JPH0141320B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to the production of extracts from the blood cells of organisms such as Limulus, polyphemus, horseshoe crabs. In particular, the present invention relates to an improved method for removing inhibitory factors found in the lysates of amoeboid cells of the genus Limulus. All of the extracts to which the present invention relates have the enzyme coagulase in common. This enzyme, in cooperation with other extract components, produces a gel or clot when it comes into contact with endotoxin. Therefore, these extracts are defined as endotoxin-clotting extracts. They are usually obtained from the blood amoeboid cells of Limulus, Polyphemus. Amoeboid cells are usually collected from blood, lysed to liberate soluble intracellular components, and centrifuged to remove suspended waste products. A suitable technique for obtaining such an extract from Limulus is described in British Patent No. 1499846. These extracts can also be obtained from other species, such as Tachypleus tridentatus. Endotoxin-clotting extracts contain two main components responsible for clot formation. These components are coagulase and coagulogen. It is believed that coagulase is activated by endotoxin and catalyzes the proteolytic hydrolysis of coagulogen into protein fragments that spontaneously polymerize to yield detectable products. Coagulase has been extensively studied and characterized. Illustrative achievements include Tai e+
al., J. Biol. Chem. 252 (14): 4773–4776 (1977)
and Solum, Thrombosis Research 2 :55-
70 (1973). Coreglogen and methods for its purification have been described by Gaffin in Biorheology 13 :273-280 (1976). Coagulogen essentially acts as a substrate for activated coagulase. Coagulogen is particularly useful as a measure of coagulase activity because it provides its own indicator, i.e., coagulase-hydrolyzed fragments form insoluble particles and sometimes semi-solid clots without the intervention of extra reagents. be. The high sensitivity of endotoxin-clotting extracts to endotoxins has led to their widespread adoption in the screening quantification of pyrogienes in human body fluids and biological agents such as pharmaceuticals and parenteral fluids. Several conventional methods have been employed to use extracts to perform endotoxin quantification.
These include methods that follow the increase in optical density of the extract or the formation of gel clots when contacted with endotoxin. Typical endotoxin quantification involves analyzing specific amounts of proteins contained in the clot. Commercial widespread use of such quantification is hampered by the variations in sensitivity encountered between different lots of Limulus extracts and the difficulty of correcting for this variation. Such titer fluctuations are thought to be based on seasonal factors, ie, depending on the time of year when Limulus blood is collected. At least one component that has been determined to be partially responsible for this variation has been characterized as an "inhibitor" of the clot reaction. This nomenclature is based on the apparent action of this inhibitor, since whenever it is present a loss of endotoxin quantification sensitivity is encountered. This inhibitor is an enzyme [Nachum et al., Journal of Invertebrate
Pathology, 32 :51-58 (1978)] or simply lipoproteins that bind endotoxin [Sullivan et al., Applied Microbiology, 28
(6): 1023-1026 (1974)]. Whatever its mode of action, this substance will henceforth be named an inhibitor. The Sullivan et al paper cited above also discloses techniques to reduce the activity of inhibitors. The method consists of shaking equal volumes of organic solvent and Limulus solute, centrifuging, and collecting the aqueous phase. This method has proven unsatisfactory because it requires toxic or highly volatile reagents and requires cumbersome phase separation. and
Residual organic solvents may remain in Sullivanetal products. Although water-immiscible solvents exhibit low solubility in water, in the case of chloroform this solubility is 0.82 parts per 100 parts of water at 20°C. The effect of this residue on the sample to be quantified may be unpredictable or undesirable. The presence of this inhibitor in the supposedly standardized extract prevented it from actually serving as a reliable standard. Furthermore, known standardized extracts are not manufactured with proper consideration of coagulogen and true coagulase levels in the product. Typical standardized Limulus lysates are described in US Pat. Nos. 3,944,391 and 4,038,147. These products are
To determine the rough titer of solute, batches of solute are prepared by simply quantifying them against systematic dilutions of endotoxin. The main difficulty with this method is that it is passive in nature, and the extract is given a single predetermined signal, e.g. the occurrence of a given nephelometric endpoint upon reaction with a given endotoxin concentration. It is not processed in any way to get it. This is because a new standard curve must be created at the user level for each new lot of solute.
