The ASV production encompasses many processes of manufacturing and quality control. Various manufacturers use various techniques according to their needs and resources. The advance countries having technology and funds in abundance uses methods to have most purified form of the antagonizing globulins and fractionating the remaining proteins through advance procedures like chromatography. But these methods are very costly and the product stands of high cost as much as 1000 to 1200 US$s/ vial. The developing countries mostly use the traditional method of precipitation and dialysis with additional purification with pepsin or papain digestion. The Sindh project is in continuous contact with Insitituto Colarido Picado of Costa Rica National University and Queen Salvobha Memorial Institute Bangkok of Mohidol University Thailand which are the leading institutes worldwide for research and development of cost effective safe ASV production. Three of the world authorities and authors of WHO guidelines for production, quality control and store of Anti Snake Venom Globulins worldwide has promised to extend technical assistance to Sindh Project free of cost. In this regard Professor Dr. Kavi Ratanabanngkoon visited Sindh for one week during July 2010.
- Animal Farming
Sindh project is using local species of animals having adequate herd immunity to the local equine diseases. It is using young and best age group (4.5 to 6 years) animals to have high quality production for a longer period. The Project is having technical collaboration of Sindh Agriculture University Tando Jam where two foreign qualified PhD Head of Department are assisting the Project in maintaining the horse farm and animal health.
The project is keeping computerized record of health and nutrition profile of each animal with complete record/ documentation of venom inoculation, bleeding and other lab operations performed on each animal. It will use scientific procedures to separate plasma and re-introduce the blood cells back in the animal so as to minimize the stress thus can enhance bleeding frequency even up to eight times in a year without any health damage to the project animals.
The project will ensure optimum animal safety by using low dose, low volume multiple site venom inoculation to minimize the risk and misery for the animals. The project will ensure application of guidelines set by Association of Animal Farming of Europe to it’s maximum. The project is introducing an advance quality control system having minimal in-vivo and maximum in-vitro experimentation wherein any experiment deem essential in animals would first be done in vitro through ELISA to minimize animal injuries and deaths.
- Venom Use
Sindh project do not favor caging and killing the snakes while milking their venom unscientifically. It will conduct venom milking sessions well in the field where the snakes after milking will again be freed in the field. However few snakes of each type will be kept scientifically for research purpose or for meeting any emergency demand of venom. The guidelines for such would be obtained from school of tropical medicine UK the biggest reference in the world. In the field all scientific formalities will be observed to avoid any venom protein damage due to temperature or any contaminating factor.
The project will utilize purified and lyophilized venom in Freunds adjuvant at multiple sites as against the commonly practiced use of raw whole venom at one site which is highly painful, abscess formatting and sometimes tend lethal. The new procedure is proven safe, effective and provides better quality ASV with minimum use of venom deem essential for animal safety.
Sindh project with the assistance of HEJ Institute Karachi University, Hi-Tech Institute, and Institute of Biochemistry Sindh University is having capability to purify venom, fractionate it’s proteins and evaluate it’s LD50 or ED50 of any product in vitro before using the same in animals. Such procedure ensures optimizing animal safety and quality antibody production. Based upon project facilitation the University of Sindh has awarded Bechalors degree on venom fractionation and has registered 4 M.Phil and 2 PhD enrolments on various themes of venomology at Institute of Biochemistry.
- ASV Production
The Sindh project intends to opt recently introduced Caprylic Acid fractionation method to produce whole IGg instead of traditional Ammonium Sulphate precipitation with pepsin and papain digestion to produce F(ab)2 or F(ab) which are costly, time consuming and cause maximum loss of antibodies (65-70%). The yield through new method is almost double (70%) than the traditional ammonium sulphate precipitation and dialysis method (35%). The caprylic acid fractionation needs few hours where as the ammonium sulphate precipitation and dialysis cycle takes many days. The caprylic acid procedure not only derives the double yield but ensures virus elimination and least anaphylactic or Anaphylactoid reactions. The Sindh project has selected filtration, nano filtration and ultra filtration through cost effective series of equipments instead of time consuming, complex and costly techniques of chromatography or electrophoresis. The new technique of Caprylic Acid Fractionation is practiced and proven effective since last many years by Instituto Clodomiro Picado Costa Rica and QSIM Institute Bankok and both has extended consent for technical assistance of Sindh project. Valid consent of Butantan Institute of Brazil for technical collaboration is also at hand.
Sindh project is quite conscious about energy shortage and frequent load shedding in country hence promise to produce lyophilized ASV also which can be kept at ordinary room temperature with more than five years shelf life as against the liquid ASV which needs uninterrupted cold chain maintenance with only two to three years shelf life.
- Quality Control.
Sindh project is already sensitized with the importance of quality control and quality assurance for production unit, manufacturing article of human consumption. It has been studying the recent global standardization protocols of GMP and cGMP. It will carefully observe these protocols even from the stage of construction of lab and will extend it to every integrated part of the ASV manufacturing from selection of raw material to finished product. It will introduce the highest protocol of lab and staff safety to every step of production and quality control. It will introduce at least 10000 P2 or even 100000 P3 environments to the production and purification areas and will not compromise with any quality diluting factor due to financial or any other reason.
