Process for removing pyrogens

Abstract: Pyrogens are removed out of a solution thereof by (1) treating the solution with a membrane and then (2) treating the resulting solution with a pyrogen adsorbent. The obtained solution is useful in the pharmaceutical field.

Description: This invention relates to a process for removing pyrogens from a pyrogen-containing solution to thereby prepare a pyrogen-free solution. Pyrogen-free solutions are used for medical purposes, for example, as a dialysate for treating those who suffer from renal insufficiency, as a replenishing fluid for an artificial kidney of filtration type, as a diluent for an instillation drug of a high concentration for bedside uses, in washing various medical instruments, in the preparation of drugs such as purified water for injection and in washing injection containers. For hematic administration it is necessary to remove pyrogens from an aqueous solution containing drug(s). . Examples of such an aqueous solution include those of various drugs which should be subjected to a pyrogen test according to The Pharmacopoeia of Japan, for example, fructose injection, physiological saline solution, dextran 40 injection, glucose injection, Ringer solution and sodium citrate injection for transfusion. A pyrogen, in particular an endotoxin which is a typical example thereof, originates from the cell wall of a gram-negative bacterium and comprises lipopolysaccharides. The molecular weight of lipopolysaccharide varies thousands to tens of thousands depending on its bacterial origin. However; lipopolysaccharides are considered to be present as an associate which has relatively hundreds of thousands to millions of molecular weight in aqueous solution. When it invades into the body, even in an ultramicro amount, it would induce serious fever and sometimes cause death. It has been recently disclosed that lipid A, which is a constituent of an endotoxin and has a molecular weight of approximately 2,000, is also pyrogenic. The Pharmacopoeia of Japan obliges us to effect a pyrogen test on rabbits in order to detect endotoxins. Since every conventional pyrogen test requires a troublesome procedure and a prolonged period of time, there has been marketed a detection reagent by which endotoxins can be readily detected within a short period of time. This reagent takes advantage of a reaction between an endotoxin and limulus hemocytes whereby the hemocytes coagulate. Further there has been established a colorimetric method with the use of a synthetic substrate based on the reaction mechanism between an endotoxin and limulus hemocytes. Recently the detection limit of this colorimetric method has been further improved from the order of ng/ml to that of pg/ml. Thus it is now possible to detect an ultramicro amount of endotoxins. Known processes for preparing a pyrogen-free solution from a pyrogen-containing stock solution include those wherein a reverse osmotic membrane or an ultrafiltration membrane is employed. A reverse osmotic membrane, which is used in desalination, has a cut-off molecular weight of 500 or below. Thus it enables to remove lipopolysaccharides constituting endotoxins as well as lipid A which is the terminal constitutent thereof. However, the reverse osmotic membrane should be operated under elevated pressure, which causes a considerable leakage of the stock solution from a damaged part of the membrane and requires a large amount of energy. On the other hand, an ultrafiltration membrane may be operated under lower pressure and thus requires less energy than the reverse osmotic membrane does. However the former is inferior to the latter in the capability of removing endotoxins. Namely, the lipopolysaccharides of a high molecular weight can be removed by using an ultrafiltration membrane of a cut-off molecular weight of, for example, 30,000. However lipid A, which is the terminal constituent of the lipopolysaccharides and has a lower molecular weight, would permeate therethrough. Therefore it is necessary to employ an ultrafiltration membrane of a cut-off molecular weight of 5,000 or below, though the permeation rate thereof is lower than that of the ultrafiltration membrane having a fractionation or cut-off molecular weight of 30,000. In addition, an ultrafiltration membrane is damaged upon prolonged operation, which might cause the leakage of some portion of the endotoxins accumulated on the whole surface thereof from the damaged part. In order