JPH0535160B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical field] The present invention provides highly purified and high-yield methods for producing human urine-derived colony formation stimulating factor (hereinafter referred to as CSF)-containing fluid.
Concerning how to collect CSF. [Prior art] CSF is a physiologically active substance that exists in extremely small amounts in human urine, and acts on bone marrow progenitor cells to promote the differentiation and proliferation of granulocytes and monocytes-macrophages, which are white blood cell constituent cells. is a glycoprotein,
It is expected to be effective as a therapeutic agent for leukopenia. Methods for purifying CSF include methods using adsorbents, methods using anion exchangers, and methods using ultrafiltration membranes. [Problems to be Solved by the Invention] By the way, since human urine contains only a very small amount of CSF, it is difficult to efficiently separate, concentrate, and purify CSF. For example, a method that adsorbs and elutes CSF by bringing minerals such as silicates and diatomaceous earth into contact with urine itself, which have been frequently used in the past,
Collection methods using various anion exchange chromatography methods are complicated to operate, and the amount of urine that can be processed at one time is limited, making it expensive to collect a large amount of CSF, which is a trace component. It has the disadvantage of becoming. In other words, for example, in chromatography using various anion exchangers, the operability is complicated, such as packing the adsorbent into the column in the column method and recovering the adsorbent by filtration or centrifugation in the batch method, and it is not efficient on an industrial scale. There are disadvantages to this method of obtaining CSF. Furthermore, there are a large amount of impurities in urine, and in conventional methods, these are likely to be recovered together with CSF, which is disadvantageous in subsequent purification. Therefore, the present invention enables high yield, high purity, low cost, and simple operation to extract CFS from solutions containing only trace amounts of CFS, such as urine, and also containing large amounts of impurities.
The purpose is to provide a method for collecting and manufacturing CSF. [Means for Solving the Problems] The object of the present invention is to apply a solution containing a human urine-derived colony formation stimulating factor to a solution containing a human urine-derived colony formation stimulating factor using a structure in which a sheet-like material made of cellulose is spirally wound in a cartridge format. This is achieved by a method for purifying human urine-derived colony formation stimulating factor, which is characterized by treatment with an anion exchanger. The solution containing CSF used in the present invention is
There is no particular limitation as long as it is an aqueous solution of CSF, and it may be, for example, urine, which has a trace amount of CSF and contains a large amount of impurities. Examples of anion exchangers include DEAE-Sephadex, QAE-Sephadex, DEAE
-cellulose, QAE-cellulose, DEAE-cepharose, DEAE-cephacel, sphaerosil-DEA, etc., but preferably a structure in which a sheet-like material with cellulose as a matrix having an anion exchange group is wound into a spiral shape. A cartridge format is advantageous. The structure is usually a carrier (e.g., cellulose) crosslinked with a vinyl polymer, into which QAE- groups, DEAE- groups, etc. are introduced as ion exchange groups (ion exchange matrix, see Figure 1).
It has a spiral shape. A preferable embodiment thereof is as shown in FIG.
2 indicates an ion exchange matrix and 2 indicates a water permeable support. The support is preferably in the form of a lattice or fabric, and its material includes, for example, polystyrene, nylon, polycarbonate, polyacetal, Teflon, stainless steel, and the like. FIG. 3 is a cross-sectional view of an embodiment of the purification method using the structure, in which CSF fluid enters from the inlet 3, moves inward from the outer periphery and is collected in the central core 4, and exits from the outlet 5. Arrows indicate the flow of CSF fluid. 6 indicates a vent, and 7 indicates a drain. Figure 4 is a cross-sectional view, entering from the outer periphery.
This shows the state in which CSF fluid is collected in the core. As conditions for using this, for example, it is preferable to pass a CSF-containing solution through a cartridge at a pH of 5 to 7 and an electrical conductivity of 20 mS/cm (25°C) or less to adsorb CSF. Under these conditions, CSF is adsorbed and most of the CSF is absorbed. Impurities pass through without being adsorbed. Next, a dilute salt solution with a pH of 5 to 7 [e.g., conductivity of 20 m
S/cm (25°C) or less of phosphate buffer]. This washing operation further removes impurities, but the CSF remains adsorbed. To elute adsorbed CSF, pH 5-9
Concentrated salt solution [e.g. conductivity 30mS/cm (25℃)
This can be easily carried out by passing the above phosphate buffer and saline solution]. In the present invention, it is preferable to further combine treatment with an ultrafiltration membrane. This treatment with the ultrafiltration membrane is performed before the treatment with the anion exchanger. Ultrafiltration membranes include those that remove low molecular weight substances from CSF (i.e., for low molecular weight fractions).
One or both of those that remove high molecular weight substances from CSF (i.e., for high molecular weight fractions) may be used, and for low molecular weight fractions, usually 1×
An ultrafiltration membrane of 10 ⁴ to 4×10 ⁴ daltons is used, and an ultrafiltration membrane of 10 ⁵ to 10 ⁶ daltons is usually used for high molecular weight fractionation. Examples of the ultrafiltration membrane include polyacrylonitrile ultrafiltration membranes and polysulfone ultrafiltration membranes. The conditions for passing a CSF-containing solution through an ultrafiltration membrane are generally as follows: PH of treatment solution: 5 to 8 Operating pressure: 1 to 5 Kg/cm ² The treatment by the ultrafiltration membrane according to the present invention Although this may be carried out at any stage of the recovery and production process, it is preferable to first perform treatment using a filtration membrane for low molecular weight fractions, and then to perform treatment using a filtration membrane for high molecular weight fractions later. The CSF thus recovered is preferably further subjected to known high-purification means (e.g., affinity chromatography, gel filtration, etc.), heating (e.g., 50 to 80°C, sterilization treatment for 1 to 10 hours, etc.), and then sterilized. , medical CSF by freeze-drying
It can be made into a formulation. Of course, it can be isolated and purified by known means without lyophilization. [Effects of the Invention] According to the present invention, CSF can be recovered with high purity and high yield. Moreover, CSF with high specific activity and pyrogenic substances removed can be obtained. Also,
It is easier and more efficient than conventional methods and has excellent operability and economy. Examples are given below to explain the present invention in more detail, but the present invention is not limited thereto in any way. Example 1 50% of fresh urine was concentrated to 1% while removing low molecular weight impurities using a polyacrylonitrile ultrafiltration membrane (molecular weight cut off: 20,000). Next, this concentrated solution was passed through Zetaprep QAE-Cartridge R250 manufactured by AMF, which had been equilibrated in advance with 0.05M phosphate buffer (PH6.5) [equivalent to a conductivity of 8.5 mS/cm (25°C)], and the CSF to be adsorbed. Next, this QAE-cartridge is 0.05M-
After thorough washing with phosphate buffer (PH6.5), 1.0M
- Elute with 0.05M phosphate buffer containing NaCl (PH6.5) [corresponding to conductivity 85mS/cm (25℃)], and
An eluate containing the following was obtained. Separately, as a comparison, fresh urine 50 was diluted 4 times and 0.05M
- DEAE equilibrated with phosphate buffer (PH6.5)
- 100 g of cellulose was added, stirred and adsorbed for 1 hour, and washed and eluted in the same manner as above to obtain an eluate containing CSF. The CSF activity of each of these eluates was measured, and the specific activity and recovery rate were determined and shown in Tables 1-1 and 1-2. The CSF activity was measured by colony formation using a single layer agar plate method using C57BL/6N mouse bone marrow cells. That is, the sample was appropriately diluted with distilled water containing 2% (v/v) bovine serum, subjected to sterile filtration (0.45 μ membrane filter), and then dispensed to 0.1 ml onto three plates. 0.3% (w/w)
agar, 20% (v/v) fetal bovine serum and
Contains ¹⁰⁵ C57BL/6N mouse bone marrow cells
After adding 1 ml of McCoy's 5A medium and mixing thoroughly,
The cells were cultured for 7 days at 37°C in a flannel with 7.5% (v/v) carbon dioxide aeration. After incubation, under the microscope field 50
The number of formed colonies was counted, and the number of formed colonies (unit) was expressed as the CSF activity. That is,
One unit of CSF activity was defined as one formed colony. As shown in Tables 1-1 and 1-2, the recovery rate of the method of the present invention is comparable to that of the conventional method, but the specific activity (purity) is improved by introducing purification using an ultrafiltration membrane.
By removing impurities in advance, the increase was more than ten times higher. In addition, while the conventional method increases the amount of liquid during adsorption due to conductivity adjustment through dilution, the method of the present invention uses pretreatment and purification using an ultrafiltration membrane, so the amount of liquid is small. In addition to being quantifiable, the conductivity can be easily adjusted, and the subsequent treatment with an anion exchanger has improved operability and work efficiency. In addition, by introducing a cartridge type system, we were able to further improve workability during chromatography using an anion exchanger.

