 72 and the respective outlet port 81-84 are aligned when open. Conduit 132 can include a length of tubing having an internal passage for delivering mixed liquid from the valve 70 to the tube 120. In one embodiment, the conduit 132 is between about 3 cm and 15 cm in length. The length of the conduit 132 is sized to allow the programmed final displacement of the syringe pump 20 to locate the middle of the final mixed 200 μL slug of the chromogenic substrate, LAL reagent, and test sample in the illuminated region of the detector cell 105. Another length of tubing 123 extends from the detector cell 105 to a waste container 140. As illustrated, the tube 120 extends a length from each side of the detector block 110. The tube 120 can extend between 5 cm and 15 cm on either side of the exterior surface of the block 110. In a preferred embodiment, the tube 120 extends about 10 cm on either side of the block 110. The tube 120 is covered with a material that prevents the intrusion of ambient light into the tube 120. In one embodiment, black heat shrink tubing covers the tube 120 outside the block 110. In another embodiment, metal tubing covers the tube 120 outside the block 110.
The detector cell 105 also includes a light source 150 for the absorbance measurement for each cell, as shown in
As shown in
In operation, the LED 154 is driven at about 15 mA (12 V with a 500 series resistor). Fiber 170 carries the light transmitted through the interference filter 163 to the block 110 of the detector cell 105. Fiber 172 carries the light transmitted through the block 110 of the detector cell 105 to the signal photodiode 182. In an embodiment, the signal photodiode 182 is kept in a separate electronics enclosure 183 from the block 110. In one embodiment, a conventional signal photodiode usable in the detector 100 is available from Siemens under part number BPW34. The detector cell 105 also includes a third fiber 176 that collects light from the bottom of the LED 154 and addresses a reference photodiode 186. As illustrated, each electronics enclosure 183 for the detector 100 contains a signal photodiode 182 and a reference photodiode 186. Fibers 170 and 176 are securely connected to the union 160 and hold the LED 154 securely in place. In one embodiment, cooperating nuts and ferrules can form this connection of the fibers 170, 176 to the union 160. The photocurrent from each of the photodiode 182, 186 is converted to voltage using dual JFET operational amplifiers 184, such as those available from Texas Instrument under part number TL082. Each operational amplifier 184 services at least one photodiodes 182, 186 within the detector 100 and each with a nominal gain of 1 V/A. In an embodiment, each operational amplifier 184 can service a pair of photodiodes 182, 186.
According to the present invention, the eight generated voltage signals (two from each detector cell 105) are acquired by signal comparing system 191 that includes a computer 197 through a 12-bit analog-digital converter 193. In one embodiment, the computer 197 includes a Pentium 11 class laptop PC through A/D PC card (PCM-DAS16D 12/AO, Measurement computing of Middleboro, Mass.). Software calculates the ratio of the reference photodiode 186 output to the signal detector photodiode 182 output for each pair of photodiodes 182, 186 and calculates its logarithm to measure absorbance. The results are then compared to detect the presence of an endotoxin. In operation, the four sets of measured absorbance values can be continuously or sequentially displayed on the PC as a scrolling record.
The LEDs 154 or laser diodes at precisely this wavelength are available from, e.g., Cree, Inc., Nichia America Corporation, or Bivar, Inc., and can also be used in the present invention. In an alternative embodiment, any light source that emits in the range of 395-405 nm can be used, thereby possibly increasing photometric sensitivity and allowing the detection of smaller concentrations of pNA.
The normalized emission spectrum of an LED used in an embodiment of the system 10 of the present invention appears in
Absorbance=0.0587 [pNA, mM]+0.0152, r2=0.9627
Incorporation of a narrow band (HBW 1 nm) interference filter dramatically improved both sensitivity and linearity.