to overcome these disadvantages, two-step filtration is commonly employed. In this method, however, the removal efficiency in the first step is high but that in the second step is low. It is therefore the object underlying the present invention to provide a process for removing pyrogens from a pyrogen-containing solution that is not accompanied by the problems as described above and which may be applied to any drug for hematic administration, from which pyrogens should be removed during the process of the preparation thereof, without limitation. According to the present invention this object is attained with a process for removing pyrogens out of a solution which comprises the steps of: (a) treating a solution containing pyrogens with an ultrafiltration membrane having a cut-off molecular weight of 10,000 to 200,000, a large pore size and a high permeation rate to water to remove pyrogens having molecular weights of ten thousands to millions and then (b) treating the solution with a pyrogen adsorbent to remove endotoxins of molecular weights of several tens of thousands or below and lipid A having molecular weights of about 2,000. Figs. 1 to 3 are each a schematic view of a preferable embodiment of the process of the present invention. Figs. 4 to 7 are each a schematic view of an example of the system of the present invention. Figs. 8 and 9 are each a schematic view of an example of the system of the present invention wherein two UF membranes are employed. In the figures, a represents a stock solution, b and e represent each a pump, c represents an ultrafiltration membrane module, and d represents an adsorbent; A represents a stock solution, B represents a regeneration solution, C represents a product solution, D is a regeneration waste, E is a waste. The treated liquid is received by a receiver. 1 represents a stock solution tank, 2 represents a regeneration solution tank, 3 represents a pyrogen adsorbent column, 4 represents a product solution tank, 5 represents an ultrafiltration membrane, 5' represents another ultrafiltration membrane, 6 represents a pump, 6' represents another pump, 7 represents a flow meter, and 11 to 20 represent each a valve. The process of the present invention is advantageous in that endotoxins of of high molecular weights can be removed by the pyrogen adsorbent when the ultrafiltration membrane might be damaged upon prolonged operation; and that the lipid A of a molecular weight of approximately 2,000 can be also stably removed throughout a prolonged operation. The process of the present invention is further available in the removal of pyrogens from an aqueous solution of a useful material of a low molecular weight of, e.g., thousands of daltons or below, capable of permeating through an ultrafiltration membrane. In this case, the ultrafiltration membrane to be employed preferably has a cut-off molecular weight of 10,000 to 200,000 and an average pore size of 2x10⁻⁹m to 10⁻⁷m (20 Å to 0.1 µm), still preferably 3x10⁻⁹m to 2x10⁻⁸m (30 Å to 200 Å). The process of the present invention is further available in the removal of pyrogens from an aqueous solution containing a drug for hematic administration. More precisely, endotoxins having higher molecular weights than that of the drug, such as associated lipopolysaccharides and bacteria from which the endotoxins originate, are removed by using a membrane of a large pore size through which the drug can readily permeate. Then endotoxins of lower molecular weights and lipid A are selectively removed with the pyrogen adsorbent. Thus all of the pyrogens contained in the drug solution can be efficiently removed. The membrane to be used in the above process should have such a pore size that the drug can readily pass therethrough while impurities including endotoxins larger than the drug and microorganisms can be removed thereby. Examples of the drug include blood preparations, enzyme preparations, recombined proteins, peptides, hormones and polysaccharides for drugs. Further various injections and infusions such as dextran injection, fructose injection, glucose injection, Ringer solution and sodium citrate injection for transfusion may be treated in the above mentioned manner. When the drug is a compound having a molecular weight of 1,000,000 daltons or above, such as hyaluronic acid, a UF-membrane of a cut-off molecular weight (which means the upper limit of the molecular weight of a sample capable of passing through the membrane; the