2Production area has five sectional areas: PRODUCTION OF PARENTERALSSpecial care should be taken during the production of parenterals products so that the final products are free from physical (particulate), chemical, microbial and pyrogenic contamination. The raw materials should be of special grades of purity (parenteral grade), the production areas should be highly cleaned and of controlled environ-ment to avoid the particulate and microbial contamina-tion of the product. The apparatus and equipment should also be thoroughly cleaned and sterilized.Production area has five sectional areas:1. Clean-up area, 2. Preparation area,3. Aseptic area, 4. Quarantine area, and5. Finishing or packaging area
3Process Flow Diagram Store room Preparation area Aseptic filling area Quarantine areaStorage&shippingClean-up areaSterilizationPackagingfinishing
4Floor Plan of an Aseptic Area with its Service Area Air lockQC areaDressing RoomSterile filling areaFilling Dept officeAirLockService areaSterile areaSterile areaSterile filling AreaAir lockSterile Supplies
5Clean-up area:Clean-up area will be constructed to withstand moisture, steam & detergents.The ceiling, walls and floor should be constructed to provide smooth, non-porous and non-absorbent surface.These areas should be adequately exhausted, so that heat and humidity will be removed for the comfort of personnel.Precautions must be taken to prevent the accumulation of dirt and the growth of micro-organisms, esp, in the presence of high humidity and heat.Aseptic conditions are not required, but must be kept clean and the microbial- load must be monitored and controlled.Precautions also must be taken to prevent deposition of particles or other contaminants on clean containers and equipment.
6Ceiling walls and floor should be sealed. Preparation area:In the preparation area, the formula is compounded and preparation is made for the filling operation such as filtration of the solutions.Although this area need not be aseptic, it should be more strictly controlled than the clean-up area.Cabinets should be of stainless steel. They should fit snugly (tightly) to walls and other furniture so that there are no catch areas for dirt to accumulate.Ceiling walls and floor should be sealed.Ceramic, plastic cement is used for the floors to get a smooth surface.(Spray- on- tile finish with epoxy resin gives a smooth and non-porous surface for walls and ceiling). All such surfaces can be washed at regular intervals to keep them thoroughly clean.
7Aseptic area:It requires construction features which have been designed for maximum security.The ceiling, walls and floor must be sealed so that they not only be washed, but also treated with an antiseptic spray before each use.All counters (cabinets)must be of stainless steel and hung from the wall (to minimize dust accumulation at the legs resting on the floor).As far as possible, tanks containing compounded product and mechanical equipment should remain outside the aseptic area and the product should be fed thro’ hose-lines.Mechanical equipment that must be located in the aseptic area must be enclosed with in stainless steel cabinets in order to seal their operating parts.
8ENVIRONMENTAL CONTROL: The standards of environmental control will vary, depending upon the area involved (clean-up, packaging, compounding or filling) and the type of product being prepared. If the product is prepared aseptically (without terminal sterilization), the entire area involved in preparation should be under the most rigid control. When the product is terminally sterilized somewhat less rigid control of compounding and filling area may be acceptable, but rigid standards of cleanliness must be maintained. High standards of cleanliness must also be maintained in clean-up and packaging areas (need not be disinfected daily). Traffic should be minimized, particularly in and out of the aseptic rooms. Unauthorized persons should never be permitted to enter the aseptic area.
9ENVIRONMENTAL CONTROL… All the surfaces, at least in the aseptic areas should be sprayed with a liquid disinfectant after thorough cleaning. Irradiation from U.V. lamps properly located will further reduce the viable microorganisms present on surfaces and in air. U.V. rays are used to sterilize the inside exposed surfaces of processing tanks, surfaces of conveyor belts or other confined surfaces.The air in the aseptic area must be free from fibers, dust and microbes. This can be achieved by filtration of air thro’ high efficiency particulate air (HEPA) filters. Even submicron range particles (up to 0.3 μm), and microorga-nisms are removed by HEPA filters (eff: 99.97%, composed of glass-wool and asbestos or electrostatic precipitators).
10The clean, aseptic air is distributed into the aseptic room at the greatest volume flow rate, there by producing a positive pressure in the room. This prevents the entry of unclean air into the aseptic room thro’ cracks temporarily open doors or other openings.The entire wall, ceiling or work benches can be fitted with HEPA filters to produce laminar air flow. In laminar-flow system the sterile air flows in parallel, with minimum turbulence, at a speed of about 30 metres /min (100 ft/min).
11The effectiveness of environmental control system must be tested routinely. Microbial contamination is tested by:air impingement on nutrient media orsettling and surface contact nutrient agar plates.Particulate matter can be tested by membrane filtration or electronic particle counters.
12PERSONNEL:The personnel working in the aseptic areas may provide particulate and microbial contamination.All employees should be in good health (free from cold, sore-throat etc.)They should wear special sterile clothings, including hoods, face masks, shoe covers and gloves.The garments should be of textiles that shed minimum fibres (ex. Dacron, nylon).The workers should also be contamination conscious and should be highly trained and skilled.Before entering the aseptic area they should strictly follow the prescribed procedures including washing the hands and arms thoroughly with a disinfectant soap.
13PROCESSING OF PARENTERALS: The steps involved in the processing of parenterals are:1. Manufacturing, storage and distribution of water for injection (WFI)2. Cleaning equipment and containers.3. Compounding the product (mixing of ingredients)4. Filtration of solutions5. Filling and sealing6. Sterilization.
