PARENTERAL DRUG DELIVERY : PARENTERAL DRUG DELIVERY Mr. Prashant L. Pingale
School of Pharmacy & Technology Management, NMIMS,
Mail ID: firstname.lastname@example.org Slide 2: Definitions related to the topic:
Sterilization & Sterile Product
Light Resistant Containers
Well closed containers
Tightly closed containers
Single dose container
Multiple dose container
Hermetically sealed container Slide 3: PARENTERALS
enteron: intestine (i.e. beside the intestine)
These are the preparations which are given other than oral routes.
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:
Free from particulate matter (must)
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)
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
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
Deltoid (upper arms)
Vastus lateralis (lateral thigh)
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.
Hydro alcoholic solutions
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
Radio opaque media
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
Neuroleptics Slide 16: Intraarticular:
Given directly into the joints
2 to 20 ml
5 inch 22 gauge
Must be isotonic
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
Chemotherapeutic agents Slide 18: Intracardial:
Directly given into the heart
0.2 to 1 ml
5 inch , 22 gauge needle
Calcium salts as a calcium channel blockers Slide 19: Intradermal:
Also called as diagnostic testing
½ inch, 25 to 26 gauge needle
Should be isotonic
Diagnostic agents Slide 20: Official Types of Injections:
1. Solutions of Medicinal
Example: Codeine Phosphate 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
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:
Water miscible vehicles
Non- aqueous vehicles
Added substances (Additives)
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:
LVP’s like Dextrose, NaCl or combination etc…. Formulation of Parenteral : 2.Solvents:
Should meet compendial requirements
Water miscible vehicles
Non aqueous vehicles
Fixed oils Formulation of Parenteral Formulation of Parenteral : Formulation of Parenteral Solvents
Solvents used must be:
No pharmacological activity of its own
Not affect activity of medicinal Formulation of Parenteral : 3. Added substances (Additives)
Added for fungistatic or bacteriostat action or concentration
Used to prevent the multiplication of micro-organisms
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
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 %
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:
Chemical effect on the total product
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.
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
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:
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)
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.
Mfg. & Expiration date
Retail price (incl. of all taxes)
Veterinary product should be so labeled Slide 36: Must check each individual monogram for:
Type of container:
Type of glass
Tests for glass containers
Powdered Glass test
Water Attack test
Special storage instructions Slide 37: Production facilities
QC tests for parenteral Slide 38: Production facilities:
Clean- up area
Finishing and packaging area Sterile area Slide 39: S
AREA CLEAN UP
AREA STERILIZATION STORAGE
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
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.
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.
Sodium chloride & Dextrose injection IP: (DNS)
0.11 to 0.9 % Sodium chloride
2.5 to 5.0 % Dextrose
Nutrient replenisher Slide 49: Sodium chloride injection IP:
0.9 % conc.
Also known as normal saline solution
Sodium lactate injection IP:
Contains 1.75 to 1.95 % w/v of sodium lactate
Electrolyte replenisher Slide 50: Mannitol injection IP:
Contains 5, 10 , 15, 20 % of mannitol
Used as :
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 :
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
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
Minerals………Electrolytes & Trace Elements Nutritional Requirements : Nutritional Requirements Amino acids
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
PPN: Peripheral Parenteral Admixture Indications for TPN : Indications for TPN Short-term use
Bowel injury /surgery
Nutritional preparation prior to surgery.
Malabsorption - bowel cancer
Prolonged Intestinal Failure
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
Clotted catheter line
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 :
waste product is removed
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
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.
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
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
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
Types of media:
Fluid thioglycollate medium
Soya-bean casein digest medium
other media 184.108.40.206.Fluid thioglycollate medium : 220.127.116.11.Fluid 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 18.104.22.168.Soya-bean casein digest medium : 22.214.171.124.Soya-bean 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 126.96.36.199.Fertility control of the media : 188.8.131.52.Fertility 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 184.108.40.206.Effectiveness of the media under test conditions : 220.127.116.11.Effectiveness 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
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 18.104.22.168.Selection of filters for membrane filtration : 22.214.171.124.Selection of filters for membrane filtration pore size of 0.45 m
effectiveness established in the retention of micro-organisms
the size of filter discs is about 50 mm in diameter 126.96.36.199.The procedure of membrane filtration : 188.8.131.52.The 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
Endogenous (inside body)
Exogenous (outside body)
Exogenous pyrogens –
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 method of storage between preparation and sterilization The endotoxin characteristics : The endotoxin characteristics thermostable
unaffected by the common bactericides
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 0.6C 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 39.8C
the differences in initial T should not differ from one another by more than 1C 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
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
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.
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
Much Less False Positives
Much Less Expensive
Alternative to Animal Model
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)
100 ml or more volume containers of single dose LV given by IV infusion
Not applicable for:
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
50 Nil Sources of particulate matter : Sources of particulate matter 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
Light blockage method Slide 146: Visual 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.
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
Electron microscopy etc… Significance of Particulate Matter monitoring : Significance of Particulate Matter monitoring Its presence may causes:
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.