same will apply hereinafter) of 1,000,000 or above may be employed. When the drug is a compound having a molecular weight of 100,000 to 1,000,000 daltons, such as immunoglobulin, an UF membrane of a fractionation molecular weight of 1,000,000 or above may be employed. When the drug is a compound having a molecular weight of 100,000 daltons or below, such as albumin, a membrane of a cut-off molecular weight of 100,000 or above may be employed. When the drug has a molecular weight of 10,000 daltons or below, a membrane of a cut-off molecular weight of 10,000 or above may be employed. Further a combination of two or more UF membranes may be employed. Figs. 8 and 9 show each a system for removing pyrogens with the use of two UF membranes. The two UF membranes in the system of Fig. 8 are located in the reverse order of those of Fig. 9. Either system may be selected depending on the drug and pyrogens contained therein. The UF-1 used in these systems is a membrane having a fractionation molecular weight which enables a sample to permeate therethrough as described above. The optimum fractionation molecular weight should be determined depending on the molecular weight of the sample to be treated. The UF-2 is a membrane having a fractionation molecular weight which prevents the sample from permeating therethrough. For example, when the sample is a compound of a molecular weight of 1,000,000 daltons or above, such as hyaluronic acid, it is possible to use a UF-2 having a cut-off molecular weight of 1,000,000 or below. When the sample is a compound of a molecular weight of 100,000 to 1,000,000 daltons, such as immunoglobulin, it is possible to use a UF-2 havihg a cut-off molecular weight of 100,000 or below. When the sample is a compound of a molecular weight of 10,000 to 100,000 daltons, such as albumin, it is possible to use a UF-2 having a cut-off molecular weight of 10,000 or below. When the sample is a compound of a molecular weight of 2,000 to 10,000 daltons it is possible to use an UF-2 havihg a cut-off molecular weight of 2,000 or below. Thus the UF-2 havihg the optimum cut-off molecular weight should be selected depending on the sample, similar to the case of the UF-1. As shown in Figs. 8 and 9, two microfiltration membranes (MF membranes) may be located before and/or after the UF membranes depending on the sample, though these MF membranes are not always required in these systems. The MF-1 is employed in order to remove macromolecules including cells of bacteria such as gram-negative ones which might be obstacles for the treatment with the UF membranes, while the MF-2 is employed for removing minute pyrogen adsorbent fragments (P.A.f.). It is preferable that each of these MF-1 and MF-2 membranes has a pore size of approximately 0.2 µm. The symbols LS, LM and LL used in Figs. 8 and 9 mean each the size of a pyrogen associate. A pyrogen generally forms associates having a certain molecular weight distribution in a solution. When treated with a membrane, therefore it is divided into fractions of associates different in molecular weight from each other. Thus these pyrogen associated are divided into the types depending on the size for convenience. Namely, a pyrogen associate of a molecular weight comparable to that of a sample is referred to as LM, while those larger and smaller than it are referred to as LL and LS, respectively. The membranes to be used in the present invention may be made of a synthetic polymer such as polysulfone (PS), polyacrylonitrile (PAN) or polyamide or a semisynthetic polymer such as cellulose acetate. This modules thereof may be selected from among, for example, hollow fiber (HF) type, spiral type, pleated type, tubular type and plate and frame type. Any pyrogen abdorbent may be used in the present invention without limitation, so long as it can efficiently adsorb pyrogens. Preferably examples thereof include those obtained by immobilizing, for example, an amino acid, iminodiacetic acid or an antibiotic on a substrate comprising, for example, agarose or cellulose. Alternately, an adsorbent obtained by binding a nitrogenous heterocyclic compound such as L-histidine to a water-insoluble carrier (cf. JP-A-183172/1982) or another one comprising hyaluronic acid and an anionic resin (cf. JP-A-67024/1979). Although the pyrogen-containing solution to be treated according to the present invention is not particularly restricted, those having an endotoxin concentration of 10⁴ ng/ml