Prof. Motohide Takahashi introduced the history of serum therapy.

Production and quality control history of mountain lionfly antivenom

Motohide Takahashi Specially Appointed Professor, Kumamoto Health Science University, Joint Research Chair of Biotoxins and Antitoxins

National Institute of Infectious Diseases, Department of Immunology, Guest Researcher

Tetanus, diphtheria, and gastroesophageal diseases are caused by toxins produced by toxin-producing bacteria that settle and invade wound sites, larynx, and other localized areas and produce toxins as the bacteria multiply. Due to the different effects of neurotoxins, cytotoxic toxins, and other toxins, different symptoms and pathologies are observed in each infected patient. However, the so-called “serotherapy” developed by Shibasaburo Kitasato and his colleagues more than 100 years ago has been used to treat patients, and has been used as an effective means to save lives, with notable results. This classic serotherapy has been used as a symptomatic treatment for patients bitten by insects (e.g., red moss spiders) and marine organisms (e.g., habu jellyfish) as well as bacterial toxins. The general method of production is advanced immunization by frequent inoculation of animals such as horses or goats with toxin or inactivated toxin (toxoid vaccine). Horse antivenom preparations made from blood plasma or serum obtained by blood collection and purification processes are used internationally. Knowledge and skilled techniques are important to manage the production of induced antitoxin because of differences in the purity of the toxin or toxoid used as antigen, the toxoidization method, individual differences in the immunized animals such as horses, etc., and the inoculation interval.

Meanwhile, apart from classical serotherapy, advances in the sciences of immunology, molecular biology, and engineering have led to the development of pharmaceuticals that are safe and of consistent quality. Camelids have special heavy-chain antibodies whose structure is different from that of normal IgG. The nanobody VHH (Variable domain of the heavy chain of heavy chain antibody) present in the plasma obtained by immunization is highly stable against heat and acid, and its small antibody size (15 KDa) is utilized in recombinant processing and technology in E. coli and other bacteria A formulation has been developed (Rituximab RITUXAN for acquired thrombotic thrombocytopenic purpura). In addition, humanized mouse monoclonal antibodies have been utilized and developed, and are being commercialized after approval under the Pharmaceutical Affairs Law for the treatment of cancer and rheumatism (e.g., Adalimumab HUMIRA, a treatment for chronic rheumatoid arthritis). In addition, preparations produced by genetic modification technology using human antibodies produced after inoculation of antigens into mice transfected with human chromosomes (antibody drugs) have also been developed.

In addition to animal blood-derived materials, in the case of the SARS and COVID-19 epidemics, plasma obtained from infected patients during their recovery period is directly transfused to patients using aseptic technique (plasma therapy), and a large amount of plasma from recovered patients is collected and only immunoglobulins are purified at a manufacturing plant and prepared in a formulation (immunoglobulins only). The method of converting blood to plasma for use (plasma preparation) is also moving toward practical application in Japan and overseas. Thus, human antibody therapy, which was developed through advances in science and engineering from serotherapy using antitoxins, is now a tool used not only for toxic diseases but also for a wide range of other diseases in the medical field.

This paper describes the Yamakagashi horse antivenom, which is still being implemented as a publicly funded research project as a serotherapy using classical horse antivenom. First, the author looks back on the 30-year history of serotherapy by reviewing the author’s involvement in the research of horse antivenom, including the utilization of public research funds and administrative responses. The author’s first encounter with horse antitoxin was during his tenure at the Chiba Serum Research Institute, where he was directly involved in the production and quality control of botulinum toxin. The production process for the production of antitoxin includes antigen preparation for horse immunization, immunization of horses, plasma collection and purification, and formulation. Botulinum horse antitoxin was manufactured in two formulations, “dried E-type botulinum antitoxin” and “dried ABEF botulinum antitoxin,” and both were controlled under the order instructions of the Blood Control Division of the then Ministry of Health and Welfare as government-owned products. My role was to purify high-concentration botulinum toxin solution by column chromatography by collecting the toxin from the culture of Clostridium botulinum type A, type B, type E, and type F bacteria, which were used for the production of both types of products. Maintenance of two types of seed strains, culture medium preparation, bacterial culture, crude toxin, purified toxin, etc. were subjected to quality control tests, and a toxin quantification test (LD50) using mice, which is a biological activity, was conducted as a confirmation test for each process. After inactivation with formalin, the toxin was prepared as an antigen for equine immunization, and inoculated into horses raised on a farm in Sakura City at the time. Chiba Serum, as a public utility company in Chiba Prefecture, is strongly involved in the production of antitoxins as well as vaccines, and in addition to botulinum antitoxin, the company also produces three types of gas toxin (Clostridium perfringens, C. novyi, and C. septicum), the He was responsible for the production of diphtheria antitoxin and pit viper antitoxin and had a mission in the medical treatment of serotherapy. Similar to the production process of botulinum antitoxin, the preparation of antigens for horse immunization of gas-ethic toxin was almost the same in terms of purification of plasma, formulation, etc., except for the details of bacterial culture, toxin purification, and toxin inactivation methods. The antigens for immunization with diphtheria antitoxin were obtained from the human formulation team that had been producing diphtheria toxoids for humans, and were used with appropriate adjuvants to immunize horses with high levels of diphtheria toxoids.

