CA1301680C - Process of preparing physiologically active proteinaceous solutions - Google Patents

Abstract

Abstract of the disclosure:

A membrane filter of 0.025 to 0.05 µ in pore size is treat-ed by passing the solution of a water-soluble high molecular substance such as albumin, dextran, polyvinylpyrrolidone, poly-sorbate 80, gelatin or the like through the membrane filter.
Employing the filter thus treated, the solution of a physiologi-cally active substance of human origin such as human growth hormone, kallikrein, trypsin inhibitor, epidermal growth factor, leucocyte interferon etc. is filtered at high recovery rate of the active substance avoiding the adsorption of the active substance onto the filter. By the filtration, harmful viruses such as Creutzfeldt-Jacob disease pathogen which may exist in the phisiologically active substance can be removed.

Description

Process for producing physiologically active substances The present invention relates to a process for removing possibility of mixing a harmful virus into a physiologically active substance originated from human urine, blood and internal organs in high recovery rate of the active substance.
In recent years, it has been reported that a disease as-ssumed to be Creutzfelt-Jacob disease (hereinafter referred to briefly as "CJD") occuued in eight patients with pituitary dwarfism in the United States of America and one case of the same disease in the United Kingdom who had been given prepara-tions of human growth hormone produced from human pituitaries being employed as a starting material (Scrip No. 995, p. 25, 1985; The Lancet, August 3, p. 244, 1985).
The causative factor of said CJD has not yet been identi~
fied up to now, but the slow virus is considered to cause the disease, with the incidence being regarded as about one per million persons.
The pathologic chracteristic of CJD is that there are observed spongiform deformity in the cerebral cortex and de-generation in various nervous systems.
Using brain homogenates of the mice having developed CJD, inactivation of the virus was attempted by means of different methods by J. Tateishi, et al., Department of Neuropathology, Faculty of Medicine, Kyushu University, Japan (Annals of Neurol-ogy, 7 (4), 890-891 (1980); Gazette of Medical Society of Japan (Nippon Ishikai Zasshi), 84 (3), 275-282 (1980).
According to the above literature, the mouse brain homo-genate containing the virus is not inactivated by either of heat treatment (at 60C for 30 minutes; at 80C for 10 minutes;
at 100C for 10 minutes), ultraviolet irradiation, one-year treatment with formalin (10 %), one-week treatment with glutar-aldehyde (2 %), one-year treatment with sodium hypochloride ~1 %), one-year treatment with iodine (3 %), one-year treatment with hydrogen peroxide (30 %), 15-hour treatment with potassium permanganate (2 mM), 15-hour treatment with 95 ~ ethanol, etc.