Initially the quality control will be shared with HEJ Institute, Sindh University and Sindh Agriculture University Tando Jam. The animal experimentation for quality control will be done at SAU Tando Jam under supervision and guidance of Dr. Abdullah Arejo among very few scientists in Pakistan having international certification for conducting animal experimentation. The vitro experimentation mostly on ELISA for quality control would be done at HEJ Institute under supervision of Dr. Abid having PhD from Germany and at Sindh University under supervision of Professor Dr. Muhammad Yar Khuhawar a renounced national scientist and Professor Dr. Allah Bux Ghanghro Director Institute of Biochemistry. Gradually the trained staff of the laboratory will introduce a well equipped quality control cell in the laboratory itself for ISO and WHO certification.
- Research & Development (R&D).
The globally accepted GMP standards define R&D an essential integrated part of any manufacturing establishment. This specialized manufacturing project needs very much a Research and Development component to enable continuous quality enhancement and updating scientific procedures.
Unfortunately no scientific and organized research is being conducted since last 150 years about the local snakes and characteristics of their venoms. Nothing structured even in most recent medical curricula to define evidence based locally appropriate snake bit management. The total reliance remained on some western medical books which are written for advance countries having no similarity of environment and resources those of developing countries. Even the snake culture in these western countries is totally different from those in eastern countries. It is one of the reasons that snake bite management is not alike elsewhere in Pakistan. Interestingly the medical first aid techniques for snake bite abandoned during 1880 are still part of our medical curriculum.
Likewise the ASV production, store and its use including it’s quality control is largely research dependent not only to have optimum standard practices but also to ensure continuous refinement and improvement of various processes.
The affiliation of the project with various Medical and General Sciences Universities enables the project to run a comprehensive research program parallel to it’s manufacturing activities. The snake farming and Lab techniques of ASV manufacturing and quality control of the project support to introduce graduate and post graduate courses of Microbiology, Biochemistry, Herpetology, and Serology at interested Universities without any additional investment for equipments and consumables. The tutorial output ordained under these educational and research activities will help the project in it’s quality enhancement. The project has assisted Institute of Biochemistry Sindh University in successful completion of graduate studies on Cobra venom and yet planed to enroll two M. Phil and one PhD scholar on venom finger printing and fractionation. Both these research premises are essential for producing a real representative ASV for the province as well as to have costly venom fractions free of cost for quality control of the project.
The R&D component of the project will help in updating various reference material on local snakes, their venom, snake bite management, ASV use and on many other affiliated aspects.
Being high technology project the training and capacity building will be an integral part of project execution and operation. The project staff will need initial skill development trainings to perform well for quality production as well as a built-in regular system of their capacity building, knowledge enrichment and time to time professional development. It will keep them acquaint with any new development of procedures or techniques if happening elsewhere in the world, as well as will enhance their professional abilities to meet ever changing project demands.
The investments made for the training will safeguard not only the entire investment in the concern but will surely reduce the project implementation and operation costs. The internationally qualified product would be an additional benefit out of such instructional support to the project staff.
The training program of project staff will encompass mostly the local trainings however training under technology transfer and those for learning advance techniques of serology will be abroad. A reasonable balance is kept just to ensure capacity building of laboratory crew parallel with the professional elevation of senior scientific staff and the project managers.
The project also intends to conduct the training of Medics and paramedics on scientifically proven and most evidence based protocol of snake bite management in Pakistan. The irrational use of ASV even without ascertaining any clinical sign of envenoming causes loss of already short ASV in Pakistan. Likewise even in presence of adequate ASV the most practicing physicians may not help to reduce morbidity and mortality of snake bite if they are having rudimentary concept of locally appropriate snake bite management protocol. The mostly referred western medical books for such do not help much because they are having treatment protocols for different types of snakes and those are applicable and successful only in highly advance care system of developed countries. Similar training of Medical staff in India proved saving 25 to 30% of total ASV use which counts conservation of many hundred thousand vials in a year.
Likewise indiscriminate killing of snakes in Pakistan is rapidly heading towards an unnoticed destruction of biological ecosystem in the country. Snakes are considered farmers friendly elsewhere in the globe and are preserved as agriculture support creatures. Snake eats mice and other rodents harmful for various crops and stored grains. Such damage of eatable articles by these rodents in Pakistan ranges from 11 to 15% of our total production.
Akin to the remaining country, in Sindh also only 20% of snakes are poisonous, the remaining 75 to 80% are non poisonous with no any injurious threat for human beings. It is usual in Sindh to kill any snake at first sight without realizing whether it is poisonous or not. Such attitude and practice rapidly dis-balancing the natural equilibrium and the number of harmful rodents usually pray of snakes is multiplying every year. It needs to teach the farmers community how to live with snakes rather to kill them at first sight. The project intends to design and impart such community orientation trainings especially in high risk districts to preserve this agriculture support creature and will teach the communities to develop their own snake moving kits without any tangible expense so as to enable safe shifting of even deadly snakes away from the human settlements. Detailed training program along-with tentative cost under the project is annexed at pages 75-80.
- Teaching and Training Material
Sindh project has caused the introduction of ever first National Protocol on Snake Bite Management and now Pakistan is at par with other developed countries having their National Protocols. It is going to start training of medics and paramedics for effective management of snake bite cases including on rational use of ASV. It is going to help Health Education cell at DGHSS to update it’s communication material on local venomous snakes, first aid management of snake bite cases and information for common masses for prevention of snake bite and how to live with snakes.
Sindh project has modified the outdated medical curriculum of community medicine and Health Education material. Now medical students will study most modern evidence based knowledge about local snakes and snake bite management.