In 1989, he moved to the National Institute of Preventive Health (JNIH), located near Meguro Station, to work in the Toxoid and Antitoxin Section of the Department of Humoral Immunity. Along with basic research, he was in charge of quality control testing of toxoids and antitoxins as part of the national certification work, which brought him back into contact with bovine antitoxins. In the room at that time, there were many records of basic research conducted by our predecessors, especially the production and development of gas erosion antitoxin, which was also conducted as a Defense Agency-commissioned research. This was strongly felt. Along with research on serotherapy of habu antitoxin, valuable materials on the prototype production and quality control of toxoids for the purpose of prophylaxis were also preserved.

In 1992, JNIH divided its research departments and operations into two separate buildings in Shinjuku-Toyama and Musashimurayama, and in 1997, the name was changed to the National Institute of Infectious Diseases (NIID). The laboratory to which the author belonged at JNIH and NIID inherited the basic research and quality control of toxoids and antitoxins, and has continuously exchanged information with the competent departments of the Ministry of Health and Welfare regarding serotherapy using bovine antitoxins as a research institute that supports the health administration.

This paper reviews the research that began in 1998-1999 (FY1998-1999) as part of the research group on the production and quality control of prototype wild boar antitoxin, which has continued to the present.

The “Research on the Development of Antitoxin Development and Stockpiling System for Health Risk Management” group of the Health and Welfare Science Special Research Project was headed by Dr. Takeshi Kurata, Director of the National Institute of Infectious Diseases, and included Dr. Yoshichika Arakawa (Division of Bacteriology and Blood Products, NIID), Dr. Motohide Takahashi (Division of Blood Products), Dr. Shuji Shimazaki (Department of Emergency Medicine, Kyorin University School of Medicine), Dr. Michihisa Toba (Japan Serpent Research Center), and Kiei Kaneshiro (Okinawa Prefectural Institute of Public Health and Environment). The overall objective of the research group was to develop a new antivenin for use in serotherapy, which is not expected to be developed in the industry because of its extremely rare occurrence. Therefore, the group decided to test and manufacture new antitoxins that could be manufactured in Japan on an emergency basis, and to establish an emergency system for those that could be imported from overseas by clarifying the route of acquisition. In addition, quality control of the secured formulations will be conducted in accordance with the Biological Preparation Standards, with a view to considering quality control methods that incorporate new molecular biological and physicochemical methods. Furthermore, the goal is to establish an organization and system to manage and store antitoxins whose quality assurance has been confirmed at several sites in Japan, and to be able to cope with emergencies. In this context, the importance of Yamakagashi antitoxin as a serotherapy was confirmed as follows, and trial production was started.

The mountain kagashi is found throughout Japan and was once thought to be a nonvenomous snake. In fact, they are venomous, and there have been reports of deaths due to dysfunction of the blood coagulation system caused by their bites. In the past, researchers at the Japan Snake Science Institute immunized rabbits and goats and produced a test version of the mountain lion antivenom, which was used for treatment as an emergency measure. Since the stock of this antitoxin has been depleted, it was decided to urgently manufacture it as part of the research group’s activities. The cooperation of the National Institute of Chemistry and Serum Therapy (KAKETSUKEN), which had been manufacturing the horse antivenom, was sought. Since KAKETSUKEN had been manufacturing pit viper and habu antitoxins that were compliant with the Pharmaceutical Affairs Law, there were discussions to prevent confusion with the items approved for production. In order to clarify that the research group was responsible for the production of the Yamakagashi antitoxin, it was decided to clearly state this on the label of the prototype so that it would be conducted within the scope of the research activities of the research group. Research collaborators (titles omitted) included Akihiko Yamamoto and Yasushi Fukuda of the National Institute of Infectious Diseases; Kunio Okuma, Kazunori Morokuma, Mutsuo Udo, and Tokuhiro Kobori of the KAKETSUKEN, and others in the antitoxin production and quality testing and Aso branch departments; Jun Sakai of the Japan Snake Science Institute; and Zenji Kawamura of the Japan Society of Tropical Medicine; and Assistant Director Kiyoto Nakai of the Blood Control Division, Ministry of Health, Labor and Welfare, and others participated as observers.

It was estimated that it would be difficult to complete the entire project within the research period from October 1998 to March 2000, when the research group started. Therefore, it was also confirmed that the process after the huma immunization would need to be continued in the next research group.

