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Published on February 3, 2009

Author: prashantlp


PARENTERAL DRUG DELIVERY :  PARENTERAL DRUG DELIVERY Mr. Prashant L. Pingale School of Pharmacy & Technology Management, NMIMS, Shirpur Campus Mail ID: Slide 2: Definitions related to the topic: Parenteral Products Sterilization & Sterile Product Pyrogen SVP LVP Light Resistant Containers Well closed containers Tightly closed containers Single dose container Multiple dose container Hermetically sealed container Slide 3: PARENTERALS para: outside enteron: intestine (i.e. beside the intestine) These are the preparations which are given other than oral routes. Injections: These are Sterile, Pyrogen free preparations intended to be administered parenterally (outside alimentary tract). Slide 4: Why Parenteral? Parenteral Route Is Used bcoz 1) Rapid action 2) Oral route can not be used 3) Not effective except as injection 4) Many new drugs particularly those derived from new development in biotechnologically can only be given by parenteral coz they are inactivated in GIT if given orally. 5) New drugs require to maintain potency & specificity so that they are given by parenteral. Slide 5: Advantages: Quick onset of action Suitable for the drugs which are not administered by oral route Useful for unconscious or vomiting patients. Duration of action can be prolonged by modifying formulation. Suitable for nutritive like glucose & electrolyte. Suitable for the drugs which are inactivated in GIT or HCl (GI fluid) Slide 6: Disadvantages: Once injected cannot be controlled (retreat) Injections may cause pain at the site of injection Only trained person is required If given by wrong route, difficult to control adverse effect Difficult to save patient if overdose Sensitivity or allergic reaction at the site of injection Requires strict control of sterility & non pyrogenicity than other formulation. Slide 7: Necessities of Parenteral preparations: Sterility (must) Pyrogen (must) Free from particulate matter (must) Clarity (must) Stability (must) Isotonicity (should) Solvents or vehicles used must meet special purity and other standards. Restrictions on buffers, stabilizers, antimicrobial preservative. Do not use coloring agents. Must be prepared under aseptic conditions. Specific and high quality packaging. Slide 8: Routes of Parenteral Administration Intradermal (23) Intramuscular (20) Intravenous (21) Subcutaneous (21) Dermis Intra arterial (20-22) Vein Artery Muscle Epidermis Subcutaneous tissue Slide 9: Parental Routes of Administration: Most Common: 1. Subcutaneous (SC; SQ ;Sub Q) 2. Intramuscular (IM) 3. Intravenous (IV) Others: 4. Intra-arterial (IA) 5. Intrathecal 6. Intraarticular 7. Intrapleural 8. Intracardial 9. Intradermal (Diagnostic) Slide 10: Subcutaneous (SC; SQ ;Sub Q): The injection is given under the skin Need to be isotonic Upto 2 ml is given Using ½ to 1 inch 23 gauge needle or smaller needle Given: Vaccines Insulin Scopolamine Epinephrine Slide 11: Intramuscular (IM): Striated muscle fibre 0.5 to 2 ml sometimes upto 4 ml 1 to 1.5 inch & 19 to 22 gauge needle is used Preferably isotonic Principle sites: Gluteal (buttocks) Deltoid (upper arms) Vastus lateralis (lateral thigh) Given: Solutions Emulsions Oils Suspension Slide 12: Intravenous (IV): Into the vein 1 to 1000 ml 1 inch ,19 to 20 gauge needle with injection rate 1ml/ 10 sec. for volume upto 5 ml & 1 ml/ 20 sec. for volume more than 5 ml. Given: Aqueous solutions Hydro alcoholic solutions Emulsions Liposome Slide 13: IV infusion of large volume fluids (100- 1000 ml) has become increasingly popular. This technique is called as Venoclysis. This is used to supply electrolytes & nutrients to restore blood volume & to prevent tissue dehydration. Combination of parenteral dosage forms for administration as a unit product is known as an IV admixture. Lactated Ringer Injection USP NaCl Injection USP (0.9 %)– (replenish fluid & electrolyte) Dextrose Injection USP (fluid & electrolyte) Slide 14: Intra-arterial (IA): Direct into the artery 2 to 20 ml 20 to 22 gauge Solutions & emulsions can be administered Given: Radio opaque media Antineoplastic Antibiotics Slide 15: Intrathecal: Also called intra-spinal Directly given into the spinal cord 1 to 4 ml 24 to 28 gauge Must be isotonic Given: LA Analgesics Neuroleptics Slide 16: Intraarticular: Given directly into the joints 2 to 20 ml 5 inch 22 gauge Must be isotonic Given: Morphine LA Steroids NSAID’s Antibiotics Slide 17: Intrapleural: Given directly into the pleural cavity or lung Used for fluid withdrawal 2 to 30 ml 2 to 5 inch, 16 to 22 gauge needle Given: LA Narcotics Chemotherapeutic agents Slide 18: Intracardial: Directly given into the heart 0.2 to 1 ml 5 inch , 22 gauge needle Given: Cadiotonics Calcium salts as a calcium channel blockers Slide 19: Intradermal: Also called as diagnostic testing 0.05 ml ½ inch, 25 to 26 gauge needle Should be isotonic Given: Diagnostic agents Slide 20: Official