【table】

[Table] Example 2 4000 ml of a CSF-containing solution was separated using a flat polysulfone ultrafiltration membrane (molecular weight cut off: 1,000,000). The time required to circulate and filter 4000 ml of solution was 30 minutes. Thereafter, ion exchange chromatography using a QAE cartridge was performed in the same manner as in Example 1. The CSF activity of the pre-filtration solution, post-filtration solution, and QAE-cartridge eluate was measured by the method of Example 1, and the total activity, specific activity, and recovery rate were determined. Furthermore, the pyrogenic substance removal effect using rabbits was measured in accordance with the 9th edition of the Japanese Pharmacopoeia. The results of these measurements are shown in Table 2. As shown in Table 2, CSF can be recovered with a high recovery rate.
Specific activity (purity) also increased. Furthermore, regarding CSF, inhibitors could be removed by the ultrafiltration membrane, resulting in increased activity. Furthermore, there was a remarkable effect in removing pyrogenic substances.

【table】 [Brief explanation of the drawing]

Figure 1 is a schematic diagram of a structure in which a sheet material having an anion exchange group is wound into a spiral shape, Figure 2 is a cross-sectional view of the structure, and Figure 3 is a spirally wound sheet material material. FIG. 4 is a vertical cross-sectional view of an embodiment of an anion exchanger in a rolled form, and FIG. 4 is a cross-sectional view thereof. C...Carrier, P...Vinyl polymer, Q...
QAE- or DEAE group, 1... ion exchange matrix, 2... water permeable support, 3... inlet,
4...core, 5...exit, 6...vent, 7...
drain.

Claims (1)

[Claims] 1. For a solution containing a human urine-derived colony formation stimulating factor, an anion exchanger in the form of a cartridge made of a spirally wound sheet-like material with cellulose as the matrix is used. A method for purifying a human urine-derived colony formation stimulating factor, the method comprising: 2. A method for purifying a human urine-derived colony formation stimulating factor according to claim 1, which further combines treatment with an ultrafiltration membrane capable of removing at least one of a low molecular weight substance and a high molecular weight substance from the colony formation stimulating factor. .