Production of Diphtheria Antitoxin: A Step-by-Step Guide | Pharmaceutical Sciences

[00:00:00]hello everyone once again I welcome to my series of lecture that is understanding pharmaceutical science with dr. Harry Hahn so today we are going to discuss about the production of diphtheria antitoxin so in this lecture I have elaborately discussed about how the production of diphtheria antitoxin is done so before getting inside the production of diphtheria antitoxin first we understand what is mean by an antitoxin so antitoxin is an immunological preparation otherwise we can call also called as anti toxic sera so it is generally an antibodies preparation if you see that certain bacteria especially is bacterias like diphtheria and the tetanus that is Clostridium titania and that diphtheria bacteria the organism is itself is not infectious to the persons whereas the toxin that is the exotoxin released by this bacteria is generally infectious in nature and it causes the major diseases in terms to us so to avoid this one to inhibit this exotoxin the body usually produce an antibodies in certain conditions if the infection is so vigorous that we have to supply this antibodies from outside us and passive immunization so at the time this diphtheria antitoxin has its rose that means especially the antitoxin has its roads so anti toxins are generally an immunological preparation which is generally antibodies which is an antibody containing preparation so these antibodies is generally obtained from the serum of an immunized animals so which is generally having an anti toxic antibacterial and antiviral characteristics so this antitoxin preparation are generally administered once there is a need of any a passive immunization is needed so in the module most cases that diphtheria is the most common infectious agent so we have discussing about the production of diphtheria antitoxin in this lecture so if you see the production of diphtheria antitoxin it includes several stops so if you see the Stubbs which is needed for the preparation of diphtheria antitoxin the first step is the selection of animal so we have to select a proper animal in which we can get an maximum amount of antibodies so generally the preferred animal is the horse so after the selection of animal the second step is the isolation and Quarantine of the animal just to make sure that the animal doesn't gets infected with other diseases and the third step is the human eyes the animal with that toxin or the toxoid so that it has an ability to get produced the antibodies so once the animal produces the antibodies the fourth step we will collect the blood so in the blood the serum pod carries the antibodies so after collecting the blood and the fifth step we process the blood that means we remove the blood cells and we get the serum in an IPO form so after getting stearin still we refine it to remove the clotting factors and finally we isolate that antitoxin that has been administered as a dosage form so these are the basic production step of an diphtheria antitoxin so if you see the steps one by one the first one is the selection of the animal so the most preferred animal for the production of diphtheria antitoxin is the horse so why the horse is preferred is that it is a considerably a big animal so we have an chance to collect a enough quantity of blood after immunization so without causing any harm to the animal and moreover it is easy to handle so because of its size it is easy to handle and moreover it is a domestic animal so it also helps to simplify administration into the neck muscle and withdrawing the blood also so then the final one if we see the red blood cells the RBC's of the hearts settles very faster enough and we get a clear serum and compared to other animals so this facilitate the separation of the serum and further processing also so that's why we prefer horses an animal for the source for the production of diphtheria antitoxin so we also prefer we can use other animal also what happens is that sometimes the horse proteins if it comes into the preparation if it is not properly purified these horse proteins can sensitize the individuals so some people see when you tray someone to horse proteins can cause us an anaphylactic shock so in these conditions we prefer other animals such as the goat so good has been ability to provide the diphtheria antitoxin also so after the selection of the animal the next step is the isolation so the isolation of the horse in this case be a isolate and keep the horse in a quarantine for summin days so during the seven days we will observe the animal and we will examine is there any infectious diseases or that so the most important infectious diseases we have to check is the glanders so this Glander is and disease generally caused by the bacteria that is acting a bacillus Mally so it is a zoonotic infectious disease can be transmitted from the horse to the humans from humans it is quite faithful in nature if the patient is not treated properly then the death can occur so it should be examined properly for the presence of this glanders so it is a simple diagnostic test we can do it similar to like a fair test for the TB generally we do the tuberculoid test so similarly we will do the test with the reagent called the Mallon so once we find that the animal is free from this glanders then we immunize with the tetanus so generally the horse are highly susceptible to this tetanus so we are administered with tetanus toxoid so that it can produce an enough quantity of antibodies against this tetanus so after this the next steps comes we need the production of the antitoxin so we have to immunize the horse so humanization is nothing but a process in which you administered that toxoid