141. Manufacturing storage and distribution of WFI: The initial processing step is the procurement of acceptable components (i.e., the components tested for their quality by the quality control dept.). Water for injection is mfd in the production dept. and is tested for its quality.WATER FOR INJECTION:Water for injection is prepared by specially designed distillation still or by reverse osmosis. The specifications for a still should include:1.Pre-purification of feed water by chemical softening, deionising or filtration to improve the quality of the distillate.2. Efficient baffle system to minimize entrainment.3. Removal of dissolved gases like (NH3) by pre-heating of the feed water.4. All surfaces that will come into contact with the vapor and condensate should be of pure tin, s.s. or borosilicate glass.
15Large quantities of WFI are obtained by using vapor compression stills and multiple effect stills. REVERSE OSMOSIS: Water is forced at lbs/psi thro’ membranes made of cellulose esters or polyamides (nylon)Storage and distribution: The storage and distribution of water for injections are as important as its production. A closed system is desirable, with air exchange thro’ a filter that removes micro-organisms, dirt and vapors from the air as the tank is filled and emptied.The tank may be held at 80o C by means of steam coil at the bottom of the tank to prevent multiplication of micro-organisms.In large plants WFI is distributed from the storage tank thro’ pipelines taking special precautions to prevent contamination.
162. Cleaning equipment and containers: The equipment & containers to be used in the process-ing of a sterile product must be thoroughly cleaned.In general, equipment used previously should be thoroughly cleaned using a detergent.Sometimes treatment with steam is required to loosen the solid residues.After cleaning, the equipment should be rinsed several times and finally rinsed with WFI.Just prior to rinse, large clean tanks and similar equipment should be rinsed thoroughly WFI.When equipment is reserved for use with only one type of product, cleaning problem will be minimized.
17Rinsing new containers: In cleaning new glass ware, the detergent treatment is usually eliminated (and thereby reducing the risk of detergent residue). Therefore, the cleaning cycle is a rinsing process.To loosen debris by rinsing, alternating hot (preferably steam) and cold treatments should be used.Finally rinsed with filtered distilled water (or water for injection).After these rinses, the remaining water from containers like ampoules is blown out by a blast of clean air (e.g., rotary rinser).Then they are sterilized by placing in clean stainless steel boxes.
18Cleaning rubber and plastic components: Rubber closures are usually washed by mechanical agitation in a tank of hot detergent solution (e.g., 0.5 % sodium pyrophosphate), followed by a series of thorough water rinses.They are finally rinsed with WFI.Sometimes, closures are subjected to an autoclave cycle to remove some of the soluble components from the closures.Then the closures should be saturated with the antiseptic which is to be used in the product. This is done by storing the washed closures in the antiseptic solution over prolonged period of time.
19STERILIZATION OF EQUIPMENT: In general, equipment, containers, closures and all other components should be sterilized after cleaning and prior to use.3. Compounding the product:The tanks or containers in which the ingredients are mixed should be thoroughly cleaned to prevent any chemical or pyrogenic contamination.The product should be compounded under clean environmental conditions (aseptic conditions are not required).Accuracy of compounding and order of mixing the ingredients are important.In large batches particular attention must be given to achieving and maintaining homogeneity of solutions, suspensions and mixtures.
204. Filtration of solutions: The primary objectives of filtration or clarification or sterilization are:Clarification (polishing), where particles up to 3 μm size should be removed.Sterilization- Particles up to 0.3 μm should be removed, whereby viable micro-organisms and spores are also removed.
21Filters: Filters are made up of sintered glass, unglazed porcelain (sintered ceramics), asbestos, stainless steel (sintered metal) or membranes made from cellulose esters (acetate or nitrate), PVC or silver.After sterile filtration the solution must be protected from environmental contamination until it is sealed in the final container.The best method is to collect the filtrate in a container which is a part of a closed system and the filtrate is fed directly from the collecting vessel to the filling machine thro’ a sterile piping.A secondary “in line” filter is included near the outlet from the filling machine to remove any particles or fibers picked up from the pipelines or equipment.
225. Filling Procedures: A liquid may be subdivided from a bulk container to individual dose containers more easily and uniformly than a solid and again viscous and sticky liquids require heavy-duty machinery.Filling equipment for liquids:A measured volume of the liquid is forced thro’ the delivery tube. The tube must freely enter the neck of the container and deliver the liquid deep enough to permit air to escape without sweeping the entering liquid into the neck or out of the container. The tube should have the maximum possible diameter to reduce the resistance to the flow of the liquid. Excessive delivery force causes splashing of the liquid and also foaming (if surface tension of liquid is low).Relatively small volumes of liquids can be delivered from the stroke of the plunger of a syringe.
23A retraction device is designed as a part of most filling machines, to prevent the wetting of the neck of the ampoule by the hanging drop at the tip of the tube after a delivery.Filling machines should be so designed that the parts thro’ which the liquid flows can be easily opened for cleaning and for sterilization.Sterile solutions of low potency dispensed in large volume (up to 1 L) do not normally require the same precision of filling that is required for small volumes of potent injectables. Therefore, bottles of solution are usually filled by gravity, pressure or vacuum filling devices.
24Gravity filling- Hand operated and the bottles are filled to graduations on the bottles. Pressure filling- semiautomaticVacuum filling- can be fully automated. Used for faster filling of large liquid volumes (vacuum is created in the bottle and filled).A slight excess (overage) is required in each container to compensate the loss that will occur at the time of administration by adherence to the wall of the container and retention in the syringe and hypodermic needle.Suspensions and emulsions are constantly agitated in the reservoir during filling to maintain the homogeneity of the product and to obtain uniformity of drug content in the filled containers.
25Filling equipment for solids: Sterile solids, such as antibiotics, are more difficult to subdivide accurately and precisely into individual dose containers than the liquids.Fine powders flow slowly and irregularly. In such cases the sterile powder is weighed in the individual containers. This is a slow process.In the case of free-flowing granular powders machine filling is employed. But stratification of particles and electrostatic charges on particles should be minimized by maintaining uniform particle size and using small electric current to neutralize the developing charge.