Makes quality control even more difficult. Commercially, a constant concentration of endotoxin is used for endotoxin quantification, even though the original untreated lot may be higher than some predetermined levels and lower than others, resulting in signal scattering. It would be desirable to be able to supply solutes from various lots that successively produce the same result. It is therefore an object of the present invention to produce an endotoxin-clotting extract which provides a reproducible, predetermined analytical signal for a given endotoxin concentration within an analytically significant range. Other objects of the present invention are to improve the methods previously used for inactivation of Limulus inhibitors without the need for toxic or harmful reagents, with lower labor and material costs, and without the possibility of interference in the product. It is a method of replacing certain residues that does not leave them behind. These and other objects of the invention will be apparent from consideration of the specification as a whole. SUMMARY OF THE INVENTION The present inventors have surprisingly discovered that the effects of inhibitory factors can be removed from a Limulus extract by adjusting the pH of the extract to about 8.5 or higher. After the desired degree of inactivation has occurred, the pH of the treated extract
is lowered to the point where coagulase becomes active.
The resulting extract is significant in that it does not contain organic solvent residues and is substantially free of inhibitory factors. The improved method of the present invention can also be employed to produce standardized extracts. This standardized method for producing an endotoxin-clotting extract involves (a) substantially inactivating the inhibitor components of the natural endotoxin-clotting extract, and (b) measuring the coagulase activity of the extract. (c) subdividing the extract into aliquots calculated to contain a predetermined coagulase activity. It is usually desirable to determine that the concentration of coagulogen in the extract is at least in excess of a predetermined amount. The standardized product produced is a quantity of endotoxin-clotting extract that is substantially free of inhibitors and includes an endotoxin-clotting extract containing a predetermined coagulase activity. Again it is desirable that the volume of extract contains at least a predetermined amount of excess of coagulogen. DETAILED DESCRIPTION OF THE INVENTION The discovery that inhibitory factors can be destroyed by PH regulation was quite surprising. Levin et al, J.Lab.Clin.Med.
75(6):903-911 (1970) discloses several attempts to remove Limulus lytic clot reaction inhibitors found in plasma. Although Levin et al teaches inactivation of the inhibitor with chloroform, precipitation with trichloroacetic acid was unsuccessful in removing or inactivating the inhibitor. By the way, inhibitory factors in plasma have previously been bound to inhibitory factors found in Limulus extract (U.S. Patent No.
No. 4107077). Furthermore, not only did Levin et al suggest that acids are not effective against inhibitors;
Others also concluded that solute acidification irreversibly inactivates coagulase [Solnm,
Thrombosis Research, 2 :55-70 (1973)]. This is, of course, the opposite of the desired result here.