Absorbance=0.3557 [pNA, mM]+0.0110, r2=0.9995
The kinetics of the LAL reaction can be temperature dependent. The temperature of the detector cell block 110 is maintained constant at 37±0.5° C. by a miniature temperature controller 190 as shown in
In a typical operational sequence, 100 μL of a test sample, 30 μL of an LAL reagent, and 70 μL of a chromogenic substrate solution were sequentially aspirated by the operation of the syringe pump 20. Suitable concentrations of the LAL reagent and chromogenic substrate are taught, for example, in U.S. Pat. No. 5,310,657. A gas, typically air, is introduced through one of the ports of valve 70 before and after aspiration of an LAL reagent, chromogenic substrate, and test sample (which form the stacked fluid slug). As a result, a buffer of gas is positioned at either side of the liquids drawn into the holding coil 50. The contiguous solutions in holding coil 50 were then thoroughly mixed by additional operations of the syringe pump 20 in which the piston 22 reciprocates back and forth within its housing 23. The gas buffers (e.g., air buffers) allow a user to obtain a completely mixed liquid segment such that precise positioning of the segment in the detector 100 does not become an issue that must be considered during the detecting process according to the present invention. Test samples can be withdrawn from a production line; this process can be automated. In an embodiment, multiple stacked fluid slugs comprising aliquots of the same test sample, each separated by a gas buffer, can be positioned within the holding coil 50.
In an embodiment, the piston 22 within the housing 23 is moved at a controlled speed that mixes the liquids within the holding coil 50 without breaking up the liquids or taking an excessive length of time. In an embodiment, the piston 22 makes between two and four complete reciprocating cycles, each cycle including a backward and forward movement, at speeds of between about 4000 and 8000 steps per second. In a preferred embodiment, the speed of the piston is about 6000 steps per second (62.5 μL/s). After the final mixing step of the piston pump 20, the combined liquid is delivered to one of the four detector cells 105 via the holding coil 50 and the selection valve 70.
In another embodiment of the system 10, a valve 70 is designed with at least sixteen ports to accommodate connections to at least six detection cells 105, endotoxin standards of three different concentrations, an LAL reagent, a chromogenic substrate, two cleaning solutions, endotoxin-free water and finally the sample port (configured with a very short line from a recirculating sample loop, as typically used in a water purification system). In this embodiment, the sequence for analysis of six different solutions could include: blank, sample, 0.005 EU/ml standard, sample spiked with a 0.05 EU/mL standard (50 μL+50 μL), 0.05 EU/mL standard, and 0.5 EU/mL standard. In this scenario, no cleaning other than air blow down is needed between samples, and cleaning is performed only after the analysis of the six solutions is over. Commonly available sixteen port selector valves can be used in this embodiment. If a greater number of solutions need to be analyzed, selector valves with as many as twenty-eight ports can be used as discussed above.
Photometric precision of the system can be evaluated by using 0-2 mM buffered pNA solutions that covers an absorbance range of 0-1 AU. Day to day reproducibility is excellent For example, a six-point calibration plot on disparate days yielded the respective linear calibration equations:
Absorbance, AU=0.4929 [pNA, mM]+0.0011, r2=0.9999 (3)
Absorbance, AU=0.4913 [pNA, mM]+0.0004, r2=0.9998 (4)
Stability of LAL Reagent and Chromogenic Substrate
Lysate-substrate reagents for use in chromogenic assays typically consist of a mixture of amebocyte lysate and substrate, which is supplied as a co-lyophilized solid in sterile containers. Immediately before use, the user or a robotic system reconstitutes the lysate-reagent by adding a prescribed amount of endotoxin-free reagent water. Equal amounts of the reconstituted reagent and a test sample are pipetted into microplate wells using standard sterile techniques, and the absorbance is monitored as a function of time. A plot of the logarithm of the time t for the starting absorbance to increase by a fixed amount (typically 0.2 AU) vs. log [endotoxin] is linear with a negative slope (color develops faster as the endotoxin concentration increases). The endotoxin concentration of a sample is determined by reference to a calibration curve generated with endotoxin standards and the same reagent batch, usually on the same microplate.
In systems such as those disclosed herein, the LAL reagent and chromogenic substrate should be reasonably stable. Preferably, these components should not need replacement more often than once a week, otherwise the purpose of automating the system is compromised. The typical combined lysate-substrate reagent is too unstable when kept at room temperature (22-24° C.); even in the absence of any endotoxin, cleavage of pNA from the substrate is rapid. The background absorbance of the blank reagent gets so high within a relatively short period, e.g., 8 hours, that it becomes unusable. In many pharmaceutical plants where sterile water is made, the ambient temperature is often higher than this, and such a reagent preparation and usage protocol will be unsuitable.