26Machines for poor-flowing powders: It consists of adjustable cavities in the rim of the filling wheel. The cavity comes under vacuum connection as the wheel passes under the hopper. The contents are held by vacuum until the cavity is inverted over the container, when a jet of sterile air discharges the dry solids.Sealing: Containers should be sealed in the aseptic area immediately adjacent to the filling machine.
27Ampoules can be sealed by either tip-sealing (bead seal) or pull-sealing. Tip sealing is done by melting the glass at the tip of the ampoule neck to form a bead of glass. Pull-seals are made by heating the neck of a rotating ampoule below the tip and then pulling away the tip to form a small twisted capillary, which is then melted and closed. Pull-sealing is a slower process, but perfect seals are obtained without leaks. Powder ampoules or other types having a wide opening must be sealed by pull-sealing.Multiple dose vials or transfusion bottles are sealed by rubber closures on to which aluminum caps are crimped.
286. Sterilization of product: A product must be sterilized by the most reliable method possible. Except in the case of thermo-labile products, sterilization in effected after filling and sealing the final containers.Aqueous preparations - AutoclavingOily products- Hot air ovenRadiation sterilization (gamma rays) involves huge capital investment.
29FREEZE- DRYING: (Lyophilization) or (Gelsiccation). Freeze drying is a process of drying in which water is sublimed from the product after it is frozen.The biologicals and pharmaceuticals which are relatively unstable in aqueous solution can be dried at low temps and preserved indefinitely in the dry state.Sublimation of ice occurs at pressures and temps below the triple point (4.579 mm Hg and o C). For pharmaceutical solutions, the temp and pressure relationship varies because of the dissolved solids. In such cases, the temp and pressure at which sublimation of the frozen solid occurs is called as the eutectic point. Freeze- drying is carried out at temps and pressures will below this point. In actual practice, freeze- drying of pharmaceuticals is carried out at temps of -10o C to -40O C and at pressures of about 100 microns (Hg).
30The stages involved in the freeze- drying are: 1. Freezing the aqueous preparation and cooling it to a temp below its eutectic temp.2. Evacuating the chamber, usually below 100 microns Hg.3. Subliming ice on a cold condensing surface at a temp below that of the product. (the condensing surface being within the chamber or in a connecting chamber).4. Controlled heating of the product (keeping the temp below its eutectic temp) to provide energy for sublimation.
31The product may be frozen on the shelf in the chamber by circulating refrigerant (usually Freon , ammonia or ethylene glycol) from the compressor thro’ coils with in the shelf. After freezing is complete, the chamber and condenser are evacuated by the vacuum pump; the condenser surface is also cooled previously by the circulating refrigerant from the large compressor.The controlled heating of the product is done by means of electric coils within the shelf or by circulating hot water. The product is dried until it contains <1% moisture.The rate of drying largely depends on the thermal conductance of the frozen product, the rate at which the vapour can diffuse thro’ the increasing depth of the dried porous material and the rate of transfer of the vapour thro’ the system to the condenser surface.
32After completion of the drying cycle, re- absorption of moisture must be prevented. Therefore, the product must be sealed as rapidly as possible after removal from the chamber under controlled low- humidity conditions.Freeze dryers may also be equipped for stoppering vials within the drying chamber. Special slotted rubber closures are partially inserted in to the neck of the vials prior to freezing the product. (The water vapour escapes thro’ the slots during the drying cycle). At the end of the drying cycle, a hydraulic plate or rubber diaphragm presses the closures firmly into the neck of the vials and seals them under vacuum.
33Fig: Essential Components of a freeze- drying system. Heavy insulationFig: Essential Components of a freeze- drying system.
34CONTAINERS AND CLOSURES: Containers and closures are in intimate contact with the product. They may react with the product during storage and thereby affect the stability of product. Therefore, containers and closures must be carefully selected.Glass containers: Glass containers have been traditionally used for sterile products. These are still preferred for injectables products. Glass is composed principally of silicon dioxide or silica, modified physiochemically by various oxides (Na, K, Ca, Mg, Al, B and Fe).
35The two general types of glass are soda- lime and borosilicate The two general types of glass are soda- lime and borosilicate. Glass that is composed entirely of SiO2 is most resistant chemically but it is relatively brittle and has high melting point. Boric oxide modifies the above properties, but it enters the structural configuration, so the borosilicate glass is having high chemical resistance. But the other oxides will not enter the structural configuration and therefore free to migrate into the product on prolonged contact, particularly into the aqueous solutions. These oxides hydrolyze to raise the pH, catalyze reactions or react chemically with the ingredients of the formulation. Glass flakes are also sometimes produced during this solution process. These interactions are highly accelerated during autoclaving.
36Chemical resistance:Chemical resistance ὰ 1/ reactivity (reciprocal of reactivity) chemical resistance of glass is tested by two official test methods1. Test on powdered glass &2. Water attack test (on whole containers).The test results are measures of the amount of alkaline constituents reached from the glass by purified water under elevated temp conditions. The water attack test is used only with containers that are exposed to sulphur dioxide fumes under controlled humidity conditions. Such treatment neutralizes the surface alkaline oxides thereby increasing the chemical resistance of glass. But this increased resistance is lost, if the container is subjected to repeated autoclaving, hot air sterilization, or hot detergent treatment.
37Vol.of acid to neutralize the extract from 10g of glass (ml) On the basis of the official test results, glass compounds are classified into four types. The greatest chemical resistance is provided by type I, so it is preferred for most sterile products.USP Glass types, test limits & selection guide:typeDescriptionTest usedlimitsGeneral useSize (ml)Vol.of acid to neutralize the extract from 10g of glass (ml)IHighly resistant Boro silicate glassPowdered glassall1.0Buffered and un-buffered aq. solutions, all other usesIITreated soda- lime glassWater attack100 (or) lessOver 1000.70.2Buffered aq. Solution with pH < 7Dry powders, oleaginous solutionsIIISoda- lime glass8.5Dry powders, oleaginous solutions.NP(non- parenteral)General purposeAll15.0Not for parenterals, for tablets, oral solutions and suspensions, ointments and external liquids.
