In the fields that handle animal cells, a wide variety of enzymatic treatments of cultured cells are being carried out. Particularly, since the extracellular matrix and the like that constitute the basement membrane can be degraded under mild conditions by using enzymes, enzymatic treatments are indispensable in the subculture of cultured cells. As the enzymes to be used herein, proteases such as trypsin and collagenases, or glycolytic enzymes such as hyaluronidases are well known.
Bacillolysin is a neutral metalloprotease produced by microorganisms, particularly the bacteria of the genus Bacillus and related genera. A protease which is one kind of bacillolysin was found from a culture of microorganism Bacillus polymyxa (the genus name has been changed to genus Paenibacillus since 1994). This protease was acknowledged to have a degrading action that is different from that of trypsin or collagenases, and for example, the protease can disperse cell clusters that cannot be thoroughly dispersed by other enzymes, into single cells without damaging the cells themselves. Furthermore, this protease is used in various fields, such as the use in the culturing of adhesive cells in a suspension system by utilizing the nature that the protease is not inhibited in the blood serum (Patent Documents 1 and 2). This protease is sold under the name “Dispase (registered trademark)” from Godo Shusei Co., Ltd., and is widely used over the world.
Currently, in the field related to regenerative medicine, for example, such as in the case of culturing epithelial cells separated from a skin tissue and preparing the cells in a sheet form, or in the case of preparing insulin-producing cells from the pancreas, this protease (Dispase) is most generally used. Furthermore, investigations are being conducted on the utilization of bacillolysin as a pharmaceutical product not only for the laboratory uses and industrial uses as described above, but also for the auxiliary uses for medical purposes, particularly at the time of removing the vitreous body during the surgery for proliferative retinopathy in the ophthalmologic field, or for the use in a prophylactic therapy for diabetic cataract (Non-Patent Document 1).
Enzymes have been utilized as pharmaceutical products for a relatively long time, but at first, the use was limited only to the utilization of proteases, amylases, lipases and the like as digestion accelerators. Many of these are orally administered, so that there has been hardly any case, to date, where purity causes a problem. However, treatment methods based on the non-oral administration of enzymes were commenced, such as a supplement therapy for metabolic disorder diseases or the use of a group of enzymes that participate in the blood coagulation system, and therefore, those enzymes serving as pharmaceutical products are now demanded to have a purity necessary for non-oral administration, or to have impurities eliminated therefrom, such as to have allergy-inducing substances or endotoxins eliminated. At the same time, high purity is also required of enzyme proteins as bulk drug substances, so as to prevent any unexpected adverse side effects in advance.
Conventionally, there are occasions in which a gel filtration method is used as a method for analyzing the purity of a protein; however, the gel filtration method lacks the resolution capability for discriminating a molecular weight difference of several thousands in proteins having molecular weights of several ten thousands. On the other hand, an electrophoresis method has high resolution capability, and is widely used as a method for analyzing the purity of a protein. Particularly, an SDS-polyacrylamide gel electrophoresis method (SDS-PAGE method) in which a sample is heated in advance together with SDS to SDS-fy the sample, and the resulting sample is electrophoresed in the presence of SDS, is considered as an excellent method that can analyze the purity of a purified protein, together with a capillary electrophoresis method of similarly performing electrophoresis in the presence of a surfactant (Non-Patent Document 2).