or below are particularly preferable. Referring now to the drawings, there are shown some preferable embodiments of the present invention. Figs. 1 to 3 schematically show each a process for preparing a pyrogen-free solution from a pyrogen-containing stock solution. The process of Fig. 1 may be employed when the packing ratio of the pyrogen adsorbent is high and the adsorbent column should be operated under elevated pressure. Namely, a stock solution a is pressurized with a pump b and passed through an ultrafiltration membrane module c to thereby give a permeating solution g . When the high packing ratio of the adsorbent d makes it impossible for the stock solution a to pass through the pyrogen adsorbent d under the membrane module pressure alone, a pump e is further provided before the adsorbent d to thereby repressurize the solution permeating through the ultrafiltration membrane module c . Then the solution is passed through the adsorbent d and thus the aimed pyrogen-free solution g is obtained. The process of Fig. 2 may be employed when the packing ratio of an adsorbent d is low and the permeation pressure is low. Namely, a stock solution is pressurized with a pump b and passed through the ultrafiltration membrane module c . The permeating solution thus obtained is then passed through an adsorbent d to thereby give the aimed pyrogen-free solution g . In the process of Fig. 3, an adsorbent is immersed in a reservoir for the solution permeating through an ultrafiltration membrane module. Namely, the solution passing through the ultrafiltration membrane module is temporarily stored in the reservoir f , similar to the process of Fig. 2, in which the adsorbent d is immersed. Thus the aimed pyrogen-free solution can be prepared in the reservoir. Therefore the pyrogen-free solution g can be used continuously or at any time. (Examples) The present process for removing pyrogens of the present invention comprises combining filtration with an ultrafiltration membrane with the adsorption with an pyrogen adsorbent. In the adsorption step, however, the capability of the pyrogen adsorbent is lowered with the lapse of time. When this capability falls below a threshold value, therefore, it is necessary to regenerate the adsorbent to thereby recover the capability thereof. However none of known systems for effectively removing pyrogens involves such a means of regenerating a pyrogen adsorbent. Accordingly, in a preferred embodiment of the present invention a means of efficiently regenerating the pyrogen adsorbent is provided. More specifically, this involves a means of removing pyrogens contained in a stock solution with the use of the pyrogen adsorbent as well as another means of regenerating said adsorbent with a regeneration solution. It is preferable that this system involves a means for preliminarily removing pyrogens contained in the regeneration solution by using an ultrafiltration membrane. As the regeneration solution, an aqueous solution of a compound of a low molecular weight is generally employed. Examples thereof include aqueous solutions of sodium deoxycholate, sodium hydroxide and sodium chloride (cf. JP-A-183712/1982). Thus pyrogens of higher molecular weights are exclusively removed from the regeneration solution by treating the same with an ultrafiltration membrane. It is further preferable that this system involves a means for washing the pyrogen adsorbent with the regeneration solution flowing in the reverse direction. Namely, the regeneration solution is passed in the reverse direction of the flow of the stock solution in normal operation. Thus the pyrogens adsorbed by the pyrogen adsorbent may be readily desorbed therefrom. It is further preferable that the stock solution and the regeneration solution are fed into the system through a common pump. The normal operation for feeding the stock solution and the regenerating operation for feeding the regeneration solution are conducted one after another. Thus it is possible to feed these solutions through the same pump, which serves for saving of one pump. It is further preferable that the system involves a means of treating the regeneration waste with an ultrafiltration membrane and then recovering the same. Thus the loss of the expensive regeneration solution can be decreased. Referring to the drawings, the system of the present invention will be described. Fig. 4 is a schematic view of an example of the system of the present invention, wherein