or the toxin into the animal so that it has an ability to produce the antibodies so in this case we administer the diphtheria toxoid into the neck muscles gradually in a step-by-step manner in the increasing order of dose for yep quite a few days I see a course up to you a few months in a gradual increasing dose so if you see that the generally the first dose is generally about 5 ml in nature and the next dose after the collection of blood are two to three times then we generally increase slowly until the end of the course if you see that we might have administered up to 600 ml of this diphtheria toxoid so we increase this dose because the animal should produce a enough quantity of the antitoxin titer so generally this enough we get at the end of the course so after we immunization the animal the horse will start producing the antibodies against AIDS so we need this antibodies that is it is collected as an antitoxin so the next step is the collection of the blood so generally yet liters of blood can be get collected from this horse at a time so it is withdrawn aseptically from the jugular vein into your bottle and the bottle generally contains the anticoagulant so the anticoagulants makes up blood into the same condition later on this bleeding is repeated twice after eight days interval so if you withdraw the blood today means even leave you a gap for eight days then you can do your second bleeding process and the animal is allowed to rest for next ten days so I mean the meanwhile the ten days the animal can recover itself to produce enough blood then the second course we can start it with another short of antigen that is another start of diphtheria toxoid then we can further we take the bleeding for thrice and we can repeat this process until they enough antibody titer level it's gated maintained generally it takes four to five courses so the course is continued for four to five until the it stops producing as necessary antitoxin titer level so after collecting this blood the next step is we are processing the blood so how we are so sing the bloody fec is the blood is stored in the refrigerator so during the storage what happened is that the red blood cell started settling down I already told the horse is preferred because the rate of settling of RBC is faster enough and compared to other species so it settles faster then the supernatant serum is siphoned away then it is put into your bottle in which the calcium chloride is added so the calcium chloride will initiate the clotting process it is one of the clotting cofactor so it causes the practice to get clotted and the clot in their clot is removed by the filtration so we get the pure serum then the further process is the refinement of this serum because the serum contains many proteins so these are many horse proteins are present in the serum this protein should be removed otherwise this proteins can cause us problem to the humans because the horse and the humans are very different species in nature and so the proteins also very different so the horse protein can work as an antigen to the human body so our body has an ability to produce antibody against this horse proteins so if it is producing it's well and good but sometimes what happens is that certain individuals will develop a hypersensitivity state after the administration of this antitoxin so because of the presence of the trace amount of this horse protein so generally the to manifestation we can see because of this horse protein is the severe anaphylactic slark and the serum sickness so out of this the severe anaphylactic stock is the very dangerous condition what happens it occurs immediately after giving the injection of the serum to the patient so the patient becomes faint and pale and it and also causes difficulty in breathing if it is left untreated now the patient can die so on this case is generally seen high in the asthmatic patient or the is jamia infantile so the second condition a little bit mild one that is the serum sickness so generally it leads certain headache and swelling of the face and joint pains in the joints and fever or rash so this will be slowly get away once the antigen has been removed from the body but still the presence of the horse protein is a very dangerous to the human so the serum should be properly refined then mele it can be used for isolation of the antitoxin so the next final step is isolation of the antitoxin so the serum contains the after we find money it contains three basic proteins one is the albumin second is the beta globulin and third is the gamma globulin so out of this the antitoxin is the beta globulin so we need to isolate beta globally from this albumin and the gamma globulin so generally the gamma globulin will have an antibacterial and antiviral antibodies so it should be removed we need only the beta globulin so if you see the method of isolation of this beta globulin from the other two proteins two methods are generally employed one is the concentration by a fractional precipitation and second is the concentration by proteolytic digestion so if you see the first one that is the fractional precipitation so this technique is developed with an objective that if you slowly precipitate the other proteins by using a suitable concentration of ammonium sulfate we can isolate the beta-globulin in a pure form so how it is done if you see that first year sufficient amount of 1/3 saturated solution of ammonium sulfate is taken then it is added to the serum which carries these proteins so this at this concentration generally the gamma globulin will gets precipitated out so it is filter and removed so now the serum contains only the two constituents that is the albumin and that bit of globulin so we have to