Surprisingly, however, when raising rather than lowering the PH of the endotoxin-clotting extract, the inhibitor is irreversibly inactivated, but the coagulase activity is not lost. The endotoxin-clotting extract which is the starting material for the improved method and composition of the present invention may be any composition containing coagulase. This composition is generally obtained by separating the soluble components of the Limulus amoeboid cells from the insoluble components, such as the cell wall, and is therefore referred to as an extract. However, if a coagulase assay method is employed that does not rely on the formation of an agglomerate or suspension of polymerized coagulogen particles, it is not necessary to remove insoluble components or lyse the cells. Furthermore, if an analytical substrate other than coagulogen is used to monitor coagulase activity, the extract need not contain coagulogen. However, it is essential that the starting material contains coagulase. These endotoxin-clotting extracts generally contain inhibitory factors. The activity of the inhibitor is determined by the present invention by adjusting the pH to the alkaline range, incubating until the desired degree of inactivation is achieved, and lowering the pH to the neutral range where the coagulase becomes active again. removed. One of the significant benefits of this method is that PH adjustment usually does not precipitate components of the extract, including inhibitory factors. Unlike the known chloroform method, there is thus no need to separate any components of the reaction mixture after inactivation of the inhibitor. A simple centrifugation or filtration procedure is often desirable if the inhibitor is inactivated at a pH above 11 and the treated extract is subsequently used in nephelometry or optical density methods for endotoxin quantification. There is. However, under many circumstances no special treatment of the extract is necessary after inactivation of the inhibitor by PH regulation. Nor is it necessary to inactivate 100% of the inhibitory factor. It is generally satisfactory that the lysate is substantially free of inhibitory factors, generally at least 75% inactivated. The degree of inactivation required will depend on the desired endotoxin sensitivity and the variability of inhibitor content in the lot being standardized. In this way, the degree of inactivation is arbitrary. Typical lysate of Limulus amoeboid cells
PH is about 6.0 to 7.0. Alkalinity of the present invention
PH adjustment can be carried out in any known manner, for example by titrating an aqueous alkaline solution into the extract while stirring well. However, instead of solutions it is also possible to use crystalline or solid basic substances. The latter method does not increase the volume of water to be removed and is therefore desirable if it is desired to subsequently freeze dry the extract. The alkaline material itself may be organic or inorganic, but dilute aqueous solutions of alkali metal hydroxides such as sodium hydroxide are preferred. Exemplary organic alkaline materials that may be used are alkylamines. The specific nature of the substance is not critical; it simply raises the PH without causing undesirable side reactions that may inactivate coagulase or coagulogen to an extent that prevents the solute from being used for endotoxin quantification. That's all you need. Buffers increase the amount of reagents needed to effect PH adjustment and are preferably not included in the extract or in the alkaline material. Since the alkaline substance must not have a detrimental effect on coagulase, endotoxin, and coagulogen if present, it is possible to choose cations that are actually advantageous for quantification. For example, since manganese and magnesium ions also act as cofactors for coagulase, manganese or magnesium hydroxides can be used as at least part of the alkaline substance. Unfortunately, the use of divalent metal cofactors is limited due to their low solubility. In all cases, whether or not the cation-containing composition contributes to alkalinity, it is preferred that the divalent cation cofactor for coagulase be added before or at the same time as the alkaline PH adjustment. However, cofactors are not essential. The extract is incubated at elevated pH until the desired degree of inactivation of the inhibitor occurs. It is desirable that all of the inhibitory factor be inactivated before incubation is stopped. The degree of inactivation depends on the incubation time, temperature and PH; for example, the longer the incubation, the higher the temperature and PH, the faster the inactivation. Optimization of these parameters is a matter of simple experimentation for those skilled in the art, and the conditions depend on the properties of each solute lot. PH to any point above about PH8.5, generally from about 8.5
11, and preferably about PH 9.0 to 10.0. Both coagulase and coagulogen are stable at this hydrogen ion concentration. However, other proteins in the extract may precipitate at a pH above about 11, so it may be beneficial to choose a lower pH and incubate for a longer time. The incubation time sufficient to inactivate the inhibitor will, of course, vary depending on the amount of inhibitor present and the degree of inactivation desired. generally 20