Types of Injections: 1. Solutions of Medicinal Example: Codeine Phosphate Injection Insulin Injection 2. Dry solids or liquid concentrate does not contain diluents etc. Example: Sterile Ampicillin Sodium 3. If diluents present, referred to as.....for injection Example: Methicillin Sodium for injection Slide 21: 4. Suspensions "Sterile....Suspension" Example: Sterile Dexamethasone Acetate Suspension 5.Dry solids, which upon the addition of suitable vehicles yield preparations containing in all respects to the requirements for sterile suspensions. Title: Sterile....for Suspension Example: Sterile Ampicillin for Suspension 6. Injectable Emulsions: Example: Propofol injection Slide 22: Formulation of Parenteral: Therapeutic agents Vehicles Water Water miscible vehicles Non- aqueous vehicles Added substances (Additives) Antimicrobials Antioxidants Buffers Bulking agents Chelating agents Protectants Solubilizing agents Surfactants Tonicity- adjusting agents General steps involved : General steps involved 1. Cleaning 2. Preparation of bulk products 3. Filtration 4. Filling of solution in or product in ampoule or vial 7. Tests for Quality control 5.Sealing 6. Sterilization Formulation of Parenteral : Formulation of Parenteral 1.Therapeutic ingredients: Insulin Antibiotics Anticancer Steroids Vaccines Antipyretic Analgesics Anti- inflammatory LVP’s like Dextrose, NaCl or combination etc…. Formulation of Parenteral : 2.Solvents: Water Should meet compendial requirements Water miscible vehicles Ethyl alcohol PEG PG Non aqueous vehicles Fixed oils Formulation of Parenteral Formulation of Parenteral : Formulation of Parenteral Solvents Solvents used must be: Non-irritating Non-toxic Non-sensitizing No pharmacological activity of its own Not affect activity of medicinal Formulation of Parenteral : 3. Added substances (Additives) Antimicrobials: Added for fungistatic or bacteriostat action or concentration Used to prevent the multiplication of micro-organisms Ex.. Benzyl alcohol ------ 0.5 – 10 % Benzethonium chloride -- 0.01 % Methyl paraben ---- 0.01 – 0.18 % Propyl paraben --- 0.005 – 0.035 % Phenol --- 0.065 – 0.5 % Formulation of Parenteral Slide 28: Preservatives: Multidose containers must have preservatives unless prohibited by monograph. Large volume parenteral must not contain preservative becoz it may be dangerous to human body if it contain in high doses. Slide 29: Antioxidants: Used to protect product from oxidation Acts as reducing agent or prevents oxidation Ex: A) Reducing agent: Ascorbic acid -- 0.02 – 0.1 % Sodium bisulphite-- 0.1 – 0.15 % Sodium metabisulphite-- 0.1 – 0.15 % Thiourea - 0.005 % B) Blocking agents: Ascorbic acid esters- 0.01 – 0.015% BHT- 0.005 – 0.02 % C) Synergistic: Ascorbic acid , Citric acid , Tartaric acid D) Chelating agent: EDTA- 0.01- 0.075 % Slide 30: Buffers: Added to maintain pH, Change in pH may causes degradation of the products Acetates, citrates, phosphates are generally used. Factors affecting selection of buffers: Effective range, Concentration Chemical effect on the total product EXAMPLES: Acetic acid ,adipic acid, benzoic acid, citric acid, lactic acid Used in the conc. of 0.1 to 5.0 % Slide 31: Chelating agents: Used to form the complex with the metallic ions present in the formulation so that the ions will not interfere during mfg. of formulation. They form a complex which gets dissolved in the solvents. Examples: Disodium edetate – 0.00368 - .05 % Disodium calcium edetate - 0.04 % Tetrasodium edetate – 0.01 % Slide 32: Stabilizers: As parenterals are available in solution form they are most prone to unstabilize Used to stabilize the formulation Maintain stable Examples: Creatinine – 0.5- 0.8 % Glycerin – 1.5 – 2.25 % Niacinamide – 1.25 -2.5 % Sodium saccharin – 0.03 % Sodium caprylate – 0.4 % Slide 33: Solubilizing agents: Used to increase solubility of slightly soluble drugs they acts by any one of the following: solubilizers, emulsifiers or wetting agents. Examples: Dimethylacetamide, Ethyl alcohol Glycerin Lecithin PEG – 40 castor oil PEG – 300 Polysorbate 20, 40, 80 Slide 34: Tonicity- adjusting agents: Used to reduce the pain of injection. Buffers may acts as tonicity contributor as well as stabilizers for the pH. Isotonicity depends on permeability of a living semipermaeable membrane Hypotonic : swelling of cells (enlargement) Hypertonic: shrinking of cells (reduction) Example: Glycerin Lactose Mannitol Dextrose Sodium chloride Sorbitol Slide 35: LABELING: Name of product Quantity of the product % of drug or amount of drug in specified volume of amount of drug and volume of liquid to be added Name and quantity of all added substances Mfg. license no. Batch no. Manufacturer/Distributor Mfg. & Expiration date Retail price (incl. of all taxes) Mfger. address Veterinary product should be so labeled Slide 36: Must check each individual monogram for: Type of container: Glass Plastic Rubber closure Type of glass Type I Type II Type III NP Tests for glass containers