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130~680 -However, it was found that only filtration treatment with a membrane filter having 0.025 ~ of pore size and extraction with chloroform:ethanol (3:1) mixture were able to permit remov-al of the CJD virus, whereas filtration treatment with a mem-5 brane filter of an increased pore size as large as 0.10 failedto eliminate the CJD
According to the literatures reported by Tateishi et al., CJD symptoms are developed by inoculating the cerebral cortex of CJD patient in highest probability as compared with other 10 cases. However, there are records describing the developement of CJD symtoms by inoculating other internal organs, blood or urine etc., which lead to the possibility that CJD virus may mix into a physiologically active substance produced by employing the above matarials as starting materials.
Therefore, it is necessary to prevent the virus which makes stubborn resistance to heat and medicines from mixing into the preparations of the physiologically active substance.
The present inventors have previously disclosed that, in a method wherein filtra-20 tion with a membrane filter of 0.025 ~ in pore size is incorpo-rated into a process of producing human growth hormone to remove CJD virus which might mix in the hormone, a process for recover-ing human growth hormone in high efficiency preventing the hormone from adsorbing onto a membrane filter by previously 25 treating the membrane filter with human serum albumin.
Subsequently, pretreatment by other substnces than human serum albumin have been researched. In result, it was found that the same effect of preventing the adsorption as that of albumin was found and more effective recovery rate was obtained 30 by pretreating the membrane with the solution of polyvinyl-pirrolidone, polyoxyethylene sorbitan mono-laurate, polysorbate 80, Poligeline*(modified gelatin, Hochst, Japan), gelatin or the like.
With regard to low molecular dextran, arginine, poly-35 ethyleneglycol, polypropyleneglycol etc., the effect of prevent-ing the adsorption was also studied, however, there was not obtained good result at the least.
* Trade Mark 1301~80 As described above, these experiments were conducted with regard to human growth hormone originated from internal organs.
The research was developed to further similar experiments with regard to other physiologically active substances. Namely, with regard to a physiologically active substance orginated from human urine such as urokinase, kallikrein, trypsin inhibi-tor, epidermal growth factor, human placenta gonodotropin and hypophysis gonadotropin; a physiologically active substance originated from human blood such as interferon-~, interferon-y, superoxide dismutase, plasminogen, ~ntitumor factor origi-nated from platelet etc., desired preventing effect of the adsoption was obtained by filtration through the membrane filter previously treated with the above-mentioned materials being able to prevent the adsorption.
The present invention is directed to a process for produc-ing a physiologically active subtance of human origin which comprises filtering a solution of the active substance through a membrane filter of 0.025 to 0.05 ~ in pore size, the membrane filter being previously treated with a solution of a water-solu-ble high molecular substnace which practically does not hinder the active substnace from passing though the membrane filter.
The physiologically active substances of human origin include proteinaceous substances originated from human urine, human blood, human internal organs etc., as exemplified in the following:
The physiologically active substances originated from human urine are, for instance, urokinase, kallikrein, trypsin inhibitor, human epidermal growth facotr, human placenta gonado-tropin, hypophisis ganadotropin etc.
The physiologically active substances originated from human blood are, for instance, interferon-~, interferon-y, superoxide dismutase, plasminogen and an antitumor factor of platelet origin.
The physiologically active substances originated from human internal organs are, for instance, a hormone such as human growth hormone etc.

These physiologically active substances of human origin are purified by a method consisted of many processes, for exam-ple, precipitation, adsorption onto and elutin from an adsobent or an ion-exchanger, and filtration. The process of present 5 invention can be incorporated into a suitable process of the the method or can be added to the method as the final process.
As the membrane filter, there may be exemplified that having 0.025 to o.o.r, ~ of pore size, which is available as a commercial product produced by Millipore Co. (Japan), 10 Sartorius Co. (West Germany~ and Nuclepore Co. (U.S.A.).
In this invention, the membrane filter is previously treat-ed with a water-soluble high molecular substance which practi-cally does not hinder the physiologically active substance from passing though the filter.
The phrase "practically does not hinder the solution of physiologically active substance from passing though the mem-brane filter" means that at least the major part of activity of the physiologically active substance is able to pass through the filter. It is desirable to recover at least about 80 ~
Of the activity in the filtrate, because the physiologically active substances of human origin are expensive in general.
As the water-soluble high molecular substnce having the above-mentioned property, there may be exemplified human serum albumin, polyvinylpyrrolidone, polyoxyethylene sorbitan mono--laurate such as Tween 20 (Nakarai Kagaku Co.), polysorbate80 such as Tween 80 (ibid.), modified gelatin such as Poligeline*
(Hochst, Japan), gelatin etc.
The pretreatment of the membrane filter can be performed conveniently by passing an aqueous solution of the high molec-ular substnace through the filter, whereby the substance isadsorbed onto the filter. The passing of the solution may be carried out by means of filtration, impregnation, soak etc.
The concentration of the high molecular substance in the solu-tion can be selected from wide range as far as the purpose of the pretreatment is not obstructed, however, employ-* Trade Mark .~ ' o ing a concentration from 0.2 to 0.5 ~ , the purpose is suffi-ciently achieved in general.
The membrane filter which has been pretreated with a water-soluble high molecular substance is washed with water to remove excess of the substance.
With use of thus treated filter, the solution of the physi-ologically active substance is subjected to filtration. The solution of the substance can be prepared in the form of an aqueous solution such as a solution in physiological saline.
The purity and concentration of the physiologically active substnce can be selected over such a range as may not impede the filtration through the specified membrane filter, but need-less to say, an enhanced degree of purity of the substance facilitates the operation more easily.
By the above procedure, the solution of the physiologically active substance can be filtered through a membrane filter in improved yield.
In the present invention, the pretreatment of a membrane filter with a water-soluble substance prevents the adsorption of the physioligically active substance onto the filter in the subsequent filtration of the physiologically active sub-stance, thereby rendering it possible to allow the physiologi-cally active substance to pass through the membrane filter in improved yield. And the filtration enables the efficient removal of the CJD virus or other filterable viruses which may exist in the physiologically active substances of human origin.
The examples are described in the following to illustrate this invention in more detaile, but this invention is not to be limited by them.

