Purified Cobra Antivenin (PCAV) was fondly dubbed as a “wonder drug” by the brain behind its standardization in 2009, Lilian S. Dancel, RMT, as it manifests observable changes in seconds once administered in a patient.
“There was a patient rushed here who turned black already as the locals were not immediately able to attend to the patient. The patient already looked dead but when we injected PCAV, one could see how the color of the patient slowly changed. That’s why people call PCAV a ‘wonder drug’,” said Dancel as she recalled a severely affected patient rushed to the Animal Bite Center from the province.
Dancel explained that the patient was rushed to the Animal Bite Center because of the patient’s lack of awareness of the existence of antivenin here in the country.
“The patient hailed from a province. That is why the patient has turned black already upon arrival here. This is because there are provinces that are not aware that we have antivenin. There are also cases where certain provinces are aware of antivenin but unfortunately do not have them in their barrios,” Dancel added.
PCAV usually caters to farmers from Bicol and Nueva Ecija where the number of recorded cases of snakebites is high. These regions are the top priority for distribution of ampoules, because these areas are considered as the rice producing or rice granary of the province. Although snakes can be beneficial in decreasing the number of mice in the rice fields, they can cost farmers their lives even with just a single bite. Given this possibility, the challenge of feeding millions becomes even harder for rice farmers.
But what does really happen when a snake bites a farmer? Most people would think that first aid would suffice as they are not aware of any cure for it. But little do we know that we actually have antivenin in the country. The Research Institute for Tropical Medicine (RITM) houses the PCAV, the cure for snakebites, specifically those caused by the Philippine Cobra (Naja philippinensis).
Producing the lifesaver
The Biologicals Production Service (BPS) has been the sole producer of antivenin in the country since the 1960s. It was later on transfered to RITM in 1999. BPS then became the Biologicals Manufacturing Division (BMD) that people know today.
Today, PCAV is packaged in 5 mL ampoules containing 4.8 mL of antivenin which is capable of neutralizing 4.8 mg of venom. PCAV is monovalent, thus it can only specifically combat the venom of the Philippine cobra. Have you ever wondered how this lifesaver is produced?
Horse blood is the primary ingredient in antivenin production. Each ampoule of antivenin contains 4.8 mL of horse blood plasma rich with 800 mouse unit of their antibodies. Horse blood is collected from production horses injected with Cobra Venom Toxoid (CVT). It is a milk-like mixture of 1% snake venom and lanoline. Preparing the CVT is the first stage of antivenin production. Venom is extracted from cobras which is later purified and detoxified for administration among horses. When horses are injected with CVT, horses generate antibodies in reaction to the venom which their bodies recognize as antigens. When horses have generated enough antibodies, their blood is collected for purification.
Horses undergo basic immunization first before being part of antivenin production. Nulliparous horses or horses that have never given birth and have not received any chemicals are screened for health issues, injected with tetanus toxoid, which cleanses horses from toxic foreign bodies, and dewormed. Only then will a horse be ready for hyperimmunization or administration of CVT. A new production horse starts with the lowest dose of CVT. It takes about a year for a production horse to generate sufficient antibodies for blood collection. A horse continues to get hyperimmunization to maintain its amount of antibodies until such time it becomes ready for bleeding. About 10 mL of blood is first collected from a horse for potency testing where it is determined whether or not a horse has generated a sufficient amount of antibodies. Otherwise, a horse is not subjected for bleeding of three liters of blood. Out of the three liters collected for each horse, plasma constitutes two liters, while the remaining one liter contains the red blood cells.
The Institute’s Veterinary Research Department – Experimental Animal Facility Annex (VRDEAFA) is the one responsible for the proper care and maintenance of horses, administration of CVT, and proper collection of horse blood. VRDEAFA administers CVT among horses twice a month and bleeds horses a month and a half after CVT administration.
CVT administration and horse blood collection are conducted in VRD-EAFA’s farm in Bay, Laguna. It houses 17 cobras, 16 of which are of the Naja philippinensis species, while the remaining cobra is of the Naja samarensis species. The venom used for CVT is not extracted from the farm in Los Baños but is outsourced from Brazal Farm in Libmanan, Camarines Sur. Once a year, 200 mL of venom is extracted from Brazal Farm. The cobras available in the Los Baños farm are used for identification of venomous and non-venomous snakes, and demonstrations for medical fellows, rotators, residents, paramedical students, and visitors including on-the-job trainees.
After horse blood collection, VRD delivers the blood to PCAV to start the actual production of antivenin. The horse blood is subjected to complete sedimentation where the red blood cells are separated from the plasma. It takes three days for the red blood cells to completely settle at the bottom. Ideally, 10 liters of plasma should be collected per batch of production. PCAV produces two batches of antivenin every month. In each batch, 350 to 400 ampoules are produced.