isolate the beta globulin so the ammonium sir fit concentration is slightly rised from one third level to of the saturation so at this level what happens is this beta-globulin started getting precipitated out so the liquid part will carry the albumin so it is filtered with the help of a filter plus so the liquid portion albumin is get removed away and we collect the filter cake which generally carries the beta-globulin in a pure form so it is collected and it is packed into your Salah fan bake ya dialysis membrane then it is kept inside a solution that is chlorinated with a running water so water runs frequently with the chlorinated so it goes through the cellophane bag so the dialysis process will happen so by this dialysis process what happened is that the beta globulin is precipitated along with ammonium sulphate so the ammonium sulphate get dissolved and it passes through the dialysis membrane and it is removed with the running water whereas the pure form of this beta globulin will be written inside the cellophane bag so this generally takes two to three days until it is completely free from the ammonium sulfate so by the presence of the chlorine is that there is no possibility of contamination of bacteria because chlorine itself acts as an antibacterial agent it is free from the pyrogen and the other bacterias so afterwards what happens we take out the cellophane bag we will take the beta-globulin which is in antitoxin then its pH is adjusted with the blood plasma then it's isotonic city also kept on near to the blood plasma level that is around 0.9 percentage solution saline solution then a preservative is added and dispensers and a multi dose container so it is free from the pyrogen and other things this further to ensure it we can filter it and then we can pack it so this is one method of we are purifying and getting an isolated form of anti toxin that is diphtheria antitoxin and the second method is the concentration by proteolytic digestion so in this method we will add a proteolytic enzyme we preferably we add a pepsin so person is an enzyme which is generally destroys the protein and just digest the proteins so to get your maximum activity of pepsin we have adjust the pH to four so it gives an optimum condition for the pepsin digestion then it is incubated at a temperature of both 37 degrees Celsius for two days your following changes slowly started occurring the first one is that the album is is completely destroyed be digested by this pepsin so it passes through the dialysis membrane so generally how it is done is that we take a cellophane bag we fill the serum then we will kept inside the solution so what happened is this albumin get digested it will gets removed away then the next one is the gamma globulin is partially digested because of the change in the pH then it is the whereas the beta globulin what happens is it gets digested but split into two parts so out of these two parts one will have an anti toxic activity that is it as an antitoxin property the other does not have but both the things will get written inside the cellophane bag he doesn't got passes out through it so after that what we will do is we will take this one then we remove the gamma globulin by filtration then we get a pure form of this beta globulin so it is further subjected to ultra centrifugation and further dialysis of the product because there is no traces of albumin and the gamma globulin should be present so we will add some inorganic salts so then did the entire preparation is concentrated at 55 degree Celsius along with addition of ammonium sulfate so this ammonium sulfate what it will do is it causes the inactive beta inactive fragments of the beta globin which is the another part which if it doesn't need it it yet natured one the innocence of inorganic salts it gets precipitated out so it is remote filter off and the ammonium sulfate what it do its it forms a precipitate with the active fragment so we are isolate the active fragment in the precipitate of ammonium sulfate then it is removed similar to the fractional distillation process or if a fractional precipitation process kept in a dialysis back in a chlorinated water tank so ammonium sulfate got away we get the pure form of the beta-globulin that is the active part so then it is just a tie so tonicity and preserved eniac are multi dose container and it is dispensed so this is how the production of diphtheria antitoxin is done so if you see the dosage and administration diphtheria antitoxin is given in two ways one has an a prophylactic purpose and second is as the treatment purpose so you feel it is given as a prophylactic purpose it is given intramuscularly about 1000 IU so if it is for treatment purpose the treatment is based upon the type of infection it has happened so if it is a pharyngeal or laryngeal diphtheria and occurs about 48 hours of duration then we have to administered intramuscularly 20,000 to 40,000 IU if it is nasal pharyngeal diphtheria then about within 48 hours we have to administer about 40,000 to 60,000 IU if it is an extensive diphtheria which has occurred for the about three or more days then we have to administer about 80 thousand to one lakh 20,000 diphtheria antitoxin intramuscularly so if you see the storage condition it is generally stored similar to other vaccines two to eight degree Celsius that is 36 degree Fahrenheit or 46 degree so and moreover it should be it should not get frigid away or do not expose to any freezing temperature it should be maintained properly so thank you very much for understanding about the diphtheria antitoxin production process so in my other lectures you can understand the production of the diphtheria toxoid as well as step stocks I thank you very much