Powdered Glass test Water Attack test Package size Special storage instructions Slide 37: Production facilities Types : Emulsion Suspension Solutions Preparation of IV fluids IV admixtures TPN Dialysis fluids QC tests for parenteral Slide 38: Production facilities: Clean- up area Preparation area Aseptic area Quarantine area Finishing and packaging area Sterile area Slide 39: S T O C K R O O M COMPOUNDING AREA CLEAN UP AREA ASEPTIC AREA QUARANTINE AREA STERILIZATION STORAGE AND TRANSPORT PACKING AND LABELLING LAY OUT OF PARENTERAL MANUFACTURING AREA Slide 40: Clean- up area: Non aseptic area Free from dust ,fibres & micro-organisms Constructed in such a way that should withstand moisture, steam & detergent Ceiling & walls are coated with material to prevent accumulation of dust & micro-organisms Exhaust fans are fitted to remove heat & humidity The area should be kept clean so that to avoid contamination to aseptic area The containers & closures are washed & dried in this area. Slide 41: Preparation area: The ingredients are mixed & preparation is prepared for filling Not essential that the area is aseptic Strict precaution is taken to prevent contamination from outside Cabinets & counters: SS Ceiling & walls : sealed & painted Slide 42: Aseptic area: Filtration & filling into final containers & sealing is done The entry of outside person is strictly prohibited To maintain sterility, special trained persons are only allowed to enter & work Person who worked should wear sterile cloths Should be subjected for physical examination to ensure the fitness Minimum movement should be there in this area Ceiling & walls & floors : sealed & painted or treated with aseptic solution and there should not be any toxic effect of this treatment Slide 43: Cabinets & counters: SS Mechanical equipments : SS AIR: Free from fibres, dust & micro organisms HEPA filters are used which removes particles upto 0.3 micron Fitted in laminar air flow system, in which air is free from dust & micro organisms flows with uniform velocity Air supplied is under positive pressure which prevents particulate contamination from sweeping UV lamps are fitted to maintain sterility Slide 44: Quarantine area: After filling, sealing & sterilization the products or batch is kept in this area The random samples are chosen and given for analysis to QC dept. The batch is send to packing after issuing satisfactory reports of analysis from QC If any problem is observed in above analysis the decision is to be taken for reprocessing or others.. Slide 45: Finishing and packaging area: After proper label, the product is given for packing Packing is done to protect the product from external environment The ideal Packing is that which protects the product during transportation, storage, shipping & handling. The labeled container should be packed in cardboard or plastic containers Ampoules should be packed in partitioned boxes. Preparations for IV Fluids: : Preparations for IV Fluids: LVP’s which are administered by IV route are commonly called as IV fluids. Purposes : Body fluids, Electrolyte replenisher Volume supplied: 100 to 1000 ml Slide 47: Precautions / necessities in mfg.: Free from foreign particles Free from micro organisms Isotonic with body fluids As they are in LVP no bacteriostatic agents are added Free from pyrogens Slide 48: Examples: Dextrose injection IP : available in 2 , 5 , 10 , 25 & 50 % w/v solution. Used for Fluids replenisher, Electrolyte replenisher Sodium chloride & Dextrose injection IP: (DNS) Contains 0.11 to 0.9 % Sodium chloride 2.5 to 5.0 % Dextrose Used for Fluids replenisher, Electrolyte replenisher Nutrient replenisher Slide 49: Sodium chloride injection IP: 0.9 % conc. Also known as normal saline solution Used as Isotonic vehicle Fluids replenisher, Electrolyte replenisher Sodium lactate injection IP: Contains 1.75 to 1.95 % w/v of sodium lactate Used as Fluids replenisher, Electrolyte replenisher Slide 50: Mannitol injection IP: Contains 5, 10 , 15, 20 % of mannitol Used as : Diagnostic aid Renal function determination As a diuretic Mannitol & Sodium chloride injection IP: Contains 5, 10 , 15, 20 % of mannitol & 0.45 % of Sodium chloride Used as : As a diuretic Slide 51: Other solutions: Ringer injection IP Ringer lactate solution for injection IP Common uses : Used in surgery patients In replacement therapy Providing basic nutrition For providing TPN As a vehicle for other drug subs. IV ADMIXTURES : IV ADMIXTURES Definition: When two or more sterile products are added to an IV fluid for their administration, the resulting combination is known as IV admixture. In hospitals, prepared by nurses by combining or mixing drugs to the transfusion fluids. The drugs are incorporated in to bottles of LV transfusion fluids. Slide 53: Care : Microbial contamination Incompatibility Physical : change in color Chemical : hydrolysis, oxidation, reduction etc.. Therapeutic: undesirable antagonistic or synergistic effect Slide 54: Methods