PARENTERAL DRUG DELIVERY

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

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).

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.

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)

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.

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.

Routes of Parenteral Administration Subcutaneous (21) Intramuscular (20) Intra arterial (20-22) Intravenous (21)

Intradermal (23)

Epidermis

Dermis

Vein Subcutaneous tissue

Artery

Muscle

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)

Subcutaneous (SC; SQ ;Sub Q):

The injection is given under the skin Need to be isotonic Upto 2 ml is given Using to 1 inch 23 gauge needle or smaller needle Vaccines Insulin Scopolamine Epinephrine

Given:

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

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

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)

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

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

Intraarticular:
Given directly into the joints 2 to 20 ml 5 inch 22 gauge Must be isotonic

Given:
Morphine LA Steroids NSAIDs Antibiotics

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

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

Intradermal:
Also called as diagnostic testing 0.05 ml inch, 25 to 26 gauge needle Should be isotonic

Given:
Diagnostic agents

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

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

Formulation of Parenteral:
1. Therapeutic agents 2. Vehicles i. Water ii. Water miscible vehicles iii. Non- aqueous vehicles 3. Added substances (Additives) i. Antimicrobials ii. Antioxidants iii. Buffers iv. Bulking agents v. Chelating agents vi. Protectants vii. Solubilizing agents viii. Surfactants ix. Tonicity- adjusting agents

General steps involved

1. Cleaning 2. Preparation of bulk products 3. Filtration

4. Filling of solution in or product in ampoule or vial

5.Sealing

6. Sterilization
7. Tests for Quality control

Formulation of Parenteral
1.Therapeutic ingredients:

Insulin Antibiotics Anticancer Steroids Vaccines Antipyretic Analgesics Anti- inflammatory LVPs like Dextrose, NaCl or combination etc.

Formulation of Parenteral
2.Solvents:
o Water
o

Should meet compendial requirements

o Water miscible vehicles

o o o

Ethyl alcohol
PEG PG

o Non aqueous vehicles

o

Fixed oils

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 microorganisms 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 %

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.

 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 esters0.01 0.015% BHT0.005 0.02 % C) Synergistic: Ascorbic acid , Citric acid , Tartaric acid D) Chelating agent: EDTA0.01- 0.075 %

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 %

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 %

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 %

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

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

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

Must check each individual monogram for: Type of container:

Glass Plastic Rubber closure

Type I Type II Type III NP

Type of glass

Tests for glass containers

Powdered Glass test Water Attack test

Package size Special storage instructions

Production facilities Types :

Emulsion Suspension Solutions

Preparation of
IV fluids IV admixtures TPN Dialysis fluids

QC tests for parenteral

Sterile area

Production facilities:
Clean- up area Preparation area Aseptic area Quarantine area Finishing and packaging area

LAY OUT OF PARENTERAL MANUFACTURING AREA

S T O C K
R O O M

COMPOUNDING AREA

ASEPTIC AREA

QUARANTINE AREA

STORAGE AND TRANSPORT

PACKING CLEAN UP AREA STERILIZATION AND LABELLING

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.

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

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

 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

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..

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:

LVPs which are administered by IV route are commonly called as IV fluids. Purposes :

Body fluids, Electrolyte replenisher

Volume supplied:
100 to 1000 ml

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

Examples:
Dextrose injection IP : available in 2 , 5 , 10 , 25 & 50 % w/v solution. Used for

Fluids replenisher, Electrolyte replenisher Contains

0.11 to 0.9 % Sodium chloride 2.5 to 5.0 % Dextrose

Sodium chloride & Dextrose injection IP: (DNS)

Used for

Fluids replenisher, Electrolyte replenisher Nutrient replenisher

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

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

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

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.

Care :
Microbial contamination Incompatibility
Physical : change in color Chemical : hydrolysis, oxidation, reduction etc.. Therapeutic: undesirable antagonistic or synergistic effect

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.

Total Parenteral Nutrition

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 (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.

 Components

of TPN solutions:

(1) Protein as crystalline amino acids. (2) Fats as lipids. (3) Carbohydrate as glucose. (4) ElectrolytesSodium, potassium, chloride, calcium and magnesium. (5) Metals/Trace elementsZinc, copper, manganese, chromium, selenium. (6) Vitamins A, C, D, E, K, thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, choline and folic acid.