Minutes to 4 hours are sufficient, with 90 minutes being preferred at a pH of 9 and 4°C. Incubation temperatures can range from about 0°C to the temperature at which coagulase is thermally inactivated. The temperature of the latter has previously been determined to be approximately 60°C. The temperature should be maintained at about 0°C to 40°C, and is preferably maintained at about 4°C by submerging the reaction vessel in an ice bath. After incubation, the PH should be returned to a level where coagulase is active. An active coagulase is a coagulase that is capable of reversibly binding endotoxin or that is capable of so binding endotoxin and that can otherwise express this binding by catalysis on coagulogen or other suitable substrates. is defined as Usually also refers to catalytic alternatives. PH readjustment is most conveniently carried out by adding acid using the techniques originally employed to raise the PH. Generally, the pH is readjusted by adding a dilute aqueous acid solution to the alkaline extract with stirring. The preferred concentration of acid is about 0.1N. The optimal final PH is the one that provides optimal sensitivity of solutes in endotoxin quantification. This is usually the same as the optimum pH for coagulase activity,
The pH is about 6.0 to 7.5 and preferably 7. The lysate is then preferably lyophilized or stored frozen. The substances used to lower the PH, as well as alkaline substances, must not be harmful to coagulase, or coagulogens if present. Inorganic acids such as hydrochloric, sulfuric or nitric acid are generally satisfactory, but organic acids can also be used. This substance can be buffered,
And in fact the buffer need not be at an acidic PH, but can be prepared for use at any desired final PH, such as PH7. Alternatively, a buffer such as Tris may be added after the pH has been adjusted to about 7. Divalent cation cofactors for coagulase can also be added to the extract at this time. Once the inhibitor activity of the endotoxin-clotting extract has been substantially destroyed, the extract is suitable for use in the production of standardized extracts. Preferably, the inhibitory factor is inactivated by PH regulation as described above. However, other methods can also be used, such as the known chloroform extraction technique. Unless the inhibitor activity is destroyed, the competitive reaction thermodynamics of the inhibitor-endotoxin reaction and the coagulase-endotoxin reaction make it impossible to standardize extracts over a range of endotoxin concentrations. If, for example, an inhibitor-containing extract is quantified for coagulase activity at a coagulase saturation amount of endotoxin, e.g. 3 mg/ml, and a predetermined enzyme activity is added to each container, regardless of the activity of the original extract lot, When filled into containers in such amounts, the product is found to contain only about 8% of the activity determined when measuring endotoxin concentrations over the range of 25 to 100 pg/ml. It is speculated that this variation is due to endotoxin being wasted by the inhibitor, resulting in an apparent endotoxin level that is lower than actually present in the original sample. When the endotoxin concentration is high enough to activate all coagulase present, ie, to saturate the coagulase, wasting excess endotoxin by the inhibitor has no effect on coagulase activity. However, if coagulase is used as an indicator of endotoxin concentration, the enzyme will be even less saturated over the expected endotoxin concentration range. In such cases interference by inhibitory factors will necessarily appear in endotoxin quantification.
Similarly, if extracts are standardized at the level of a first unsaturated endotoxin and quantified at the level of a second endotoxin, extracts containing the inhibitor will give different results than those without it. will give. This discordant result is believed to result from the balance of coagulase and inhibitor thermodynamics. This may be because the activation of coagulase is favored at the first endotoxin level, while the activity of the inhibitor is favored at the second level. If the intended method of quantification is based on the hydrolysis of coagulogen by coagulase, the standard extract should be prepared in excess so that endotoxin quantification is not limited by coagulogen over the expected endotoxin concentration range. It should also contain the core globogen concentration. If coagulase is standardized simply by measuring enzyme activity at one point of endotoxin concentration, and the amount of coagulogen present is insufficient to indicate true coagulase activity at otherwise high endotoxin levels. , results at such high levels would indicate less endotoxin than is actually present. This failure can have serious negative implications in the diagnosis of bacteremia or quality control screening of parenteral products. It was found that the coagulogen concentration is generally proportional to the coagulase activity in the natural Limulus extract. For this reason, in many cases it is undesirable to do anything other than ensure that sufficient coagulogen is present, and in such cases it may not be necessary to add supplemental coagulogen to the extract. However, it is possible that inadvertent exposure of such extracts to external endotoxins during collection and processing operations can deplete coagulogen and thereby create a need for coagulogen supplementation. For this purpose, it should be determined that the core glogen concentration is at least at a predetermined level or is in great excess of the predetermined concentration. The predetermined amount of coagulogen depends on the expected endotoxin concentration in the sample to be quantified. If the sample is expected to be only slightly contaminated, between 1 and 100 pg/ml, approximately
A core glogen content of 150 μg is generally satisfactory. Coagulogen concentrations in excess of 100 μg per ml of solute typically make the product usable for most endotoxin-contaminated samples of biological material. The minimum amount of coagulogen is regulated to a low level by the detection limit of endotoxin by coagulase quantification, and this can be expected to improve in the future as this quantification method is further developed. On the other hand, the maximum amount that can be used is limited only by the maximum solubility of the coagulogen in aqueous solution, and is limited only by the maximum solubility of the coagulogen in aqueous solution, and even at exceptionally high endotoxin concentrations, e.g.