We have found that maintaining the LAL reagent and chromogenic substrate in whatever form (e.g., liquid or lyophilized) separately until their combination at the point of use increases stability of these components. Data demonstrating how calibration changes as a function of the reagent age are summarized in
In the case of reagents maintained separately until point of use, the reaction time decreases between days 1 and 2 then increases on day 3 past the original day 1 values such that the biggest change occurs between days 2 and 3. Thereafter reaction time increases monotonously. The maximum change in the reaction time during the 8 day period is 31% at the low endotoxin end and 19% at the high endotoxin end. The general direction of change in the reaction time as a function of aging is highly reproducible.
For endotoxin measurement, calibration with standards preferably is tested with the same batch of reagents at about the same time the sample measurement is conducted. Therefore it is not necessary that one make a calibration plot on day 1 and be able to use that plot for a week—with each set of samples, a set of calibration standards will be run. Over a week of use (under the above respective storage conditions), both sets of reagents provide an LOD that is more than adequate for monitoring of regulatory compliance. Separate reagents, if refrigerated, allow operation over a much longer period of time.
pH and Temperature for the Chromogenic LAL Assay
According to the literature, the optimum pH for the activation of the LAL reagent is 7.5, while that for the enzymatic cleavage of pNA from the substrate is 8.2-8.5 (Tsuji et al., Appl. Env. Microbiol. 48, 550-55, 1984; Bussey & Tsuji, J. Parenter. Sci. Technol 38, 228-33, 1984; Dunér, J. Biochem. Biophy. Meth., 26, 131-42, 1993). In a single mixed solution, the optimum pH is 7.7-7.8; the sensitivity is constant in this region (Dunér, 1993). The optimum temperature for the chromogenic LAL assay has been investigated by several researchers and reported to be 37° C. (Bussey & Tsuji 1984; Dunér, 1993). We found that these reported optima apply to the systems disclosed herein as well.
Cleaning Procedures
The present invention also provides cleaning procedures to prevent cross contamination between liquids having the same or different endotoxin concentrations. Contamination is a significant consideration when designing an automated system that is exposed to different concentrations of endotoxin. Even when the system 10 is initially thoroughly cleaned and endotoxin-free, contamination can develop over use due to sample carryover. Contamination is a particular problem because of the propensity of endotoxin to adsorb onto surfaces.
In one embodiment, the system 10 is washed after each set of mixed liquids has been placed into one of the detector cells 105 of the detector 100. In operation, after the mixed liquids pass from the holding coil 50 through the valve 70 and into a detector cell 105, the holding coil 50 and portions of the valve 70 are washed a plurality of times with LAL reagent water for the next sample if the next sample is a repeat of the previous sample. If more than one cell needs to be cleaned, the valve 70 is programmed to rotate to each selected cell and liquid is similarly blown to waste. In one embodiment, the holding coil 50 is washed three times. If a sample or standard with a different, particularly a lower endotoxin concentration is being mixed, then a more stringent cleaning protocol, such as those described below, can be used.
More stringent cleaning protocols can use depyrogenating solutions to remove pyrogens from pharmaceutical processing equipment. One such detergent product, specifically formulated for removing endotoxins (PyroCLEAN™, available from ALerCHEK, Inc.), was indeed effective in removing the endotoxin residuals. However, excessive washing protocols can be required to remove the depyrogenating agent from within the system 10. In another embodiment, strong bases and organic solvents that denature proteins, and aqueous and ethanolic NaOH solutions can be used for the removal of endotoxins. Certain of these bases, solvents and solutions can be used alone and in combination at room temperature and with solutions preheated to temperatures at, and between, about 37° C. and about 60° C., for various residence times and in combination with a nonionic fluorosurfactant, such as Zonyl FSN from DuPont.
The chosen cleaning agent should be in contact with the surface to be cleaned for a predetermined and extended period of time sufficient to remove endotoxin. This time may differ with each cleaning agent. Elevating the temperature of the cleaning agent may increase its effectiveness. In addition, the effectiveness of NaOH solutions in the concentration range of 0.2 to 2 M perceptibly increases with concentration.