for safe & effective use of IV admixture: Proper training to nurses & pharmacist Instruction regarding labeling Information for stability & compatibility to the hospital pharmacy dept. Information for the formulation skills to the pharmacist. Slide 55: TPN stands for Total Parenteral Nutrition. This is a complete form of nutrition, containing protein, sugar, fat and added vitamins and minerals as needed for each individual. Total Parenteral Nutrition (TPN) may be defined as provision of nutrition for metabolic requirements and growth through the parenteral route. Total Parenteral Nutrition Total Parenteral Nutrition (TPN)(Intravenous Nutrition) : Total Parenteral Nutrition (TPN)(Intravenous Nutrition) TPN refers to the provision of all required nutrients, exclusively by the Intravenous route. Parenteral Nutrition (PN)can be used to supplement ordinary or tube feeding. Slide 57: Components of TPN solutions: (1) Protein as crystalline amino acids. (2) Fats as lipids. (3) Carbohydrate as glucose. (4) Electrolytes–Sodium, potassium, chloride, calcium and magnesium. (5) Metals/Trace elements–Zinc, copper, manganese, chromium, selenium. (6) Vitamins A, C, D, E, K, thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, choline and folic acid. Slide 58: TPN might be necessary if: a patient is severely undernourished, and needs to have surgery, radiotherapy or chemotherapy; a patient suffers from chronic diarrhea and vomiting; a baby's gut is too immature; a patient's (their "gastrointestinal tract") is paralysed, for example after major surgery. Why it is necessary? Slide 59: Normal Diet TPN Protein…………Amino Acids Carbohydrates…Dextrose Fat……………..Lipid Emulsion Vitamins………Multivitamin Infusion Minerals………Electrolytes & Trace Elements Nutritional Requirements : Nutritional Requirements Amino acids Glucose Lipid Minerals Vitamins Water and electrolytes Trace elements Total Parenteral NutritionElectrolytes : Total Parenteral NutritionElectrolytes Slide 62: TPN is normally used following surgery, when feeding by mouth or using the gut is not possible, When a person's digestive system cannot absorb nutrients due to chronic disease, or, alternatively, if a person's nutrient requirement cannot be met by enteral feeding (tube feeding) and supplementation. When is it necessary? Slide 63: Short-term TPN may be used if a person's digestive system has shut down (for instance by Peritonitis), and they are at a low enough weight to cause concerns about nutrition during an extended hospital stay. Long-term TPN is occasionally used to treat people suffering the extended consequences of an accident or surgery. Most controversially, TPN has extended the life of a small number of children born with nonexistent or severely birth-deformed guts. Slide 64: GENERAL INDICATIONS Patient who can’t eat Patient who won’t eat Patient who shouldn’t eat Patient who can’t eat enough “If the gut works, use it.” Slide 65: NOMENCLATURE TPN: Total Parenteral Nutrition IVH: Intravenous Hyperalimentation TNA: Total Nutrient Admixture TPN: Total Parenteral Nutrition 3-In-1 Admixture All-In-One Admixture PPN: Peripheral Parenteral Admixture Indications for TPN : Indications for TPN Short-term use Bowel injury /surgery Bowel disease Severe malnutrition Nutritional preparation prior to surgery. Malabsorption - bowel cancer Long-term use Prolonged Intestinal Failure Crohn’s Disease Bowel resection Slide 67: The preferred method of delivering TPN is with a medical infusion pump. A sterile bag of nutrient solution, between 500 mL and 4 L is provided. The pump infuses a small amount (0.1 to 10 mL/hr) continuously in order to keep the vein open. Feeding schedules vary, but one common regimen ramps up the nutrition over a few hours, levels off the rate for a few hours, and then ramps it down over a few more hours, in order to simulate a normal set of meal times. Slide 68: The nutrient solution consists of water, glucose, salts, amino acids, vitamins and (more controversially) sometimes emulsified fats. Long term TPN patients sometimes suffer from lack of trace nutrients or electrolyte imbalances. Because increased blood sugar commonly occurs with TPN, insulin may also be added to the infusion. Often though, an insignificant amount of insulin is added, sometimes 10 units or less in 2 liters of TPN. In actuality, the patient will probably get less than that. Occasionally, other drugs are added as well, sometimes unnecessarily. Complications of TPN : Complications of TPN Sepsis Air embolism Clotted catheter line Catheter displacement Fluid overload Hyperglycaemia Rebound Hypoglycaemia Slide 70: Dialysis is the process in which substances are separated from one another due to their difference in diffusibility (distribution) thr’ membrane. The fluids used in dialysis are known as dialysis fluids. DIALYSIS FLUIDS Slide 71: General uses : Renal failure waste product is removed Maintain electrolytes Also called as haemodialysis or intraperitoneal dialysis