ml can theoretically be determined by the formation of coagulogen hydrolysis products, especially if they are removed from the reaction mixture as the hydrolysis proceeds. The selection of a specific predetermined coagulogen content, since coagulogen contents of 100 to 400 μg/ml or more do not adversely affect the quantification, and lower coagulogen concentrations are regulated by the state of the art regarding the detection of coagulogen hydrolysis products. is currently a matter of choice. Core Glogen is Analytical Biochemistry,
91:509-515 (1978) according to the Munford endotoxin quantitative method. Instead of quantifying unknown endotoxin as specified in this method, a fixed endotoxin concentration is
For use with labeled coagulogens, unknown coagulogens and coagulases. After the incubation period, the radioactivity bound to the clot is measured as in a typical competitive radioimmunoassay. Large amounts of coagulogen displace labeled coagulogen from coagulase-mediated precipitation, and the opposite occurs at relatively low levels of coagulogen. Thus, the higher the clot-bound radioactivity, the lower the core glogen concentration. This method can be quantified through the use of purified coagulogen standards. If the coagulogen concentration is lower than the predetermined concentration, it may be necessary to increase the coagulogen level in the extract. For example, a coagulogen-rich extract or purified coagulogen can be mixed with the extract to achieve the desired coagulogen concentration increase. Alternatively, coagulogens can be concentrated by ultrafiltration. The production of standardized endotoxin-clotting extracts requires the measurement of coagulase activity. This can be accomplished by any known method for quantifying coagulase. These methods are also often used to measure endotoxin concentrations. However, known, preferably saturating concentrations of endotoxin are used here. Saturated endotoxin concentration is generally around 1 mg/ml
in excess of endotoxin, preferably 3
mg/ml. Since relative rather than absolute enzyme activity is more appropriate for standardization purposes, this quantitative result can be converted to arbitrary enzyme units. Once the coagulase activity of the extract is determined, the extract is proportioned into the endotoxin quantification reaction mixture in an amount calculated to produce the selected enzyme activity. For example, the treated extract can be added directly into the endotoxin quantification reaction mixture in a volume calculated to give a predetermined activity. Since it is rather rapid for the analyst to always add a fixed volume of extract, it is necessary to add a predetermined activity to the container for storing the extract when reconstituted to a fixed volume of aqueous solution. can be filled with the calculated amount of extract liquid. Proportional filling saves the user the effort of performing a proportional restoration. The extract is then lyophilized. The invention will be more fully understood by reference to the following examples. Example 1 Limulus, Polyphemus
Two lots of amoeboid cells (Polyphemus polyphemus) were collected separately and lysed by the method of GB 1499846. One lot was used as the reference solute. The remaining lot was used as starting material for the production of standardized solutes according to the method of the invention. This was called Lot 8-113. Both lots were first quantified for coagulase activity. A 50mM Tris buffer containing PH7.5 and _10mMgCl2 was used as the following buffer. E.
E. coli endotoxin (055:B5, Difco, Laboratories) was dissolved in buffer. Coagulogen was adjusted to a concentration of 8.0 mg/ml in buffer. The method consists of precipitating the crude solute with 80% saturated ammonium sulfate before redissolving and passing through a Sephadex column, then eluting the column with phosphate buffer and further purifying the eluate on a carboxymethyl cellulose column. Except for this, the Solum method mentioned above was used.
3.0mg/ml endotoxin solution 0.1ml, solute 0.1ml
and 2.7 ml of buffer were mixed and incubated for 8 minutes. 360nM after incubation and
The change in optical density at 37°C was recorded. Enzyme 1
The unit was arbitrarily defined as 0.001 absorption unit change in optical density at 360 nM. Reference lot and lot 8-113 have enzyme activities of 665 and 440, respectively.
