1. PARENTERAL DRUG DELIVERY

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

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).

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
sodium that they are given by parenteral.

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)

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.

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.

8. Routes of Parenteral Administration
Subcutaneous (21) Intravenous (21)
Intramuscular (20)
Intradermal (23)
Intra arterial (20-22)
Epidermis
Dermis
Vein
Subcutaneous Artery
tissue
Muscle

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)

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

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

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

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)

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

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

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

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

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

19. Intradermal:
Also called as diagnostic testing
0.05 ml
½ inch, 25 to 26 gauge needle
Should be isotonic
Given:
Diagnostic agents

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

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

22.  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

23. 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

24. Formulation of Parenteral
1.Therapeutic ingredients:
 Insulin
 Antibiotics
 Anticancer
 Steroids
 Vaccines
 Antipyretic
 Analgesics
 Anti- inflammatory
 LVP’s like Dextrose, NaCl or combination etc….

25. Formulation of Parenteral
2.Solvents:
o Water
o Should meet compendial requirements
o Water miscible vehicles
o Ethyl alcohol
o PEG
o PG
o Non aqueous vehicles
o Fixed oils

26. 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

27. 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 %

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.

29. Antioxidants:
 Used to protect product from oxidation
 Acts as reducing agent or prevents oxidation
 Ex:
 A) Reducing agent:
o Ascorbic acid -- 0.02 – 0.1 %
o Sodium bisulphite-- 0.1 – 0.15 %
o Sodium metabisulphite-- 0.1 – 0.15 %
o Thiourea - 0.005 %
 B) Blocking agents:
o Ascorbic acid esters- 0.01 – 0.015%
o BHT- 0.005 – 0.02 %
 C) Synergistic:
o Ascorbic acid , Citric acid , Tartaric acid
 D) Chelating agent:
o EDTA- 0.01- 0.075 %

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 %

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 %

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 %

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

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

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

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

37. Production facilities
Types :
Emulsion
Suspension
Solutions
Preparation of
IV fluids
IV admixtures
TPN
Dialysis fluids
QC tests for parenteral

38. Sterile area
Production facilities:
Clean- up area
Preparation area
Aseptic area
Quarantine area
Finishing and packaging area

39. LAY OUT OF PARENTERAL MANUFACTURING AREA
S COMPOUNDING ASEPTIC QUARANTINE
T AREA AREA AREA
O
C STORAGE
K
AND
R TRANSPORT
O
O PACKING
M CLEAN UP
STERILIZATION AND
AREA
LABELLING

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 sodium that to avoid
contamination to aseptic area
The containers & closures are washed & dried in this area.

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

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

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

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..

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.

46. 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

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

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

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

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

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.

52. 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.

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

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.

55. 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.

56. 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.

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.

58. 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.

59. Normal Diet TPN
Protein…………Amino Acids
Carbohydrates…Dextrose
Fat……………..Lipid Emulsion
Vitamins………Multivitamin Infusion
Minerals………Electrolytes & Trace Elements

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

61. Total Parenteral Nutrition
Electrolytes
Electrolyt Daily Requirement Standard Concentration
e.
Na 60-150 meq 35-50 meq/L
K 40-240 meq 30-40 meq/L
Ca 3-30 meq 5 meq/L
Mg 10-45 meq 5-10 meq/L
Phos. 30-50 mM 12-15 mM/L

62. 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.

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.

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.”

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

66. 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

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.

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.

69. Complications of TPN
Sepsis
Air embolism
Clotted catheter line
Catheter displacement
Fluid overload
Hyperglycaemia
Rebound Hypoglycaemia

70. 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.

71. General uses :
Renal failure
 waste product is removed
 Maintain electrolytes
 Also called as haemodialysis or intraperitoneal dialysis
Transplantation of kidney
Poisoning cases

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.

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.

74. COMPOSITION
Composition of Concentrated Haemodialysis Fluid BPC
Dextrose monohydrate ----------- 8.0 gm
Sodium acetate --------------------- 19.04 gm
Lactic acid --------------------------- 0.4 ml
Sodium chloride ------------------- 22.24 gm
Potassium chloride --------------- 0.4 gm
Freshly boiled & cooled water -q.s. 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.