38Test for Hydrolytic resistance I.P: The test is carried on the unused containers. The number of containers to be examined and the volume of test solution to be used are given in the table.Normal capacity of containerNumber of containers to be usedVol. of test solution to be used for titration5 ml (or) lessAt least 1050 ml> 5 ml, up to 30 mlAt least 5> 30 mlAt least 3100 mlMethod: Each container is rinsed at least twice with water at room temp. just before the test, each container is rinsed with freshly distilled water. The containers are filled to be brim with freshly distilled water. They are emptied and the average overflow volume is calculated.
39Ampoules are filled with freshly-distilled water to the maximum volume compatible with sealing them. Bottles are filled to 90% of their calculated over flow volume and covered with borosilicate glass dishes previously rinsed in freshly distilled water. The containers are placed in autoclave. The autoclave is closed and the air in the autoclave is displaced by passing steam for 10 min. Temp is raised from 100 to 121o C over 20 min. and it is maintained for 60 min. then the temp is reduced to 100o C over 40 min. The containers are cooled in a bath or running water. Within 1 hr of removing from the autoclave the following titration is carried out.
40The liquid from the containers is combined and the specified volume is measured into a conical flask. 0.5 ml of methyl red solution is added for each 50 ml of liquid. Then it is titrated with 0.01 N HCl. End point is compared with a blank made from the same volume of freshly distilled water. The difference between the titrations represents the volume of 0.01 N HCl required by the test solution. Then the volume of 0.01 N HCl required for each 100 ml of test solution is calculated. It should not exceed the value given in the table.According to I.P the glass compounds are classified as follows:Type I (Borosilicate glass or neutral glass)Type II (Treated soda- lime glass)Type III (soda- lime glass)- generally not used for injectable preparation, except where suitable stability test data indicates that type III is satisfactory.
41Physical characteristics: One of the important physical characteristics of a glass container is the protection of light sensitive products from U.V radiation. U.V rays can be completely filtered out by the use of amber glass (colour is due to Fe2O3). If the product contains ingredients whosedecomposition is catalyzed by iron, amber glass cannot be used. In such cases, the product should be protected from U.V. rays by means of an opaque carton surrounding a flint (colourless) glass container.
423. Transparency to facilitate inspection of the contents. In addition to other physical characteristics, glass containers should have:1. Sufficient physical strength to withstand the rigors (high pressure differential) of autoclaving and the mechanical shocks during processing, transportation and storage.2. A low coefficient of thermal expansion to withstand the thermal shocks during washing and sterilization.3. Transparency to facilitate inspection of the contents.4. Uniform physical dimensions to facilitate handling by the mechanical machinery used for automatic production operations.
43Glass containers are sometimes coated with silicones to produce a hydrophobic surface, thereby to minimize the adherence of costly suspensions and emulsions.The size of single dose containers is limited to 1000 ml multiple dose containers to 30 ml (to limit the no. of withdrawals & thereby to minimize the risk of microbial contamination of the remaining contents).The risk of microbial and viral contamination can be further minimized by the use of prefilled disposable syringes.Double chambered vials designed to contain a sterile powder (or freeze-dried product) in the lower chamber and the solvent in the upper chamber separated by a rubber disc.
44Rubber Closures: Rubber closures are used to seal the openings of cartridges, vials and bottles. Rubber closures should provide a material soft and elastic enough to permit the withdrawal of a dose without loss of the integrity of the sealed container.Composition and reactivity:Rubber closures contain several ingredients:Natural rubber (latex) and/ or a synthetic polymer (neoprene or poly-isoprene),A vulcanizing agent (↑ strength of cross-linking b/w rubber molecules) – sulphur,An accelerator – several org compds like 2- mercaptobenzothiazole (MBT),Activator – ZnO, zinc stearate (for MBT)Fillers – carbon black or lime-stoneAntioxidants (phenyl beta - naphthylamine) &Lubricants (Zinc stearate or talc)
45There are two compatibility problems: Ideally, closure s should be completely non-reactive with the product with which they are in contact. But the rubber closures should be tested for compatibility with the preparation.There are two compatibility problems:1. Leaching of ingredients from the rubber closure and reaction with the ingredients of the product.2. Removal of ingredients from the product by sorption or by vapour transfer thro’ the closure.These problems can be minimized by plastic coatings or Teflon liners.Coring (Shedding fragments when hypodermic needle is inserted) should be minimal. This can be controlled by using a needle of minimum gauge and its insertion with its bevel side up at an angle < 45O C.
46Testing of closures: Method of testing for lot to lot uniformity of rubber closures are: 1.Physicochemical tests on aq. extracts: pH, turbidity, residue on drying, iodine number and heavy metal content.2. Biological tests on saline, PEG 400 and cotton seed oil extracts. (acute and chronic toxicity in mice and rabbits).Plastic containers:Containers are made from thermoplastic polymers (become soft at elevated temps, but can be repeatedly heated without change of properties). E.g. polyethylene, polypropylene, PVC, nylon (polyamide), Teflon (PTFE, polytetrafluoroethylene).Plastic containers may contain plasticizers, fillers, antioxidants etc. (not chemically bound in the formulation) which may migrate into the product.Most of the thermoplastic polymers can be autoclaved except polystyrene and LDPE.