i30~680 Reference Example 1 1. Experimental materials Membrane filters produced by Millipore Co. of Japan:
Pore size Catalogue No.
0.01 ~ (VSWP02500) 0.050 ~ (VMWP02500) 0.10 ~ (VCWP02500) Memhrane filters produced by Sartorius Co. of West Germany:
Pore size Catalogue ~o.
O.OlS ~ (11318) 0 05 ~ (11328) 0.10 ~ (11358) Mmebrane filters produced by Nuclepore Co. of U.S.A.A:
Pore sizeCatalogue No.
0.015 ~(110601) 0.03 ~(110602) 0.05 ~(110603) 0.08 ~(llb604) 2. Experimental method The following experiment was carried out with 80 mQ
(a.60 IU/mQ) of a human growth hormone solution (a physiological saline solution):
Using a 10-ml disposable syringe, 7 ml of the human growth hormone solution for each filter was filtered through 25 a filter of 25 cm in diameter being fixed onto a filter holder.
Throughout the experiment, each filter was autoclaved in advance (at 120C for 30 minutes), followed by passing 5 ml of physiological saline and the test solution (human 30 growth hormone solution), successively, through each membrane.
The human growth hormone was quantitatively determined by radioimmunoassay (RIA.Kit manufactured by D~lnabot Co.).

3. Experimental results The results are shown in Table 1.

i30~6~

Table 1 Membrane filter Recovery (~) of HGII*activityin filtrate Porduced by Millipore Co.
0.025 11 62.4 0 05 ~, 69 . 0 0.10 ll 6~.3 Produced by Sartorius Co.
0.01 ~ Difficult -to be filtered 0.05 ~ 72.9 0.10 ~ 74.6 Produced by Nuclpore Co.
0.015 ~ Difficult to be filtered 0.03 ~ 87.1 0 05 ~ . 101.7 0.08 ~ ` 99.6 15 Solu-tion prior to filtration100.0 through a membrane filter:
Note: *, I~GH stands ~or human growth hormone (the same is to be applied hereinafter).
Example 1 1. Experimental materials Membrane f~lters produced by Millipore Co.:
Pore size Catalogue No.
0.025 ~ (VSWP 02500) o.05 ~ (VMWP 02500) 0.10 11 (VCWP 02500) Membrane filters produced by Sartorius Co.:
Pore size Catalogue No.
0.05 ~ (11328) 0.10 ~ (11358) 20 ~ human serum albumin (Chemical & Serum Therapy Research Laboratories (a juridical person), Lot. A 325) 2. Experimental me-thod 1;~01680 - As a test solution, there was used 40 ml of the human growth llormone solution. Each filter was fixed onto a filter holder and then autoclaved (at 120C for 30 minutes), and the filtration procedure was carried out by use of 5 ml of 5 0.2 % human serum albumin solution (prepared by dilution physiological saline) to perform the coating of the membrane filters.
Subsequently, the filters were washed twice with physiological saline (5 ml x 2) to remove excessive human 10 serum albumin, and then the filtration experiment was carried out with 7 ml of tlle human growth hormone solution.
The humna growth hormone was quantitatively determined by radioimmunoassay (RIA Kit manufactured by Dynabott Co.).
The filtrate of the human growth hormone solution 15 was investigated for contamination of -the human growth hromone with human serum albumin by means of SDS-poly-acrylamide slab gel electrophoresis.
3. Experimental results The resul-ts are shown in Table 2.
2 0 Table 2 Membranefilter Recovery (~) ofHGH*activityinfiltrate . . _ . _ . . _ . . _ . . _ Produced by Millipore Co.
0.025 ~ 96.3 0.05 ~ 97.0 o . 10 ~ 99 . 8 Produced by Sartorius Co.
o o5 ~ 96.0 0.10 ~ 97 4 Solution prior to filtration 100.0 30 through a membrane filter:
Analysis by SDS-polyacrylamide slab gel electrophoresis indicated that there was no contamination of the HGH solution (filtrate) with human serum albumin.