After separating the plasma, it will then be subjected to purification. For this process, PCAV employs the Ammonium Sulfate Precipitation Technique. Ammonium Sulfate acts as the primary agent to remove the toxins from the horse blood plasma. This process subjects the horse plasma to two rounds of precipitation and filtration. The first round involves the removal of unwanted protein. Meanwhile, the second round is being done to precipitate the specific antibodies which is the IgG. After purification steps, follow Dialization Process to remove ammonium sulfate.
Once done, horse blood plasma undergoes formulation and sterilization for dispensing and filling, with PCAV taking in the form of ampoules. After accomplishing the dispensing and filling stage, all ampoules are now subjected to the final round of quality control testing. In this stage, ampoules are checked whether they are pyrogenic or not. Pyrogenic ampoules do not proceed to the next stage of production as these can cause patients very high fever which can be fatal. If ampoules have passed quality control, these will be delivered to the labelling department for final packaging for storage and distribution among hospitals and health centers.
Barriers to antivenin production
At present, the most pressing challenge PCAV is facing is the inadequate supply of horse blood plasma for antivenin production. According to Dancel, PCAV is now working with only 7 to 8 liters of horse blood plasma instead of the regular 10 liters of plasma. The current situation traces to VRD-EAFA’s dilemma in their farm in Laguna.
Currently, there is a decrease in number of horses used for bleeding because several horses have become “unfit” for production as VRD-EAFA Section Head, Dr. Marc Louie Monzon has claimed. “There are 31 horses here in the farm, but only 15 out of those horses are used for bleeding. Out of that 15, many horses are unfit for production. As of now, I can say that the toxoid is really strong that even if you feed the horses well, their bodies still remain unfit. That is why
some horses here used for production are thin),” said Dr. Monzon.
The remaining horses are not used for production because they are not mature enough for bleeding. Following the Animal Welfare Act of 1998, pregnant horses are also excluded from CVT administration and bleeding as it may cause harm to pregnant horses and to the offspring they are carrying.
Ideally, pregnant horses should not be a common sight in farms dedicated for laboratory purposes. Dancel says that castrated animals are preferred in such farms to avoid breeding. But in VRD’s farm, animals freely graze on the fields which makes breeding a definite possibility.
Due to insufficient funds, VRD’s farm lacks sturdy stables to separate male horses from female horses and adult horses from young horses. Given this situation, VRD-EAFA has resulted to alternative solutions.
“The horses multiply naturally but we try to control it…so we leave the horses tied outside their stables,” Dr. Monzon said.
Dr. Monzon said that the proposal for the improvement of the facility is already in the works. The proposal includes the construction of additional stables, a stable water source, a systematic drainage flow, a small animal laboratory, and an office.
Future for PCAV
PCAV is currently exploring the possibility of transitioning from Ammonium Sulfate Precipitation Technique to using another venom purification technique called the Caprylic Acid Precipitation Technique. Through the latter, the horse blood plasma is subjected to only one round of precipitation and filtration. Moreover, studies also show that it can generate higher yield of antibodies and combats infective viruses better than ammonium sulfate. The only disadvantage is that it is harder to filter as it is oil-based which means that it hardens when it is exposed to low temperatures.
But as per Dancel, ammonium sulfate is still capable of producing a higher antibody titer yield than caprylic acid to purify the IgG contained in the blood plasma. Dancel has experimented combining both ammonium sulfate and caprylic acid for venom purification. Her initial claim is that the antibody titer yield is twice the yield generated by ammonium sulfate. Dancel has finished the first trial of the experiment and shall continue to the third trial to validate her work.
Aside from new venom purification techniques, PCAV is also hoping to expand antivenin production to be able to provide ampoules to more hospitals and health centers.
“If we have already expanded antivenin production, we should distribute ampoules. Much like how vaccines are distributed to every region. As of now, antivenin distribution only works upon request [and submission of utilization report] because we are not capable of supplying in a large scale,” Dancel explained.
Lastly, since PCAV only produces monovalent antivenin specifically targeted to the venom of the Philippine cobra, PCAV aims to procure venom from other species of snakes particularly the Naja samarensis and Naja sumatrana species for the development of a polyvalent antivenin. While a polyvalent antivenin is weaker than a monovalent antivenin, it is still advantageous as it becomes essential in instances when it is difficult to identify which species of snake has bitten a certain victim. PCAV is also open to procuring venom from snakes endemic to other countries for exportation of antivenin.
by Anel Azel Dimaano
College of Development Communication
University of the Philippines Los Baños
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