47Plastic containers are light in weight and non breakable, but they have high permeability for water- vapour (Polystyrene- high oxygen permeability).Uses: Flexible polyethylene containers- for eye drops.Flexible PVC bags- for I.V. solutions.Tests for evaluating toxicity of plastic materials:1. Implanting small pieces of the plastic material IM- in rabbits.2. Injecting eluates using NaCl inj, with and without alcohol I.V.- in mice and eluates using PEG 400 and same oil intraperitoneally in mice.3. Injecting all four eluates subcutaneously in rabbits.
48QUALITY CONTROLThe three general areas of quality control are:Incoming stock,Manufacturing (processing) andThe finished product.Control of incoming stock involves:Routine tests on all ingredientsPyrogen tests on WFI.Glass tests on containers andIdentity tests on rubber closures.Process control involves tests and observations made throughout the manufacturing process.e.g. conductivity measurements during the distribution of WFI.Checking of fill volume in the container Recording of cycle time and temp for thermal sterilization of productChecking the identity of labels for the product.
49Product control includes the final assays and tests. In addition to the usual chemical & biological tests, a parenterals product is subjected to various other tests like:Leaker testClarity testPyrogen test andSterility test.Leaker test: The leaker test is intended to detect incompletely sealed ampoules so that they may be discarded. Tip-sealed ampoules are more likely to contain leakers than the pull-sealed ampoules. In addition, small cracks may also develop around the seal or at the base of the ampoule due to improper handling.
50Leakers are usually detected by producing a negative pressure within an incompletely sealed ampoule, usually in a vacuum chamber, while the ampoule is completely immersed in a deeply coloured dye solution (0.5- 1% methylene blue). The vacuum should be sharply released after 10 min, whereby the dye solution is forced into the incompletely sealed ampoule. The procedure is repeated three times.Leakers can be more effectively detected when the ampoules are immersed in a dye solution during the autoclaving. After autoclaving, the dye solution with the ampoules immersed in it should be allowed to cool sufficiently for the dye to be drawn in to the faulty ampoules as their contents contract.
51Clarity test: The clarity of a solution is usually evaluated by individual examination of each externally clean container under a good light, baffled against reflection into the eyes. The contents are examined against a black and white back ground with the contents set in motion with a swirling action. A moving particle is much easier to see than a stationary one, but care must be taken to avoid introducing air bubbles, which are difficult to distinguish from dust particles. Finally heavy particles are inspected by inverting the container.Electronic particle counters were also used but they could not differentiate between an air bubble and a dust particle.
52Test for large volume injections (Infusion fluids) (U.S.P): A specified volume of the product is filtered thro’ filters with an average pore size of 8μm,followed by microscopic examination of the filter disc. There should be not more than 50 particles of 10 μm and larger per ml of liquid. (Particulate matter produces pathological lesions or tissue damage in vital organs).Pyrogen test:The presence of pyrogens in parenteral preparations is detected by a qualitative biological test based on the fever response of rabbits. Rabbits are used as the test animal because they show a physiologic response to pyrogens similar to those of human beings. If a pyrogenic substance is injected into the vein of a rabbit, an elevation of temp occurs within a period of three hours.
53The injection is made into the ear vein and the fever response is measured by recording the rectal temps by inserting a thermometer into the rectum at a depth of 6-9 cm or thermocouples can be used.The test is first carried out on 3 rabbits, which are put into their cages for 1 hr before the test begins. Temps are recorded at regular intervals of not more than 30 min, beginning at least 90 min before injection (rabbits showing abnormally high temps are rejected) and continuing for 3 h afterwards. The injection given into the marginal ear vein. If its volume is >10 ml, it is warmed to o C previously. The syringes and glass vessels should be pyrogen free (heated at 210O C for 3-4 h).
54The fever response of each rabbit is found by subtracting the mean of the temps recorded in the 40 min immediately preceding injection from the highest temp recorded after the injection. Then the responses of the three rabbits are added to give the ‘summed response’. F.R.= highest temp after inj - mean of normal tempsInterpretation of results:If the sum of the responses of the group of three rabbits does not exceed 1.4o C and if the response of any individual rabbit is less than 0.6o C, the preparation being examined passes the test.If the response of any rabbit is 0.6o C or more, or if the sum of the responses of the three rabbits exceeds 1.4o C, the test is continued using five other rabbits.If not more than three of the eight rabbits show individual responses of 0.6 o C or more, and if the sum of responses of the group of 8 rabbits does not exceed 3.7O C, the preparation being examined passes the test.
55Ind. Res. should not exceed 0.6 o C No. of rabbitsS. R. should not exceedInd. Res. should not exceed 0.6 o C31.4o CNone83.7o Cn.m.t. 3Preliminary test (Sham Test):One to three days before the proper test, a ‘sham’ test is performed using animals that have not taken part in a test during the previous 2 wks; each is given 10 ml/kg of body wt of pyrogen free isotonic saline. Rabbits showing a temp variation of >0.6 o C are excluded from from pyrogen tests until they behave normally. This pre-testing also introduces the animals to the test conditions.
56LAL TEST (Limulus Amebocyte Lysate Test): Many medicinal agents (if present) interfere with the test results because of their antipyretic or other interfering effects. Therefore, the pyrogen test is performed on all vehicles used for injection, but only on those finished products that do not interfere with the test and those having a greater possibility of being contaminated with pyrogen and those injected in large volumes. The pyrogenic effect is less than with IM injection than with IV injection.LAL TEST (Limulus Amebocyte Lysate Test):In vitro test depends on the gelling property of the lysate of the amebocytes of the horse-shoe crab (Limulus polyphemus) in the presence of an endotoxin; when incubated at 37o C for 60 min.Test for sterility: All parenteral products must pass the sterility tests
57PREPARATION OF HEAT STERILIZED INJECTIONS Ex. 1: Histamine acid phosphate inj. B.P (6 ampoules)Method of sterilization: Heating in an autoclave Type of injection: Single dose of small volume.Strength: mg in 1 ml.Two alternative methods of sterilization are given in the pharmacopoeia- heating in an autoclave or filtration. But heating process is more reliable,First method is chosen. No bactericide (as an aid to sterilization or as a preservative) is required (single dose injection).