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

76. COMPOSITION
Composition of Fluid Intraperitoneal Dialysis IP
1985
Sodium chloride ------------------- 5.56 gm
Sodium acetate --------------------- 4.76 gm
Calcium chloride ------------------- 0.22 gm
Magnesium chloride -------------- 0.152 gm
Sodium metabisulphite ---------- 0.15 gm
Dextrose (anhydrous) ----------- 17.30 gm
Purified water -----------q.s.----- 1000 ml
Sterilize by autoclave immediately after
preparation.

77. STERILIT Y TESTING FOR
PARENTERAL PRODUCTS

78. 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.

79. 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

80. 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

81. 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

82. 1.2.Culture conditions
Appropriate conditions for the growth of any surviving
organism should be provided by the culture media selection.

83. 1.2. Culture conditions
Factors affecting growth of bacteria
Phases of bacterial growth
Culture media for sterility testing

84. 1.2.1. Factors affecting growth of bacteria
Nutrition
Moisture
Air
Temperature
pH
Light
Osmotic pressure
Growth inhibitors

85. 1.2.2. Phases of bacterial growth
 Lag phase (A)
 Log (logarithmic or
exponential) phase (B)
 Stationary phase (C)
 Decline (death) phase (D)

87. 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

88. 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 -35°C

89. Fluid thioglycollate medium

90. 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 -25°C

91. Soya-bean casein digest medium

92. 1.2.3.3.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

93. 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;

94. 1.3.The test method for sterility of the
product
Membrane filtration
Direct inoculation of the culture medium

95. 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

96. 1.3.1.1.Selection of filters for membrane
filtration
pore size of 0.45 µm
effectiveness established in the retention of micro-organisms
appropriate composition
the size of filter discs is about 50 mm in diameter

97. 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).

99. 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

100. Scheme for sterility test by membrane filtration Scheme for sterility test by direct inoculation

101. 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

102. 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

103. 1.5. Sampling
Selection of the samples
Sample size

104. Minimum number of items to be tested

105. 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

106. 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.

107. PYROGENS AND PYROGEN TESTING

108. I Love The Rabbit!

109. 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

110. 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

111. Generalized structure of endotoxins
Generalized structure of Endotoxins

114. Sources of pyrogen contamination
solvent - possibly the most important source
the medicament
the apparatus
the method of storage between preparation and sterilization

115. 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

116. Tests for pyrogenic activity
Test for pyrogens = Rabbit test
Bacterial endotoxins

117. 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.

118. 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

119. 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

120. 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)

122. 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

123. 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

124. 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

125. The result of pyrogen test:
No.of Rabbits Individual Tempt. Test
Tempt. rise Rise in
(°c) group (°c)
3 rabbits 0.6 1.4 Passes
If above not passes 0.6 3.7 Passes
3+5 = 8 rabbits
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.

126. 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

127. Limulus polyphemus = horseshoe crab

128. 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

129. 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.

130. 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

131. 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

132. 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.

133. Gel-cloth technique (Methods A, B)
allows detection or
quantification of endotoxins Gel Clot
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

134. 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 λ (labeled lysate sensitivity).

135. 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.

136. 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

137. 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

138. 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

139. Particulate Matter Monitoring

140. 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.

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

142. Permitted limits of particulate matter
Particle size in micrometer Max.No.of particles
(equal to or larger than) per ml
10 50
25 5
50 Nil

143. 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.

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…

145. Methods of monitoring particulate matter
contamination
 Visual method
 Coulter counter method
 Filtration method
 Light blockage method

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.

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.

148. Identification of Particulate Matter
Microscopy
X- ray powder diffraction
Mass microscopy
Microchemical tests
Electron microscopy etc…

149. Significance of Particulate Matter
monitoring
Its presence may causes:
Septicemia
Fever & blockage of blood vessels
Quality of product may affect

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.