JPS6160050B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a method for recovering CSF with high purity and high yield from a solution containing human urine-derived colony formation stimulating factor (hereinafter referred to as CSF). CSF is a physiologically active substance that exists in extremely small amounts in human urine, and is a glycoprotein that acts on granulocyte precursor cells in the bone marrow and promotes the differentiation and proliferation of granulocytes and monocytes-macrophages, which are constituent cells of white blood cells. Therefore, it is expected to be effective as a therapeutic agent for leukopenia. [Prior art] By the way, human urine contains only a very small amount of CSF, so it is difficult to efficiently separate CSF.
Difficult to concentrate and purify. For example, the conventional methods of adsorbing and eluting CSF by bringing minerals such as silicates and diatomaceous earth into contact with the urine itself, and methods of recovering CSF using various anion exchange chromatography methods are difficult to operate. This method is complicated, and the amount of urine that can be processed at one time is limited, so it has the disadvantage of being expensive to recover a large amount of CSF, which is a trace component. 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. [Disclosure of the Invention] Therefore, the present invention provides a method for extracting CSF from a solution containing only a trace amount of CSF such as urine and a large amount of impurities with high yield, high purity, low cost, and simple operation. The purpose of this invention is to provide a method for recovering and manufacturing the present invention.
That is, a CSF characterized in that a solution containing CSF is passed through an ultrafiltration membrane capable of removing at least one of a low-molecular substance and a high-molecular substance from the CSF.
This is achieved by the manufacturing method. The solution containing CSF used in the present invention is not particularly limited as long as it is an aqueous solution of CSF, and for example, a solution containing a trace amount of CSF and a large amount of impurities, such as urine. It may be. 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, one or both of them may be used.
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: 5-8 Operating pressure: 15Kg/cm ² The treatment using the ultrafiltration membrane according to the present invention is CSF
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. In the present invention, it is preferable to further combine treatment with an anion exchanger, preferably chromatography treatment with an anion exchanger, and the treatment with an anion exchanger is preferably carried out by the above-mentioned ultrafiltration. This is done after the membrane treatment. Examples of anion exchangers include DEAE-cephadex, DEAE-cepharose, DEAE-
Examples include cephacel and sphaerocil-DEA. The CSF thus collected is preferably
Furthermore, it can be made into a medical CSF preparation by subjecting it to known high-level purification means (for example, affinity chromatography), heating (for example, 50 to 80°C) sterilization, and the like, followed by freeze-drying. Of course, it can be isolated and purified by known means without lyophilization. Example 1 50ml of fresh urine was separated using a polyacrylonitrile ultrafiltration membrane (molecular weight cut off: 40,000). CSF activity was measured for fresh urine, membrane permeate, and concentrate, and the total activity, specific activity, and recovery rate are shown in Table 1. As shown in Table 1, CSF could be recovered in high yield, and the specific activity (purity) also increased. Next, 1000 ml of concentrated solution was added with DEAE-
20 g of cellulose was added and stirred for 1 hour to adsorb protein components in the concentrate. Non-adsorbed components were separated by filtration, and the collected adsorbed DEAE-cellulose was packed into a column, and then thoroughly washed with 0.05M phosphate buffer (PH7.0±0.1). Next, add 0.05M phosphate buffer (PH7.0) containing 1.0M sodium chloride.
±0.1) to obtain an eluate containing CSF. Separately, as a control, 0.05 M
Equilibrated with phosphate buffer (PH7.0±0.1)
Add 100g of DEAE cellulose, stir and adsorb for 1 hour, wash and elute in the same manner as above,
An eluate containing CSF was obtained. The CSF activity of each of these eluates was measured, and the specific activity and recovery rate were determined and shown in Tables 2-1 and 2-2. Table 2-1 and Table 2-2
As shown in Figure 2, the recovery rate of the method of the present invention is about the same as that of the conventional method, but the specific activity (purity) is increased by more than ten times due to the introduction of purification using an ultrafiltration membrane. It will be understood that the amount of anion exchanger used in the process is smaller than in the conventional method, and the ease of operation is improved by reducing the amount of liquid.

【table】

[Table] CSF activity was measured by colony formation using a monolayer 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, filtered for sterilization (0.45μ membrane filter), and then transferred to three trays.
Dispense to 0.1ml, add 0.3%w/w agar, 20%
After adding 1 ml of McCoy's 5A medium containing (v/v) fetal bovine serum and ¹⁰⁵ C57BL/6N mouse bone marrow cells and thoroughly mixing, the mixture was heated at 37°C in a 7.5% (v/v) flan vessel with carbon dioxide aeration. The cells were cultured for 7 days. After culturing, the number of formed colonies was counted under a microscope field, with aggregates consisting of 50 or more cells being counted as colonies, and the CSF activity was expressed by the number of formed colonies (unit). That is, one unit of CSF activity was defined as one formed colony. Example 2 4,000 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. The CSF activity of the pre-filtration solution, post-filtration solution, and filtration residual solution 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 of the membrane was measured in a rabbit pyrogenic substance test in accordance with the 9th edition of the Japanese Pharmacopoeia. The results of these measurements are shown in Table 3.

[Table] As shown in Table 3, CSF was recovered with a high recovery rate, and the specific activity (purity) also increased. Furthermore, with respect to CSF, inhibitory substances could be removed by the ultrafiltration membrane, resulting in an increase in activity. Furthermore, there was a remarkable effect in removing pyrogenic substances.

Claims (1)

[Claims] 1. A human urine-derived colony formation stimulus characterized by passing a solution containing a human urine-derived colony formation stimulation factor through an ultrafiltration membrane capable of excluding at least one of a low molecular weight substance and a high molecular weight substance from the colony formation stimulation factor. Method of manufacturing factors.