Transplantation of kidney Poisoning cases Slide 72: Haemodialysis: To remove toxins from blood In haemodialysis, the blood from artery is passed thr’ artificial dialysis membrane, bathed in dialysis fluid. The dialysis membrane is permeable to urea, electrolytes & dextrose but not to plasma proteins & lipids So excess of urea is passed out from blood thr’ dialysis fluid. Slide 73: After dialysis blood is returned back to the body circulation thr’ vein. A kidney unit may require more than 1200 litres of solution / week. So haemodialysis fluid is prepared in conc. Form then it is diluted with deionised water or dist. water before use. Slide 74: Composition of Concentrated Haemodialysis Fluid BPC Dilute 1 liter of conc. solution with 39 liters of water to make 40 litres. Storage: store in warm place as it is liable to convert into crystals on storage. COMPOSITION Dextrose monohydrate ----------- Sodium acetate --------------------- Lactic acid --------------------------- Sodium chloride ------------------- Potassium chloride --------------- Freshly boiled & cooled water -q.s. 8.0 gm 19.04 gm 0.4 ml 22.24 gm 0.4 gm 100 ml Slide 75: Intraperitoneal Dialysis: Peritoneal cavity is irrigated with dialysis fluid. Peritoneum acts as a semi permeable membrane Toxic subs. excreted by kidney are removed. Requirements: Sterile Pyrogen free Slide 76: Composition of Fluid Intraperitoneal Dialysis IP 1985 Sterilize by autoclave immediately after preparation. COMPOSITION Sodium chloride ------------------- Sodium acetate --------------------- Calcium chloride ------------------- Magnesium chloride -------------- Sodium metabisulphite ---------- Dextrose (anhydrous) ----------- Purified water -----------q.s.----- 5.56 gm 4.76 gm 0.22 gm 0.152 gm 0.15 gm 17.30 gm 1000 ml STERILITY TESTING FOR PARENTERAL PRODUCTS : STERILITY TESTING FOR PARENTERAL PRODUCTS 1. Sterility testing - definition : 1. Sterility testing - definition Sterility testing attempts to reveal the presence or absence of viable micro-organisms in a sample number of containers taken from batch of product. Based on results obtained from testing the sample a decision is made as to the sterility of the batch. Sterility testing - : Sterility testing - is made after the product exposition to the one of the possible sterilization procedures can only provide partial answers to the state of sterility of the product batch under test is inadequate as an assurance of sterility for a terminally sterilized product Major factors of importance in sterility testing : Major factors of importance in sterility testing The environment in which the test is conducted The quality of the culture conditions provided The test method The sample size The sampling procedure 1.1.Environmental conditions : 1.1.Environmental conditions avoid accidental contamination of the product during the test the test is carried out under aseptic conditions regular microbiological monitoring should be carried out 1.2.Culture conditions : 1.2.Culture conditions Appropriate conditions for the growth of any surviving organism should be provided by the culture media selection. 1.2. Culture conditions : 1.2. Culture conditions Factors affecting growth of bacteria Phases of bacterial growth Culture media for sterility testing 1.2.1. Factors affecting growth of bacteria : 1.2.1. Factors affecting growth of bacteria Nutrition Moisture Air Temperature pH Light Osmotic pressure Growth inhibitors 1.2.2. Phases of bacterial growth : 1.2.2. Phases of bacterial growth Lag phase (A) Log (logarithmic or exponential) phase (B) Stationary phase (C) Decline (death) phase (D) 1.2.3.Culture media for sterility testing : 1.2.3.Culture media for sterility testing capable of initiating and maintaining the vigorous growth of a small number of organisms sterile Types of media: Fluid thioglycollate medium Soya-bean casein digest medium other media thioglycollate medium : thioglycollate medium composition described in next slide. specific role of some ingredients primarily intended for the culture of anaerobic bacteria incubation of the media: 14 days at 30 -35°C Slide 89: Fluid thioglycollate medium casein digest medium : casein digest medium primarily intended for the culture of both fungi and aerobic bacteria specific role of some ingredients incubation of the media: 14 days at 20 -25°C Slide 91: Soya-bean casein digest medium control of the media : control of the media are they suitable for growth of each micro-organism? 