58Preparation of solution: The medicament is dissolved in water for injection or freshly distilled apyrogenic water (sterilized immediately after preparation)The container (ampoules must comply with the limit test for alkalinity of glass because Histamine acid phosphate is an acid salt.Overage: For single-dose injections in ampoules (small volume) a slight excess must be filled to compensate for the losses that occur during withdrawal and administration of the dose (overages for viscous and mobile injection of varying doses are given in a B.P.)Formula: For a 1 ml ampoule the overage is 0.1 ml for 6 ampoules a total volume of 6.6 ml solution is required.
59To allow for losses during preparation 10 ml is prepared. B.P. amounts amount usedHistamine acid phosphate mg mgWater for injection to ml to ml10 mg is not weighable quantity. Therefore, 50 mg is weighed, dissolved in a suitable volume of solvent and finally made up to 50 ml.Labels: Histamine acid phosphate is not a poison. If colourless ampoules are used a warning of the need for protection from light must be given on the box.Label for Ampoule:Histamine acidPhosphate Injection B.P.1 mg in 1 mlLabel for BoxSix ampoulesHistamine acid Phosphate Injection B.P.1 mg in 1 mlSterilized: (1) Name: (of patient)Name of the manufacturer or hospitalPROTECT FROM LIGHT
60Cutting: The neck of the ampoule is cut at a suitable length depending on the length of the filling needle. The tip of the filling needle must enter the ampoule body because deposition of solution in the neck may cause an air-lock and over-flow, and charring may occur during the sealing. Further, the ampoules should be short enough to fit into their box (if too short sealing is difficult, as it widens towards the base).Annealing: The mouth of each ampoule is rotated in a Bunsen flame to melt down the rough edge. (To prevent the rubbing off the glass fragments into the ampoule by the mount of the filling needle.
61Washing: If a special washing unit is not available a sink can be used Washing: If a special washing unit is not available a sink can be used. The ampoule is held inverted over a sink and a syringeful of freshly- distilled apyrogenic water is forcibly injected into it, so that the jet hits the base. The residual water is shaken out and the process repeated. Then the ampoule is inverted in a small beaker to drain.Drying: The inverted ampoules are dried (in the beaker) in the drying oven. (Wet ampoules may lower the strength of the solution and the medicament may diffuse thro’ the moisture film into the neck and then charring occurs on sealing).The cooled ampoules are placed upright in a suitable stand and each is covered with small glass tube.(e.g. Durham tube) to protect from dust. (On large scale the dried ampoules are transferred to a lidded metal box for clean storage).
62The solution is prepared by dissolving 50 mg of medicament in 50 ml of water by gentle heating. Filling: Filled with a syringe taking care to measure the volumes accurately and to keep the neck of the ampoule free from liquid.1. A little more than 1.1 ml of solution is carefully drawn into the syringe.2. The syringe is inverted to allow the air to rise towards the needle and the plunger is pushed up to expel the bubble and leave exactly 1.1 ml in the barrel.3. The needle is wiped with a cellulose film disc (will not give fibers).4. The ampoule is inverted over the needle. Both the ampoule and syringe are reverted together, and the liquid is expelled slowly.5. Finally the needle the needle tip is touched against the constriction at the bottom of the neck to remove the last drop of liquid and then the needle is withdrawn without touching the neck.
63Sealing: The sealing is done in the non-luminous flame of the Bunsen burner, or twin- jet burner. Preliminary Inspection: The ampoules are tested for any leaks in the seals by shaking and examined for particles. Any unsatisfactory ampoules should be rejected and extra ampoules are prepared.Sterilization: Leaker test can be carried out during auto-claving by immersing the ampoules in a solution of amaranth.Final inspection: The ampoules are rinsed in a non-ionic detergent solution and then in distilled water to remove absorbed on the glass, again examined for freedom from particles.Uses: 1. Gastric function test2. For the treatment of histamine headaches.Labeling and packing: The labels are checked carefully and stuck carefully. The ampoules are put into the box without finger-marking their necks.
64Ex.2: Preparation of 500 ml of dextrose injection B.P Type of injection: Single dose of large volume.Sterilization method: heating in an autoclaveStrength: 5% w/v of dextrose (anhydrous)Bactericides are not allowed in this type of injection overage is unnecessary.Therefore, the preparation involves only solution of the medicament 25 g in the solvent 500 ml WFI.Labels: Two special labels are neededa. warming against the use of the contents on more than one occasion; this is desirable on all infusion fluids.b. A direction to store in a cool place.
65500 mlDextrose Injection B.P.5% w/vStore in cool placeSterilized: (2) Name of patientName of manufacturer or HospitalWarning: Discard Any Unused PortionPreparation of solution: Dextrose monohydrate is not sufficiently pure for the preparation of injections and an autoclaving its solution becomes brown due to caramelisation. Therefore, dextrose (anhydrous) should be used.The weighed quantity of dextrose is transferred to a conical flask (500 ml capacity) containing 250 ml of water for injection and dissolved by gentle warming. Volume made up to about 400 ml.The solution is filtered thro’ G-4 filter by applying vacuum and sufficient solvent is passed thro’ the filter to make up the volume.
66Precaution: While preparing the solution, it is particularly important, not to add the solvent to the weighed amount of dextrose, because a slowly soluble sticky mass may be formed and if any amount of this sticky mass remains on the bottom surface of the flask that may be charred during heating.Then the solution is sterilized by autoclaving (30 min). The outer surface of the bottle is polished by dipping into non- ionic detergent (to remove drip marks from the autoclave). Inspected for any particles in the solution and labeled.