In a preferred protocol, a room temperature cleaning protocol is performed. This protocol includes a treatment with about 2 M NaOH being in contact with the interior of the system 10 for a predetermined period of time. The predetermined period of time can be between about five and twenty minutes. In a preferred embodiment, the predetermined period is about ten minutes. In a preferred embodiment, 300 μL of about 2 M NaOH solution is aspirated by the pump 20 through an open port, for example port 85, of the selection valve 70. This NaOH solution is introduced into each detector cell 105 and the holding coil 50. After the NaOH solution has been in contact with the system for ten minutes, the NaOH solution is blown out by the air introduced from the pressurized air source in response to the opening of the solenoid 60. The pressurized air causes the NaOH solution to be removed from the holding coil 50 and each detector cell 105.
Next, the protocol includes repeated treatments of an ethanol:water solution (e.g., about 50% ethanol) and a water wash. In a preferred embodiment, about 300 μL of 1:1 ethanol and water mixture is introduced into each detector cell 105, the valve 70, and the holding coil 50 through a respective port, such as port 85, of the valve 70, and then blown off after about one minute of residence in the system 10. The ethanol:water wash step is then repeated three times, followed by a final wash step with endotoxin-free water. The endotoxin-free water is allowed to reside in the system 10 for about one minute. The system 10 can be cleaned and/or primed prior to use. Priming can utilize the steps and protocols discussed below with respect to the cleaning.
An alternative cleaning protocol is described in Example 3.
In any of these cleaning protocols, the wash liquid can be removed from the system 10 by opening the solenoid valve 60 and introducing compressed air blows into the holding coil 50 and the valve 70. The air blasts drive off the liquid within the holding coil 50, the valve 70 and a detector cell 105 to waste. The pressurized air can be introduced for a predetermined period of time. Such a period of airflow introduction can be between ten seconds and one minute. In a preferred embodiment, the period of airflow introduction can be about 30 seconds.
Performance and Limit of Detection
While there is some difference in the exact reaction times from one reagent batch to another, the typical time required to reach a 20 ΔmAU threshold in systems described above ranges from 7650 s for a blank to 4850 s for 0.005 EU/mL endotoxin. This constitutes a substantial difference and permits good resolution at low concentrations.
All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference.
The above discussions do not limit the invention. Although the disclosure describes and illustrates preferred embodiments of the invention, it is to be understood that the invention is not limited to these particular embodiments. Many variations and modifications will now occur to those skilled in the art. For example, the active components of the system, including the pump 20, the three-port valve 30, the selection valve 70 and the detector 100 are controlled by one or more computers via appropriate analog and/or digital communications protocols.
Sample Analysis
Different tap water samples, diluted 50 to 100-fold with endotoxin free water, were analyzed at least in duplicate, both by an embodiment of the system described above and by a commercial microplate reader-based instrument. It has been reported that Mg2+ accelerates, while Ca2+ inhibits, the color development in the chromogenic LAL reaction. See Dewanjee et al., J Nucl. Med. 31, 243-45, 1990. The local tap water contains ˜1.5 mM each of Ca2+ and Mg2+ and also contains nearly 1300 ppm of total dissolved solids. Recovery of the added endotoxin ranged from 87 to 124%. This would suggest that at least at low levels, presence of common ionic impurities do not cause any significant problems in measuring endotoxins.
The data generated by the two instruments are plotted in
Lysate Properties
The physical properties of the QCL-1000 lysate were studied to provide information on longevity once reconstituted and the ability to store and dispense the lysate. Once reconstituted, the storage temperature of the lysate could be an important variable in determining the useful life of the reagent cartridge. In a temperature controlled lyophilizer, the temperature of the reconstituted lysate was slowly decreased to −4° C. with little change in appearance or viscosity. Depending on the cooling ability of the on-line device, low temperature storage could be used if this would extend the useful life of the lysate.
Shelf life studies on the reconstituted reagents were also performed. The endotoxin standards (0.05 EU/ml, 0.5 EU/ml, 5.0 EU/ml), substrate and LAL reagent water were all stored at room temperature and protected from light exposure. Two samples of lysate were used, one stored at 2-8° C. and one stored at 10° C. The latter temperature point was chosen as an achievable set point for a Peltier cooling device incorporated in the on-line system.
Based on the data in
Another issue was the length of time that the standard curve could be used. The following tables analyze the running % coefficient of variation (CV) for lysate stored at 10° C.