Why it is necessary?

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.

Normal Diet TPN

ProteinAmino Acids CarbohydratesDextrose Fat..Lipid Emulsion VitaminsMultivitamin Infusion MineralsElectrolytes & Trace Elements

Nutritional Requirements
Amino acids Glucose Lipid Minerals Vitamins Water and electrolytes Trace elements

Total Parenteral Nutrition Electrolytes

Electrolyt Daily Requirement Standard Concentration e. Na 60-150 meq 35-50 meq/L K Ca Mg 40-240 meq 3-30 meq 10-45 meq 30-40 meq/L 5 meq/L 5-10 meq/L

Phos.

30-50 mM

12-15 mM/L

When is it necessary?

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.

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.

GENERAL INDICATIONS
Patient who cant eat Patient who wont eat Patient who shouldnt eat Patient who cant eat enough If the gut works, use it.

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

Short-term use

Bowel injury /surgery Bowel disease Severe malnutrition Nutritional preparation prior to surgery. Malabsorption - bowel cancer

Long-term use

Prolonged Intestinal Failure Crohns Disease Bowel resection

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.

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
Sepsis Air embolism Clotted catheter line Catheter displacement Fluid overload Hyperglycaemia Rebound Hypoglycaemia

DIALYSIS FLUIDS
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.

General uses :
Renal failure
waste product is removed Maintain electrolytes Also called as haemodialysis or intraperitoneal dialysis

Transplantation of kidney Poisoning cases

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.

 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.

COMPOSITION
Composition of Concentrated Haemodialysis Fluid BPC

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

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.

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

COMPOSITION

Composition of Fluid Intraperitoneal Dialysis IP 1985

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

Sterilize by autoclave immediately after preparation.

STERILITY TESTING FOR PARENTERAL PRODUCTS

1. Sterility testing - definition

Sterility testing attempts to reveal the presence or absence of viable microorganisms 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 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

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
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

Appropriate conditions for the growth of any surviving organism should be provided by the culture media selection.

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

Nutrition Moisture Air Temperature pH Light Osmotic pressure Growth inhibitors

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

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

1.2.3.1.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 -35C

Fluid thioglycollate medium

1.2.3.2.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 -25C

Soya-bean casein digest medium

1.2.3.3.Fertility control of the media

are they suitable for growth of each microorganism? '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

1.2.3.4.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

Membrane filtration Direct inoculation of the culture medium

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

1.3.1.1.Selection 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

1.3.1.2.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

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

Scheme for sterility test by membrane filtration

Scheme for sterility test by direct inoculation

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

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

Selection of the samples

Sample size

Minimum number of items to be tested

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

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.

PYROGENS AND PYROGEN TESTING

I Love The Rabbit!

Pyrogens
Pyrogenic - means producing fever Pyrogens - fever inducing substances

Having nature

Endogenous (inside body) Exogenous (outside body) mainly lipopolysaccharides bacterial origin, but not necessary

Exogenous pyrogens

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

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

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

Test for pyrogens = Rabbit test Bacterial endotoxins

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?

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

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 23C

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 0.1C)

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

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

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:

No.of Rabbits Individual Tempt. rise (c) Tempt. Rise in group (c) Test

3 rabbits If above not passes 3+5 = 8 rabbits

0.6 0.6

1.4 3.7

Passes Passes

If above test not passes perform the test again

If above test not passes, the sample is said to be pyrogenic or go thr the sources of contamination of pyrogen.

Bacterial endotoxins

to detect or quantify endotoxins of gramnegative 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

Mechanism of LAL

the test is based on the primitive bloodclotting 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 bleeding adult crabs reagents 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)

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 37C, 1 hour remove the tube - invert in one smooth motion (180) - read (observe) the result pass-fail 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 peptidechromogen complex

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)

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 Clot

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

Gel-cloth technique (Methods A, B)cont.

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)

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)

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

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 dont 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 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

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.

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

Particle size in micrometer (equal to or larger than)
10 25 50

Max.No.of particles per ml

50 5 Nil

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.

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

Visual method Coulter counter method Filtration method Light blockage method

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.

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

Microscopy X- ray powder diffraction Mass microscopy Microchemical tests Electron microscopy etc

Significance of Particulate Matter monitoring

Its presence may causes:

Septicemia Fever & blockage of blood vessels

Quality of product may affect

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.