~0~ 680 Example 2 1. Experimental materials:
(1) Membrane filter (0.025 ~ , Cat. No. VSWP 02500) [Millipore Co. Ltd.]
(2) Human growth hormone ["Crorum", Cerono Co. Ltd., Switzerland]
(3) Human serum albumin (Chemo- & Serum-therapy Research Foundationl (4) Dextran (M.W. 5000-7000) lNakarai Kagaku Co. Ltd.]

(5) PVP-25 (Polyvinylpyrrolidone 25) [Nakarai Kagaku Co.
Ltd.]

(6) L-aginine hydrochloride [Nakarai Kagaku Co. Ltd.]

(7) PEG-4000 (Polyethyleneglycol 4000) [Wako Junyaku Co.
Ltd.]

(8) PEG-5000 (Polyethylene-polypropyleneglycol 5000) [Nishio Kagaku Co. Ltd.]

(9) Polygeline ~Modified gelatin) ~Hochst, Japan]

(10) Human immune globulin (Globelin-I; Nippon Seiyaku Co. Ltd.]

(11) Gelatin (pure powder) [Nakarai Kagaku Co. Ltd.]

(12) MDS (Methyl dextran sulfate) [Kowa Co. Ltd.]

(13) Tween 20 (Polyoxyethylenesorbitan mono-laurate) [Nakarai Kagaku Co. Ltd.]

(14) Tween 80 (Polysorbate 80; Polyoxyethylene sorbitan mono-oleate) [Nakarai Kagaku Co. Ltd.]
2. Experimental method:
Membrane filters were fitted onto respective filter hold-ers of 25 mm in diameter and autoclaced at 120C for 30 minutes.
Respective 0.5 ~ aqueous solution of materials (3) to (14) were prepared. Each of the membrane filters was pretreated by filtering 5 ml each of the solutions through the filter, thereby the filters were coated with respective materials.
Subsequently, each membrane filter was washed two times with physiological saline (5 ml x 2) to remove excess of the coating materials, and then filtration tests were conducted * Trade Mark , ~301680 employing 7 ml each of human growth hormone solutions l0.6 IU/ml).
The activity of human growth hormone was assayed with a radioimmunoassay kit (Dainabot Co. Ltd.).
3. Results:
The test results are shown in Table 3.

Table 3 Pretreating solution Recovery rate (%) of HGH
activity in filtrate (HGH
activity before filtration = 100 %) Physiological saline 52.6 0.5 % solution of human 15 serum albumin 98.0 0.5 % solution of dextran 56.1 0.5 % solution of PVP-25 103.0 0.5 % solution of arginine 54.5 0.5 % solution of PEG-4000 51.0 0.5 % solution of 74.3 0.5 % solution of Polygeline 88.6 0.5 % solution of human immune globulin ---*
0.5 % solution of MDS 40.1 0.5 % solution of Tweem 20 97.0 0.5 % solution of Tween 80 94.1 0.5 % solution of gelatin 93.5 * Pretreatment was immpossible because of difficulty in filtration.
Example 3 1. Experimental materials:
(1) Membrane filter: (0.025 ~, Cat. No. USWP 02500) [Millipore Co. Ltd.]
(2) Urokinase (Lot 01016) [Nihon Chemical Research Co.
Ltd.]
Materials from (3) Human serum albumin to (13) Tween 80 are the same as those in Example 2.