67INJECTIONS INVOLVING SPECIAL PROCEDURES (HEAT STERILISED)A. Use of minimum size of container:e.g., Adrenaline injection B.P. (0.1% w/v or 1 in 1000).In this injection the oxidation of Adrenaline acid tartrate (adrenaline HCl was used previously, which is less stable than acid tartrate) are prevented by-a) adding a reducing agent (0.1% sodium metabisulphite as antioxidant).b) using well-filled, well closed containers- ampoules are preferredc) protection from light.d) maintaining the pH in between (pH > 3.6 is harmful).Therefore, the the glass containers should comply with the test for alkalinity.Use: In the treatment of status asthmaticus (repeated attacks of asthma) and other allergic emergencies (hypersensitivity reactions) Dose: ml, SC inj.
68e.g. Apo morphine injection (3 mg in 1 ml). B. Nitrogen filling:e.g. Apo morphine injection (3 mg in 1 ml).Ascorbic acid injection (10% w/v)Apomorphine injection (HCl salt): Sodium metabisulphite is used as an antioxidant. The air above the solution in the ampoule is replaced by nitrogen; the ampoules are immediately sealed and sterilized by heating in an autoclave.It should be protected from light.Use: Emetic (2-8 mg SC or Im injection).Warning: The injection should not be used if the solution turns green.
69Ascorbic Acid injection: Sodium bicarbonate is used to adjust the pH between The air in the ampoule is replaced by nitrogen before sealing and sterilized by heating with a bactericide (chlorocresol) or by filtration. Ascorbic acid decomposes rapidly even in slightly alkaline solution. Therefore, the containers should comply with the limit test for alkalinity of glass. The injection should be protected from light.For very easily oxidized substance, such as phentolamine methanesulphonate (adrenaline antagonist used to control excessive variation of blood pressure), the preparation of solution, filtration and filling should be done under nitrogen atmosphere.
70C. Preparation of a suspension: e.g.: Bismuth oxychloride injection B.P.C.This is a suspension of bismuth oxychloride in water for injection. It also contains 0.9% of sodium chloride, to produce isotonicity and 0.1% chlorocresol as a bactericide.No suspending agents are included and therefore the heavy bismuth oxychloride must be in a very fine state of subdivision. In order obtain uniformity of contents in the filled ampoules, the suspension should be continuously stirred during filling. The ampoule should be labeled ‘shake well before use’ and on the box ‘for intramuscular use only’.Use: (By IM injections) in the treatment of syphilis.
71A super saturated solution: e.g. Calcium gluconate injection B.P. (10% w/v)Solubility is 1 in 30 but readily forms a super saturated solution. This injection is a super saturated solution. The solution is filtered hot and sterilized by heating in an autoclave. The tendency for the formation of crystals is reduced by replacement of up to 5% of the gluconate with Calcium-d saccharate (i.e. acts as stabilizer). The ampoules should be inverted during autoclaving, in order to dissolve dry solid particles formed in the tips of the ampoules during sealing. Otherwise these solid particles may act as nuclei and encourage crystallization on storage. (Multi dose containers are unsuitable because particles from the closure may act as nuclei for crystallization).Warning: As the injection is often given intravenously (10-20 ml) the injection should be discarded if it shows crystallization.The containers should comply with the limit test for alkaline of glass.Uses: Calcium deficiency states like hypocalcaemic tetany and in lead poisoning.
72E. A soap solution:e.g. Ethanolamine oleate injection B.P.C.This injection is a solution of the soap, ethanolamine oleate. It is made from ethanolamine and oleic acid. The oleic acid is insoluble in water and the slightest excess causes turbidity in the solution. If excess ethanolamine is used the pH may be outside the limits. Therefore, the two liquids must be weighed very carefully. A weighing boat can be used, from which the liquid can be easily transferred and thoroughly rinsed. Foaming should be controlled during the preparation and filling of the solution by performing the operation gently.
73When ethanolamine oleate is injected into a varicose vein (dilated vein, the valves of which cannot prevent reverse flow of blood), it acts as a sclerosing agent. (hardening) (2 to 5 ml). It also contains benzyl alcohol to keep the solution bright and clear, and also has a slight local anaesthetic action. Benzyl alcohol also acts as a preservative when supplied in a multiple dose vial and no further addition of a bactericide is required.F. pH adjustment:e.g.: Mersalyl injection B.P.:This injection is sterilized by heating with a bactericide. Phenylmercuric nitrate must be used as the bactericide (since the injection contains a mercury derivative).The injection contains theophylline (5%, also a diuretic) as a stabilizer. It prevents the liberation of toxic mucus ions by forming a complex with mersalyl.Formulae:Mersalyl AcidTheophyllineSodium hydroxide (10% solution) for pH adjustment0.002% w/v phenyl mercuric nitrate solution to produce final volume (solvent)
74The mersalyl acid and the theophylline are suspended in the solvent and dissolved by adding slowly, a 10% solution of NaOH in the solvent. The solution is adjusted to pH 8 with the alkali solution (first adjusted to full blue colour of boromo-thymol blue on a tile, pH 7.6 and then to the greenish colour of thymol blue pH 8.0, finally checked by a pH meter). The solution is clarified and adjusted to volume Contact with metal must be avoided during preparation of the injection. (Toxic mercury ions will be displaced from the complex). It must be packed in ampoules because extractives from the rubber closures of multiple dose containers may cause decomposition.Use: It is employed as a powerful diuretic in the treatment of certain cardiac diseases (IM, ml).Strength: 10% of mersalyl (i.e. of the sodium salt)
75G. Saturation with carbon dioxide: e.g. Sodium bicarbonate injection B.P (5%)The sodium bicarbonate is dissolved in water for injection, filtered, adjusted to volume and CO2 is passed into the solution for about a minute. The solution is distributed into gas-tight containers which are then sterilized by heating in an autoclave.When aqueous solution of NaHCO3 is heated above 100o, some of the bicarbonate is decomposed to carbonate NaHCO Na2CO3 + CO2 + H2OThe reaction is reversible so that on cooling in a gas- tight container, any sodium carbonate formed is reconverted into sodium bicarbonate.