'Growth promotion test for aerobes, anaerobes and fungi' ; inoculation of media tubes with a MO incubation (T, t) the media are suitable if a clearly visible growth of the micro-organisms occurs of the media under test conditions : of the media under test conditions are culture conditions satisfactory in the presence of the product being examined? comparing the rate of onset and the density of growth of inoculated MO in the presence and absence of the material being examined growth control; 1.3.The test method for sterility of the product : 1.3.The test method for sterility of the product Membrane filtration Direct inoculation of the culture medium 1.3.1. Membrane filtration : 1.3.1. Membrane filtration Appropriate for : (advantage) filterable aqueous preparations alcoholic preparations oily preparations preparations miscible with or soluble in aqueous or oily (solvents with no antimicrobial effect) solutions to be examined must be introduced and filtered under aseptic conditions All steps of this procedure are performed aseptically in a Class 100 Laminar Flow Hood of filters for membrane filtration : of filters for membrane filtration pore size of 0.45 m effectiveness established in the retention of micro-organisms appropriate composition the size of filter discs is about 50 mm in diameter procedure of membrane filtration : procedure of membrane filtration sterilization of filtration system and membrane filtration of examined solution under aseptic conditions (suitable volume, dissolution of solid particles with suitable solvents, dilution if necessary…) one of two possible following procedures: the membrane is removed, aseptically transferred to container of appropriate culture medium passing the culture media through closed system to the membrane, incubation in situ in the filtration apparatus (sartorius, millipore). 1.3.2.Direct inoculation of the culture medium : 1.3.2.Direct inoculation of the culture medium suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium volume of the product is not more than 10% of the volume of the medium suitable method for aqueous solutions, oily liquids, ointments an creams Slide 100: Scheme for sterility test by membrane filtration Scheme for sterility test by direct inoculation Advantages of the filtration method : Advantages of the filtration method wide applications a large volume can be tested with one filter smaller volume of culture media is required applicable to substances for which no satisfactory inactivators are known neutralization is possible on the filter subculturing is often eliminated shorter time of incubation compared with direct inoculation 1.4. Observation and interpretation of the results : 1.4. Observation and interpretation of the results Examination at time intervals during the incubation period and at its conclusion When the sample passes the test and when fails? When the test may be considered as invalid? There is low incidence of accidental contamination or false positive results 1.5. Sampling : 1.5. Sampling Selection of the samples Sample size Minimum number of items to be tested : Minimum number of items to be tested Instead of the conclusion - Guidelines for using the test for sterility : Instead of the conclusion - Guidelines for using the test for sterility Precautions against microbial contamination The level of assurance provided by a satisfactory result of a test for sterility as applied to the quality of the batch is a function of: The homogeneity of the batch The conditions of manufacture Efficiency of the adopted sampling plan Guidelines … : Guidelines … In the case of terminally sterilized products: physical proofs, biologically based and automatically documented, showing correct treatment through the batch during sterilization are of greater assurance than the sterility test Products prepared under aseptic conditions: sterility test is the only available analytical method only analytical method available to the authorities who have to examine a specimen of a product for sterility. Slide 107: PYROGENS AND PYROGEN TESTING Pyrogens : Pyrogens Pyrogenic - means producing fever Pyrogens - fever inducing substances Having nature Endogenous (inside body) Exogenous (outside body) Exogenous pyrogens – mainly lipopolysaccharides bacterial origin, but not necessary Structure of endotoxins : Structure of endotoxins Produced mostly by gram-negative bacteria Endotoxin - complex of pyrogenic lipopolysaccharide, a protein and inert lipid; lipid part of the lipopolysaccharide is the main pyrogenic agent; polysaccharide part increases solubility Generalized structure of endotoxins : Generalized structure of endotoxins Generalized structure of Endotoxins Sources of pyrogen contamination : Sources of pyrogen contamination solvent - possibly the most important source the medicament the apparatus the method of storage between preparation and sterilization The endotoxin characteristics : The endotoxin characteristics thermostable water-soluble unaffected by the common bactericides non-volatile These are the reasons why pyrogens are difficult to destroy once produced in a product Tests for pyrogenic activity : Tests for pyrogenic activity Test for pyrogens = Rabbit test Bacterial endotoxins Test for pyrogens = Rabbit test : Test for pyrogens = Rabbit test the development of the test for pyrogens reach in 1920 a pyrogen test was introduced into the USP XII (1942) The test consists of measuring the rise in body temperature in healthy rabbits by the intravenous