This document discusses parenteral drug delivery, which refers to drug administration through routes other than the oral route, such as injections. It provides details on various types of injections including intravenous, subcutaneous, intramuscular, and others. It describes why the parenteral route is used, advantages and disadvantages, necessary characteristics of parenteral preparations, production facilities, formulation, labeling requirements, and quality control tests. Common parenteral preparations discussed include intravenous fluids, intravenous admixtures, total parenteral nutrition, and dialysis fluids.

1. Parenteral drug delivery
Ravish Yadav

2. 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).

3. 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.

4. • 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)

5. • 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.

6. • 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.

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

8. 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)

9. 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

10. •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)

11. 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

12. • 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

13. • 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

14. • 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

15. • Intraarticular:
• Given directly into the joints
• 2 to 20 ml
• 5 inch 22 gauge
• Must be isotonic
• Given:
• Morphine
• LA
• Steroids
• NSAID’s
• Antibiotics

16. • 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

17. 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

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

19. 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

20. 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

21. • 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

22. 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

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

24. Formulation of Parenteral
2.Solvents:
oWater
oShould meet compendial requirements
oWater miscible vehicles
oEthyl alcohol
oPEG
oPG
oNon aqueous vehicles
oFixed oils

25. 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

26. 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 %

27. • 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.

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

29. • 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 %

30. • 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 %

31. • 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 %

32.  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

33. • 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

34. •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

35. 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

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

37. • Production facilities:
• Clean- up area
• Preparation area
• Aseptic area
• Quarantine area
• Finishing and packaging area
Sterile area

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

39. •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.

40. • 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

41. • 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

42. • 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

43. • 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..

44. • 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.

45. 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

46. • 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

47. •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

48. •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

49. • 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

50. • 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.

51. 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.

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

53. • 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.

54. • 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

55. 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.

56. •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.

57. • 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?

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

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

60. Total Parenteral Nutrition
Electrolytes
Electrolyt
e.
Daily Requirement Standard Concentration
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

61. • 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?

62. •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.

63. • 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.”

64. • 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

65. 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

66. •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.

67. •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.

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

69. • 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

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

71. • 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.

72. • 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.

73. Composition of Concentrated Haemodialysis Fluid BPC
Dilute 1 liter of conc. solution with 39 liters of water to make 40 litres.
Storage: store in warm place as it is liable to convert into crystals on
storage.
COMPOSITION
Dextrose monohydrate -----------
Sodium acetate ---------------------
Lactic acid ---------------------------
Sodium chloride -------------------
Potassium chloride ---------------
Freshly boiled & cooled water -q.s.
8.0 gm
19.04 gm
0.4 ml
22.24 gm
0.4 gm
100 ml

74. • 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

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

76. STERILITY TESTING FOR PARENTERAL
PRODUCTS

77. 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.

78. 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

79. 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

80. 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

81. 1.2.Culture conditions
• Appropriate conditions for the growth of any surviving organism
should be provided by the culture media selection.

82. 1.2. Culture conditions
• Factors affecting growth of bacteria
• Phases of bacterial growth
• Culture media for sterility testing

83. 1.2.1. Factors affecting growth of
bacteria
•Nutrition
•Moisture
•Air
•Temperature
•pH
•Light
•Osmotic pressure
•Growth inhibitors

84. 1.2.2. Phases of bacterial growth
• Lag phase (A)
• Log (logarithmic or
exponential) phase (B)
• Stationary phase (C)
• Decline (death) phase (D)

86. 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

87. 1.2.3.1.Fluid thioglycollate medium
• composition described in next slide.
• primarily intended for the culture of anaerobic bacteria
• incubation of the media:
• 14 days at 30 -35°C

88. Fluid thioglycollate medium

89. 1.2.3.2.Soya-bean casein digest
medium
• primarily intended for the culture of both fungi and aerobic bacteria
• incubation of the media:
• 14 days at 20 -25°C

90. Soya-bean casein digest medium

91. 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

92. 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;

93. 1.3.The test method for sterility of the
product
• Membrane filtration
• Direct inoculation of the culture medium

94. 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

95. 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

96. 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).

98. 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

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

100. 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

101. 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

102. 1.5. Sampling
• Selection of the samples
• Sample size

103. Minimum number of items to be tested

104. 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

105. 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.

106. PYROGENS AND PYROGEN TESTING

107. I Love The Rabbit!

108. 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

109. 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

110. Generalized structure of endotoxins
Generalized structure of Endotoxins

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

114. 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

115. Tests for pyrogenic activity
• Test for pyrogens = Rabbit test
• Bacterial endotoxins

116. 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.

117. 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

118. 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

119. 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)

121. 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

122. 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

123. 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

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

125. 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

126. Limulus polyphemus = horseshoe
crab

127. 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

128. 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.

129. 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

130. 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

131. 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.

132. 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

133. 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

134. Particulate Matter Monitoring

135. 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.

136. 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

137. 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.

138. • 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…

139. Methods of monitoring particulate matter
contamination
• Visual method
• Coulter counter method
• Filtration method
• Light blockage method

140. • 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.

141. •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.

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

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

144. • 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.