The initial candidates for immunized animals were goats and horses, from which large amounts of primary plasma could be obtained. However, because the number of snakes captured and the amount of toxin collected were sufficient, and because all domestic antitoxin products are equine antitoxin products, the choice was made to use horses, based on their track record in human administration and zoonosis. We began full-scale activities and began to materialize a plan for capturing mountain lions (at the request of a contractor), collecting venom glands, extracting and purifying the toxin, highly immunizing horses, and securing, purifying, and formulating plasma. Immunization of two horses with inactivated toxoid and the toxin itself was initiated. Antibody response was measured by quantifying antitoxin antibody titers (neutralizing antibodies) that specifically neutralize the toxin. Sera highly immunized after several inoculations were compared with titers of previously produced goat antitoxin, and comparable titers were obtained.

Blood collection from immunized horses, purification of plasma, and formulation were continued as part of the “Research on Quality Control of Antitoxin Products in Japan and Overseas for Stable Supply” group from 2000 to 2002. The following is a summary of the major studies conducted by Motohide Takahashi, Yoshinobu Horiuchi (National Institute of Infectious Diseases), Norihisa Goto (NIID), Tsugio Sasaki (NIID), Kunio Okuma (NIID), Shigekazu Maruyama (Chiba Serum Institute), Yasushi Kuwahara (DENKA SEIKEN Co., Ltd.) and others. The research group obtained sea snake antitoxin, pit viper antitoxin, and botulinum toxin from overseas factories, which are preparations that are expected to be urgently handled or depleted in Japan. Safety tests included a sterility test, a febrile substance negativity test (endotoxin test), and an abnormal toxicity negativity test, and those formulations for which potency tests were available in the domestic standards were tested against the standards to clarify their quality through limited formulations and tests. In addition, since Hymphaena antitoxin is commercially available in China, Japan, and Korea, candidate standard products were selected after discussions among national testing officials in the three countries, and standardization tests were conducted to produce an Asian regional standard product.

The production of the mountain oak antitoxin, which was conducted in parallel with the activities of this research group, relied on the facilities, knowledge, and technology of the National Institute of Biochemistry, which produces horse antitoxin, and lyophilized products were produced. For details of the production method and quality tests, please refer to the following paper.
Experimental Manufacture of Equine Antivenom against Yamakagashi (Rhabdophis tigrinus). Kazunori Morokuma et al, Jpn. J. Infect. Dis., 64, 397-402, 2011.

Although the lyophilized specimens have excellent stability, there was discussion of the need to check for quality deterioration after 10 years of prototype production, and it was decided to conduct this study. Although the research group for the production and quality control of the mountain lionfly antitoxin had been completed, it was decided to conduct a limited number of test items from 2013 as activities of the research group related to the horsefly antitoxin. The study is being conducted in cooperation with KAKETSUKEN, a manufacturer of antitoxin products. The currently approved lyophilized bovine antitoxin products have a shelf life of 10 years. It is known that the humidity content of the dried product increases with storage over time, and we have confirmed that the humidity content is less than 3% of the biological product standard. Changes in dissolution time and insoluble foreign matter after dissolution are also inspected as quality check items. As shown in the table, the humidity content immediately after manufacturing was around 0.3%, but has now increased to approximately 0.6%, and the dissolution time has been delayed, and a potency test will be conducted in FY2020.

The MHLW and AMED research funded by the Ministry of Health, Labour and Welfare from FY2013 to FY2018 “Research on quality control of antitoxins and therapeutic methods using antitoxins”: Principal investigator Dr. Toru 123 (St. Luke’s International University) conducted clinical research by manufacturing prototypes of the antivenin for the Japanese spotted spider, conducting non-clinical studies on lyophilized specimens, and conducting clinical studies on the use of the antivenin. The research and verification included the development of an assurance system to prevent adverse reactions at the time of a bite, and the study of smooth transport of antitoxin and stockpiling locations in the event of a bite. For details of the research, please refer to the report of the Principal Investigator. Furthermore, in the ongoing “Research contributing to higher quality of antitoxin preparations and therapeutic systems using antitoxin preparations” (FY2019-2021 AMED drug project: Principal Investigator: Manabu Ato, National Institute of Infectious Diseases), as mentioned above, since it has been over 20 years since the former antivenin was manufactured, not only the usual quality test items but also potency In addition, the production of the next generation of the mountain lion antivenom is also under consideration. Please refer to the AMED research report for the details and status of these studies.

In the potency tests in Table 3, the toxin neutralization capacity per vial for anticoagulant activity immediately after manufacture in FY2000, and in FY 2013 and FY 2017, showed that a large number of toxins could be neutralized. used, but no toxin stability studies were conducted. We plan to analyze the past study records and the results of the study to be conducted in FY2020 to investigate the cause of the difference in anticoagulation only, even though the same test toxin was used to measure anticoagulation and anti-hemorrhage.

Figure 1: Bovine immunization methods and antitoxin induction.

Figure 2: Completed prototype (lyophilized Yamakashi horse antitoxin)

Table 1: List of domestically manufactured huma antitoxin products used for serotherapy

ー Overview of potency test methods ー

Table 2: Results of quality control tests.

Table 3: Ongoing Quality Verification Testing