76The preliminary saturation of the solution with CO2 ensures complete reconversion and it also converts the carbonate impurity in the NaHCO3 into bicarbonate (even AnalaR sample may contain 1% of carbonate) and if the solution is carbonate free, its pH should be 8.35 or less. Leakage of CO2from an imperfectly sealed container will not result in complete reconversion and the pH of the solution will be higher, the solution must not be used with in 2 hrs after cooling to room temp, which ensures a reconversion of about 95%.Used mainly in the treatment of acidosis associated with diabetic coma by slow I.V injection (500 ml/ 30 min) because a 5% solution is strongly hypertonic solution (1.4% solution is isotonic).
77H) Compound Sodium Lactate injection: Syn : Hartman’s solution for injectionRinger- lactate solution for injectionFormula:Lactic acid mlNaOH gDilute HClNaCl gKCl gCalcium Chloride gWFI up to mlThe sodium hydroxide is dissolved in (200 ml) some of the WFI, the lactic acid added and the solution heated in an autoclave at 115O- 116OC for 1 hr. The solution is cooled, dil HCl is added carefully until a few drops of the solution gives a definite orange colour with phenol red (pH 7.0).
78The other ingredients are dissolved in WFI; the two solutions mixed, and adjusted the volume with the solvent. The solution is filtered, filled into the containers and immediately sterilized by heating in an autoclave.Prolonged heating with sodium hydroxide is required for the complete conversion of the lactide (present in lactic acid) into sodium lactate.Use: It is administered intravenously in large volumes to replace water lost from the body (dehydration) by vomiting and diarrhea. For children it is given orally (due to the difficulty of finding a vein for injection dehydrated patients).For oral use water for injection may be replaced by purified water and the solution should be sterilized (since is given to infants in a weak condition). The solution should be labeled as compound sodium lactate solution with a warming ‘FOR ORAL USE ONLY’.
79INSULIN INJECTIONS:Insulin injection: syn –InsulinInsulin is prepared by extracting the finely divided mammalian pancreas (either fresh or frozen) with acidified alcohol (or alkali alcohol). The extract is concentrated under vacuum and after defatting, the crude insulin is precipitated by NaCl or picric acid. It is re-precipitated from NaOH solution at a pH of 5.2 (iso-electric point). It purified by crystallization from a buffered solution containing ZnCl2 (purified insulin contains at least 22 units/ mg.Preparation of the injection: The pure crystalline insulin is dissolved in WFI, containing a small amount of glycerin (for maintaining isotonicity) and sufficient HCl to give a pH of (optimum stability) to the final solution. A suitable bacteriostatic in added and the solution sterilized by filtration. It is supplied in multi-dose vials containing either20, 40, or 80 units/ ml.
80Storage: Should be stored at low temp as possible above its freezing point. The containers must comply with the limit test for alkaline of glass.Label: The number of units /ml must be stated on the label of the container. The date of mfr and expiry date should be given (Therapeutic substance).Sustained action or Delayed action insulins:Protamine zinc insulin injection:This is a sterile suspension of insulin combined with suitable protamine (from the sperm of a large sea fish salmon- trout) and zinc chloride, buffered with phosphate buffer to pH a standard sterile solution of insulin is mixed with aseptically with the calculated quantities of the protamine sulphate, ZnCl2 and glycerin after adding a bacteriostatic and buffer, sealed in sterilized containers.
81Globin zinc insulin injection: syn: Globin zinc insulin; globin insulin.This is a sterile solution of insulin with the addition of a suitable globin and a small amount of ZnCl2 (pH ). the globin is obtained from the RBC of the OX by removal of the chromogen fraction.The insulin zinc suspensions:With protamine zinc insulin the initial effect is low. Therefore a mixture of unmodified insulin with protamine zinc insulin should be used (possibility of accidental contamination with one another)Isophane insulin: (Syn: Neutral pH insulin, NPH insulin) Contains a smaller quantity of protamine.Gives rapid onset of action (prompt action)
82Zinc insulin complex, without proteins: Prompt and prolonged action Zinc insulin complex, without proteins: Prompt and prolonged action. Suspension buffered by acetate buffer. (Became popular in Denmark as lente insulins).By varying precipitating condition different particle sizes can be obtained.Suspensions of smallest particle sizes (amorphous) - more rapid actionSuspensions of largest particle sizes (crystalline) - more prolonged actionInsulin zinc suspension (amorphous) – semi lente insulinInsulin zinc suspension – lente insulin (mixture of amorphous and crystalline forms 30:70)Insulin zinc suspension (crystalline) - ultra lente insulin.
83Insulin – no separate colour Protamine zinc insulin – pink Standard colour code:A standard series of coloured packs are used to indicate the strength and type of insulin preparation.e.g. the end face of the carton of insulin zinc suspension (40 U/ ml)Insulin – no separate colourProtamine zinc insulin – pinkglobin zinc insulin – orangeInsulin zinc suspension (amorphous) – vermilion (brilliant red)Insulin zinc suspension – mauveInsulin zinc suspension (crystalline) – brilliant yellowIsophane insulin – white.20 U/ml – buff80 U/ml – greenMauve (pale violet )typeBlue (strength)