injection of a sterile solution of the substance under the test. Why the Rabbit? : Why the Rabbit? Reproducible pyrogenic response Other species not predictable Rabbit vs. dog as model? Rabbits: false positives Dogs: false negatives Similar threshold pyrogenic response to humans Rabbit Pyrogen Test : Rabbit Pyrogen Test Rabbits must be healthy and mature New Zealand or Belgian Whites used Either sex may be used Length of use >48 hours within negative result >2 weeks within a positive result Must be individually housed between 20 and 23°C Rabbit test - : Rabbit test - selection of animals (healthy, adult, not less than 1,5 kg,…) housing of animals (environmental problems: presence of strangers (unknown place), noise, T, …) equipment and material used in test (glassware, syringes, needles) retaining boxes (comfortable for rabbits as possible) thermometers (standardized position in rectum, precision of 0.1°C) Rabbit test : Rabbit test Preliminary test (Sham Test) intravenous injection of sterile pyrogen-free saline solution to exclude any animal showing an unusual response to the trauma (shock) of injection any animal showing a temperature variation greater than 0.6C is not used in the main test Rabbit test - : Rabbit test - main test: group of 3 rabbits preparation and injection of the product: warming the product dissolving or dilution duration of injection: not more than 4 min the injected volume: not less than 0.5 ml per 1 kg and not more than 10 ml per kg of body mass determination of the initial and maximum temperature all rabbits should have initial T: from 38.0 to 39.8C the differences in initial T should not differ from one another by more than 1C Rabbit test : Rabbit test Interpretation of the results: the test is carried out on the first group of 3 rabbits; if necessary on further groups of 3 rabbits to a total of 4 groups, depending on the results obtained intervals of passing or failing of products are on the basis of summed temperature response The result of pyrogen test: : The result of pyrogen test: If above test not passes, the sample is said to be pyrogenic or go thr’ the sources of contamination of pyrogen. Bacterial endotoxins : Bacterial endotoxins to detect or quantify endotoxins of gram-negative bacterial origin reagent: amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). The name of the test is also Limulus amebocyte lysate (LAL) test Limulus polyphemus = horseshoe crab : Limulus polyphemus = horseshoe crab Mechanism of LAL : Mechanism of LAL the test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located with the crab's amebocyte blood cells endotoxins initiation of an enzymatic coagulation cascade proteinaceous gel Commercially derived LAL reagents : Commercially derived LAL reagents bleeding adult crabs into an anticlotting solution washing and centrifuging to collect the amebocyte lysing in 3% NaCl lysate is washed and lyophilized for storage activity varies on a seasonal basis and standardization is necessary. Test performance (short) : Test performance (short) avoid endotoxin contamination Before the test: interfering factors should not be present equipment should be depyrogenated the sensitivity of the lysate should be known Test: equal V of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube incubation at 37°C, 1 hour remove the tube - invert in one smooth motion (180°) - read (observe) the result pass-fail test LAL test : LAL test Three different techniques: the gel-clot technique - gel formation the turbidimetric technique - the development of turbidity after cleavage of an endogenous substrate the chromogenic technique - the development of color after cleavage of a synthetic peptide-chromogen complex LAL test : LAL test 6 methods with different steps of accuracy of LAL test results: Method A: gel-clot method: limit test Method B: gel-clot method: semi-quantitative test Method C: turbidimetric kinetic method Method D: chromogenic kinetic method Method E: chromogenic end-point method Method F: turbidimetric end-point method In the event of doubt or dispute, the final decision is made upon Method A unless otherwise indicated in the monograph. Gel-cloth technique (Methods A, B) : Gel-cloth technique (Methods A, B) allows detection or quantification of endotoxins clotting of the lysate in the presence of endotoxins. 