The assay variability never exceeds 25%, even when several days have elapsed between samples. The standard curve can easily be said to be useable during any one 24 hour period.
Because all components were aging, an experiment was done to determine if the lysate was the sole source of the increasing reaction times. Using fresh lysate, the data shown in
During the aging of the lysate, flocculant material appeared suspended in the vials. This material was present at a higher concentration in the lysate stored at 2-8° C. A product of the coagulation reaction, the flocculant material did not interfere with the overall reaction times (
Because fluids typically are dispensed and metered in an automated system of the invention using a time and pressure technique, viscosity of the fluids is relevant. During the shelf life experiments, the viscosity of LAL lysate appeared to increase over time. Viscosity measurements taken in a Zeitfuchs cross-arm viscometer showed that the viscosity of LAL lysate increased by 3.5% over the course of the experiments. Density of LAL lysate increased by 3%. (“WFI” is “water for injection,” preferably endotoxin-free water.)
Variations in the volume of fluids introduced into the reaction chamber could also affect the results of the assay. An experiment was performed in which the volume of LAL lysate and chromogenic substrate in the 100 μl of reformed K-QCL reagent was varied and used to detect endotoxin standards. The paired replicates had volume variations where 10% of the reagent was either added or removed. The only replicates that had a % CV greater than 3% were those involving variations in the lysate volume of ±10%. With the 0.5 EU/ml standard, the variation was 3.44%. In the case of the 5 EU/ml standard, the % CV of the paired replicates was 9.44%. Dispensing accuracy need only have a % CV of 5% or less to have a negligible affect on the assay.
Comparison of Endotoxin Removal Protocols
Using a quartz cuvette, 600 μl of K-QCL reagent was added to 600 μl of 0.05 EU/ml endotoxin standard. The reaction was then allowed to proceed at 37° C. until Δ0.305 OD had occurred. Referencing a standard curve generated in a microplate experiment, the back prediction indicated an endotoxin concentration in the cuvette of 0.042 EU/ml. A series of blanks were then run to determine if there was a chemical means of removing the endotoxin from the cuvette. One-hour incubations at 37° C. of each of the cleaning solutions was carried out, followed by a rinse with WFI. Solutions of 0.1M NaOH, 0.5M NaOH and 1% deoxycholic acid were tested, and none of the solutions gave satisfactory results. In most cases, the reaction times for the blank solutions did not vary significantly after the cleaning procedure.
At this point, the cuvette was depyrogenated in a production oven. Following this, a set of standards was run, followed by a wash of the cuvette with WFI. A blank was then run, with a reaction time nearly identical to the last standard. See
The next cleaning solution that was tried was 0.05% triethylamine (TEA), using the same procedure outlined above. The blank following the washing procedure reacted significantly slower than the lowest standard and was predicted to have a value of 0.01 EU/ml when compared to the standard curve. An assay performed in a microplate to test the inhibitory properties of TEA showed that spike recoveries were down approximately 50% in a solution of 0.05% TEA. While the full-strength solution is mildly inhibitory, thorough washing should reduce it to a level where there is no interference with the LAL assay. See
The next set of experiments applied the procedure developed in the cuvette to the Pyrex glass reaction chamber. Various standards were incubated in the chamber at 37° C. for 30 minutes, and the solution was then removed from the chamber. A 200 μl sample was read in a microplate at 405 nm.
This experiment generated a reasonable endpoint standard curve. The first blank was measured after running a full set of standards and then cleaning with TEA. A set of standards was then run, followed by the standard cleaning procedure. The following blank was then run. In this case, the endpoint absorbance was lower than that generated by the lowest standard, but not by a significant amount. The blank following the chemical cleaning procedure also produced a higher OD than the first blank run.
An interesting note is shown in the table below. Here, a wash using only DI water was performed after the 5.0 EU/ml standard, followed by a blank. In the reaction chamber, simple rinsing effectively removed a significant percentage of endotoxin, unlike the experiments done in the cuvette. Following this blank the cleaning protocol with TEA was employed, resulting in a further decrease in the background OD produced by the blank test solution.
This application claims the benefit of and incorporates by reference co-pending provisional application Ser. No. 60/376,268 filed Apr. 30, 2002.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/13519 | 4/30/2003 | WO | 6/30/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60376268 | Apr 2002 | US |