2. Experimental method:
The membrane filters were fitted onto respective filter :holders of 25 mm in diameter and autoclaved at 120C for 30 minutes.
Then, the membrane filters were coated with the experimen-tal materials of (3)-(13) respectively, in the same manner as in Example 1, and excessive coating materials were removed by washing with physiologically saline.
Subsequently, filtration tests were conducted employing 7 ml each of urokinase solutions (10,000 IU/ml).
The activity of urokinase was assayed by Fiblin Plate Method (Clin. Chim. Acta 13, 680-684 (1966)).
3. Results:
The results are shown in Table 4.
Table 4 Pretreating solution Recovery rate (%) of urokinase activity in filtrate (urokinase activity in tested solution = 100 %) 20 Physiological saline 47.7 0.5 % solution of human serum albumin 90.3 0.5 % solution of dextran 70.5 0.5 % solution of DVP-25 96.4 25 0.5 % solution of arginine 49.7 0.5 % solution of PEG-4000 65.1 0.5 % solution of PPG-5000 66.6 0.5 % solution of Poligeline 92.3 0.5 % solution of human 30 immune globulin ---*
0.5 % solution of MDS 51.8 0.5 % solution of Tween 20 91.0 0.5 % solution of Tween 80 90.2 0.5 % solution of gelatin 93.0 * Pretreatment was immpossible because of difficulty in filtration.

- ~2 -Example 4 1. Experimental materials:
(1) Human urine kallikrein [Nihon Chemical Research Co., Ltd.]
(2) Human urine trypsin inhibitor [Nihon Chemical Research Co., Ltd.]
(3) Human urine epidermal growth factor [~ihon Chemical Research Co., Ltd.]
(4) Human leucocyte interferon-ol [Nihon Chemical Research Co., Ltd.]

2. Experimental method:
With regard to the above materials, experiments were conducted in the same manner as Example 2.
The method for assaying the activities of the above materi-als are as follows;
Human urine kallikrein: By fluorescent synthetic substrate method (Journal of Biochemistry 82, 1495 (1977)).
Human urine trypsin inhibitor: By the inhibitory activity method of Tanaka et al. (Biochimica et Biophysica Acta 705, 192, (1982)), employing ox trypsin Type I produced by Sigma Co., Ltd.
Human epidermal growth factor: By the enzyme immnoassay method of Hayashi et al. (Biochemistry International 10, 856 (1985)).
Human leukocyte interferon-c~: By 50 % CPE suppression method employing FL-cell-VSV system ("The interferon System"
by W.E. Stewart II, Springer-Verlag).
3. Results:
The results are shown in Table 5.

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Claims (3)

1. A process for preparing a physiologically active proteinaceous substance of human origin characterized in that a membrane filter of 0.025 to 0.05 µ in pore size is previously treated with a 0.2 to 0.5% solution of a water-soluble high molecular weight substance selected from the group consisting of human serum albumin, polyvinylpyrrolidone, polyoxyethylene sorbitan mono-laurate, polysobate 80, and modified or unmodified gelatin and then filtering a solution of the active proteinaceous substance through said pre-treated filter to remove a virus which may be present in said proteinaceous substance.

2. A process according to claim 1 wherein the physiologi-cally active substance of human origin is urokinase, kallikrein, trypsin inhibitor, human epidermal growth factor, human placenta gonadotropin, hypophysis gonadotropin, interferon-.alpha., interferon-.gamma., superoxide dismutase, plasminogen, antitumor factor of plate-let origin or human growth hormone.

3. A process according to claim 1 wherein the previous treatment of the membrane filter is carried out by passing the solution of a water-soluble high molecular substance through the filter.