1.Preparatory testing Confirmation of the labeled lysate sensitivity Tests for interfering factors Invert Tube in Smooth Motion Gel-cloth technique (Methods A, B)-cont. : Gel-cloth technique (Methods A, B)-cont. 2. Limit test (method A) procedure described on page. 24 a firm gel - positive result. an intact gel is not formed - negative result. the interpretation of the results 3. Semi-quantitative test (method B) quantification of bacterial endotoxins in the test solution by titration to an end-point. procedure is similar as in the limit test The results are expressed as concentration of endotoxin as less, equal or greater than  (labeled lysate sensitivity). Turbidimetric technique (Methods C, F) : Turbidimetric technique (Methods C, F) photometric test to measure the increase in turbidity end-point test (Method F): quantitative relationship between the endotoxin concentration and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. kinetic test (Method C): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of turbidity development. Chromogenic technique (Methods D, E) : Chromogenic technique (Methods D, E) measuring the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate end-point test (Method E): is based on the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period kinetic test (Method D): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of color development Instead of the conclusion - Guidelines for test for bacterial endotoxins : Instead of the conclusion - Guidelines for test for bacterial endotoxins the absence of bacterial endotoxins in a product implies the absence of pyrogenic component if you wish to replace rabbit test you should prove that you don’t have interfering factors if rabbit pyrogen test is replaced by endotoxin test, the last one should be validated methods from C to F require more instrumentation, but they are easier to automate test for bacterial endotoxins is preferred over the test for pyrogens Advantages of LAL test : Advantages of LAL test Fast - 60 minutes vs. 180 minutes Greater Sensitivity Less Variability Much Less False Positives Much Less Expensive Alternative to Animal Model cheaper, more accurate than other is performed in the pharmaceutical laboratory specific for endotoxins of gram-negative origin particularly useful for: Radiopharmaceuticals and cytotoxic agents Products with marked pharmacological or toxicological activity in the rabbit (e.g. insulin) Blood products which sometime give misleading results in the rabbit Water for injection where LAL test is potentially more stringent and readily applied Particulate Matter Monitoring : Particulate Matter Monitoring Definition: : Definition: Unwanted mobile insoluble matter other than gas bubbles present in the given product. It may be dangerous when the particle size is larger than R.B.C. & may block the blood vessel. This type of products are immediately rejected from the batch. Slide 141: The limit test for particulate matter is prescribed in I.P. 1996 (A- 125) Applicable for: 100 ml or more volume containers of single dose LV given by IV infusion Not applicable for: Multidose injections Single dose SVP Injectable solutions constituted from sterile solids Permitted limits of particulate matter : Permitted limits of particulate matter Particle size in micrometer Max.No.of particles (equal to or larger than) per ml 50 25 5 50 Nil Sources of particulate matter : Sources of particulate matter Contamination Contaminant Intrinsic contamination: Originally present in products e.g. Barium ions may react or leach with Sulphur ion which are already present in formulation may produce barium sulphate crystals. Slide 144: Extrinsic contamination: Material comes from outside or environment e.g. coming off the material from body & cloths of person Entry of particle from ceiling , walls & furniture May be in the form of cotton, glass rubber, plastics, tissues, insect fragments, bacterial contamination, dust, papers etc… Methods of monitoring particulate matter contamination : Methods of monitoring particulate matter contamination Visual method Coulter counter method Filtration method Light blockage method Slide 146: Visual method: Simple method Filled container are examined against strong illuminated screen by holding neck & rotating it slowly or inverted it to keep out the foreign matter. Coulter counter method: It is used for detection of particles less than 0.1 micrometer in diameter. Based on electrode resistance. Sample is evaluated between two electrode & if particle found the resistance of electrode is increased. Slide 147: Filtration method: It is used for counting the particles in hydraulic fluids. Sample passed thr’ filter Material is collected on filter Evaluated under microscope. Disadvantage: Skilled & trained person is required Light blockage method: Used for hydraulic oils Allows stream of fluid under test to pass between a bright white light source & photoiodide sensor. Identification of Particulate Matter : Identification of Particulate Matter Microscopy X- ray powder diffraction Mass microscopy Microchemical tests Electron microscopy etc… Significance of Particulate Matter monitoring : Significance of Particulate Matter monitoring Its presence may causes: Septicemia Fever & blockage of blood vessels Quality of product may affect Slide 150: As per USP LVP : NMT 50 particles/ ml (size 10 or more than 10 micrometer) & 5 particles/ ml (size more than 25 micrometer) SVP: 10,000 particles/ container of size 10 micrometer or greater & NMT 1000 particles/ container greater than 25 micrometer.

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