A is a stock solution; B is a regeneration solution; C is a product solution; and D is a regeneration waste. The stock solution in a stock solution tank 1 is fed into a pyrogen adsorbent column 3 via a pump 6 and a flow meter 7. After pyrogens contained in the stock solution are adsorbed by the pyrogen adsorbent, the resulting product solution is stored in a product solution tank 4. When the capability of the pyrogen adsorbent is lowered to a threshold value, the feed of the stock solution is ceased and the pyrogen adsorbent is regenerated. The regeneration solution, from which pyrogens have been preliminarily removed with an ultrafiltration membrane 5, is stored in a regeneration solution tank 2 and fed into the pyrogen adsorbent column 3 via the pump 6 and the flow meter 7. After regenerating the pyrogen adsorbent, the resulting regeneration waste is discharged out of the system. Fig. 5 is a schematic view of another example of the system of the present invention. In this example, a pyrogen adsorbent is regenerated by a reverse washing method. A stock solution is fed into a pyrogen adsorbent column 3 via a pump 6. After removing pyrogens therefrom, the resulting product solution is stored in a product solution tank 4. When the pyrogen adsorbent is to be regenerated, the feed of the stock solution is ceased and a regeneration solution is fed into the pyrogen adsorbent column in the reverse direction via a pump 6'. Fig. 6 is a schematic view of still another example of the system of the present invention. In this example, the procedure of Fig. 5 is followed except that a stock solution and a regeneration solution are fed via a common pump. The stock soltuion flows through the sequence of stock solution tank 1 → valve 11 → pump 6 → valve 13 pyrogen adsorbent column 3 → valve 19 → valve 14 → product solution tank 4, while the regeneration solution flows through the sequence of regeneration solution tank 2 → valve 12 → pump 6 → valve 18 → pyrogen adsorbent column 3 → valve 20 → valve 17. Fig. 7 is a schematic view of still another example of the system of the present invention. In this example, a regeneration waste is filtered through an ultrafiltration membrane and then recovered. A stock solution flows through the sequence of stock solution tank 1 → valve 11 → pump 6 → valve 13 → pyrogen adsorbent column 3 → valve 14 → product solution tank 4, while a regeneration solution flows through the sequence of regeneration tank 2 → valve 12 → pump 6 → valve 13 → pyrogen adsorbent column 3 → valve 17 → ultrafiltration membrane 5'. The filtrate is returned to the regeneration solution tank 2 while the residue is discharged out of the system as a waste E. Thus the present invention in a preferred embodiment provides systems for removing pyrogens from a solution whereby a pyrogen adsorbent can be efficiently regenerated. To further illustrate the present invention, the following Examples and Comparative Examples will be given. Comparative Example 1 A stock solution of an endotoxin concentration of 250 ng/ml was continuously treated with a polyacrylonitrile ultrafiltration membrane module having a cut-off molecular weight of 30,000 at a filtration efficiency of 90 % for one week. Endotoxins were detected by subjecting the filtrate to two test methods, i.e., the limulus test with the use of Limulus HS Test Wako (mfd. by Wako Pure Chemicals Co., Ltd.) having a detection sensitivity of 0.01 ng/ml and the colorimetry with the use of Endospecy®, having a detection sensitivity of 1 pg/ml. As shown in the following Table, no endotoxin was detected by any of both methods until the second day. But endotoxins were detected by the colorimetry from the third day. On the fifth day, endotoxins were detected by both methods. Comparative Example 2 Polyacrylonitrile ultrafiltration membrane modules having a cut-off molecular weight of 80,000 were employed in two steps and the endotoxin removal efficiency of each module was evaluated. A stock soltuion of an endotoxin concentration of 250 ng/ml was treated with these modules. After the completion of each step, endotoxins contained in the treated solution were detected with use of Endospecy®. As shown in the following Table, the removal efficiency in the first step was 99.99 % while that in the second step was 80 %. Endotoxin conc. Removal efficiency First step: inlet after permeation 2.5 x 10⁵ pg/ml 99.99 % 25 pg/ml Second step: inlet after permeation 25 pg/ml 80.0 % 5 pg/ml