Units/ml are shown. A portion of each lysate lot was then distributed into vials. To distribute the same coagulase activity into each vial, reference lot and lot 8-113 were added to the 2 ml and 2.65 ml containers, respectively. The contents of the vial were then lyophilized. The inhibitor activity of each lot was then inactivated by the novel method of the present invention. 2 vials of reference lot and 2 vials of solute to be standardized at 2.5
ml sterile water. One vial of each lot is
The pH was adjusted to 9.0 with 0.1N NaOH and incubated at 4°C for 90 minutes. Next, check the pH of each vial.
It was lowered to 7.5 by stirring with 0.1NHCl at 4°C. Add 2.5 ml of buffer to each vial and transfer the lysate to endotoxin concentrations of 0, 25, 50 and
E above at 100 pg/ml. quantified against coli endotoxin. The quantitative method is substantially that described in British Patent No. 1499846. Table 1 below compares the optical density at 650 nM of treated and control solutes. Table: As can be seen from these results, both alkaline treatment and coagulase proration increased the sensitivity of the lysate to endotoxin and eliminated the fluctuations encountered with natural lysates. By adjusting the pH,
The standard lot has a coagulase activity almost equal to that of the reference lot, 97.9 to 99.7% of that of the reference lot.
It continued to show in the order of 8-
113 lots have endotoxin concentrations of 25, 50 and
At 100 pg/ml, only 72.6 of the reference lot activity
%, 73.9% and 82.3% apparent activity. Example 2 The procedure of Example 1 was repeated for the reference lot and another solute lot designated lot 8-112. As in Example 1 of Lot 8-113,
The coagulase activity of lot 8-112 was distributed to the same level as the reference lot. However, in this implementation the inhibitor was inactivated by chloroform extraction. See Chloroform Lot and Lot 8-
112 in a ratio of 1 part chloroform to 2 parts solute and stirred well and incubated at 4°C for 60 minutes. The solute-chloroform mixture was centrifuged to remove the precipitate and undissolved chloroform, and then Example 1
It was treated and quantified in the same manner as above. The results are shown in the table below. Table: The chloroform extraction technique yielded solutes that exhibited greater variation between determinations at various endotoxin concentrations than was encountered with the PH adjustment method of Example 1. That is, lot 8-112 was 14.4% and 10.2% more active and 7.3% less active than the reference lot at endotoxin concentrations of 100, 50, and 25 pg/ml, respectively. However, this example illustrates that the chloroform extraction method can also be used to produce the standardized endotoxin-clotting extract of the present invention.

Claims (1)

[Scope of Claims] 1. A method for inactivating an inhibitory factor component of an endotoxin-clotting extract, comprising adjusting the pH of the extract to 8.5 or higher so as to inactivate the inhibitory factor;
The above method, characterized in that the pH is then lowered to a level at which coagulase is active. 2. The endotoxin-clotting extract is Limulus amoeboid cell lysate, and the inactivation of the inhibitor component involves raising the pH of the lysate to about 8.5 to 11 and increasing the pH of the lysate until the desired degree of inactivation occurs. incubating the solute; and incubating the solute.
The method according to paragraph 1, which is carried out by lowering the pH to about 7. 3. The method of item 2, wherein the inactivation is carried out without forming precipitates. 4. A method for producing a standardized endotoxin-clotting extract, comprising: (a) adjusting the pH of the extract to 8.5 or higher so as to inactivate the inhibitor components of the extract;
(b) measuring the coagulase activity of the extract; (c) evaluating the coagulogen content of the extract; and (d) controlling the extract. Said method, characterized in that it is subdivided into portions calculated to contain a predetermined coagulase activity. 5. The method according to item 4, wherein the subdivision is performed by dispensing a volume of the extract into each container, each container having a predetermined coagulase activity. 6. The method according to item 5, wherein the extract is divided into small portions by freeze-drying after the dispensing. 7. The method according to item 4, wherein the endotoxin-clotting extract is a Limulus amoeboid cell lysate. 8. The method according to paragraphs 4 to 7, wherein the core glogen level is adjusted in excess of a predetermined concentration.