Example 1 The systems of Figs. 1 to 3 were employed. In each system, a stock solution of an endotoxin concentration of 250 ng/ml was continuously treated with a polyacrylonitrile ultrafiltration membrane module of a cut-off molecular weight of 30,000 at a filtration efficiency of 90 %. Then the treated solution was passed through a pyrogen adsorbent comprising L-histidine immobilized on agarose. Subsequently endotoxins contained in the solution thus treated were detected colorimetrically. As shown in the following Table, no solution contained any detectable endotoxin. Example 2 The procedure of Example 1 was repeated except using a physiological saline solution containing 50 ng/ml of endotoxins. The obtained results were the same as those of Example 1. Example 3 The procedure of Example 1 was repeated except using a fructose injection containing 50 ng/ml of endotoxins. The obtained results were the same as those of Example 1. Example 4 A test was conducted by using a human serum albumin (HSA) solution of an HSA concentration of 5 % (determined from the UV absorbance at 280 nm), which had been adjusted to pH 6.5 and to an ion strength (µ) of 0.01 with a phosphate buffer solution, as a sample. According to the limulus test by using Limulus HS-Test Wako and a Toxinometer ET-201 (both mfd. by Wako Pure Chemicals Co., Ltd.), this solution contained approximately 1300 ng/ml of endotoxins. 100 ml of the sample soltuion was concentrated five-fold by treating the same with a pyrogen-free (1 pg/ml or less) polyether sulfone hollow fiber UF module (cut-off molecular weight: 100,000; FUS-1081, mfd. by Daicel Chemical Industries, Ltd.). The penetrating solution thus obtained contained 3.2 % of HSA (HSA recovery: approximately 40 %) and approximately 3 ng/ml of endotoxins. Then the permeating HSA solution was passed through a column packed with the same pyrogen adsorbent as the one used in Example 1 (i.e. 0.9 cm x 27.5 cm) at a flow rate, SV, of 2 (approximately 0.6 ml/min). The eluate thus obtained contained approximately 3 % of HSA (HSA recovery after column treatment: approximately 94 %) and approximately 0.016 ng/ml of endotoxins. Therefore the efficiency of removing pyrogens from the sample according to the process of the present invention was approximately 99.998 %. When the HSA in a sample is contaminated with bacteria, it is preferable to incorporate an MF-1 (microfiltration membrane) into the system for removing pyrogens (cf. Fig. 8). When there is a possibility that minute pyrogen adsorbent fragments (P.A.f.) might be eluted from the pyrogen adsorbent column, it is further preferable to incorporate an MF-2 (microfiltration membrane) into the system as the final filter.

Claims: A process for removing pyrogens out of a solution which comprises the steps of: (a) treating a solution containing pyrogens with an ultrafiltration membrane having a cut-off molecular weight of 10,000 to 200,000, a large pore size and a high permeation rate to water to remove pyrogens having molecular weights of ten thousands to millions and then (b) treating the solution with a pyrogen adsorbent to remove endotoxins of molecular weights of several tens of thousands or below and lipid A having molecular weights of about 2,000. The process as claimed in claim 1, in which said membrane has an average pore size of 2 x 10⁻⁹m to 10⁻⁷m (20 Å to 0.1 micrometer). The process as claimed in claim 1, in which the solution contains pyrogens and a pharmacologically effective ingredient and the membrane has such a pore size that endotoxins having higher molecular weights than the ingredient may not pass through, but the ingredient may do so. The process as claimed in any of claims 1 to 3, further comprising the step of regenerating the pyrogen adsorbent with a regeneration solution. The process as claimed in claim 4, in which the regeneration solution is treated with an ultrafiltration membrane to remove pyrogens therefrom prior to contacting it with the pyrogen adsorbent.

Inventors: ASE TOMONOBU, HONDA ZENJIRO, KAGOTANI MASAHIRO, MINOBE SATOSHI, NAGAMATSU SHINJI, NISHIMURA TAKASHI, SATO TADASHI, WATANABE TAIZO, ASE, TOMONOBU, HONDA, ZENJIRO, KAGOTANI, MASAHIRO, MINOBE, SATOSHI, NAGAMATSU, SHINJI, NISHIMURA, TAKASHI, SATO, TADASHI, WATANABE, TAIZO
Applicants: DAICEL CHEM, TANABE SEIYAKU CO, DAICEL CHEMICAL INDUSTRIES, LTD., TANABE SEIYAKU CO., LTD.
Application year: 1988
Main classification: A61K9
IPC codes: B01D61, B01D15, A61K9, C02F1, A61L2