industrial pharmacy

1. Industrial Pharmacy
(Phar 3131)
Unit 6

2. Sterile Products Manufacturing

3. Introduction
Types of sterile products
Formulation consideration
Manufacturing of sterile products
Production facilities
Production Procedures
Sterilization and
Quality control
Contents

4. Sterile Products
 Sterile products are pharmaceutical preparations that
are free from any viable MOs, including bacteria,
fungi, and viruses.
 These products are manufactured and handled under
controlled conditions to maintain their sterility and prevent
any potential contamination that could pose a risk to
patients or users.
 These includes parenterals, ophthalmic products &
irrigating solutions.
 The most common route used to deliver parenteral
medication is through Intravenous, lntraspinal, Intra
muscular, subcutaneous & lntradermal etc.

5.  All components & process involved in the preparation of this
product must be selected & design to eliminate
contamination of all types whether physical ,chemical and
microbiological origin.
Parenteral: Greek words Para (Outside)
Enteron (Intestine)
 These includes any method of administration that does not involve
passage through the digestive tract.
 These preparations are administered through the skin or mucus
membranes into internal body compartments.
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6. Unique future of Parenteral products
 All products must be sterile.
 All products must be free from pyrogen
 Injectable solutions must be free from visible particulate
matter.
 Products should be isotonic depending on route of
administration

7. Advantages of the Parenteral Route
 The IV route is the fastest method for delivering systemic drugs
preferred administration in an emergency situation/ Quick onset
It can provide fluids, electrolytes, and nutrition.
patients who cannot take food or have serious problems with
the GI tract
It provides higher concentration of drug to bloodstream or tissues
Advantageous in serious bacterial infection.
IV infusion provides a continuous amount of needed medication
infusion rate can be adjusted.
to provide more or less medication as the situation dictates
V
omiting and unconscious patients
No first pass metabolism effects





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8. Disadvantages of the Parenteral Route
Traumatic injury from the insertion of needle
Potential for introducing:
Toxic agents
Microbes
Pyrogens
Impossible to retrieve if adverse reaction occurs
injected directly into the body
Correct syringe, needle, and technique must be used
Rotation of injection sites with long-term use
Prevents scarring and other skin changes
Can influence drug absorption
Trained person required
Aseptic conditions is necessary in production, compounding
sterile product. Maintaining this condition is to expensive
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9. 1) Injections
 Small-volume parenterals (SVPs): Volume < 100 ml or = to 100 ml.
 Large-volume parenterals (LVPs): Volume > 100 mL
2) Single Dose and Multiple Dose Containers
 Single Dose Container:
 Size is limited to 1000 ml
 Intended for single use only
 Multiple Dose Container:
 Size is limited to 30 ml
 Contains suitable antimicrobial preservative
 Minimizes the risk of contamination resulting from multiple penetrations of the
closure
Types of Parenterals

10. 3). Other Types of Parenterals
 Dry powder for injection or for suspensions
 It requires reconstitution with a suitable diluent before
administration.
Examples include antibiotics, vaccines, and certain hormonal
medications.
 Depos (long-acting formulation)
 Depos are often used for medications that require less frequent
administration.
Examples include long-acting contraceptives and antipsychotic
medications.
 Emulsion (e.g., vitamin K and TPN - Total Parenteral Nutrients)
Examples include vitamin K injections for hemorrhagic disorders and
Total Parenteral Nutrition (TPN) solutions for patients unable to
consume adequate nutrients orally.

11.  Ophthalmic solutions
Sterile solutions specifically formulated for ocular use.
Used for eye conditions such as infections, glaucoma, or dry
eyes.
Administered as eye drops or ointments.
Irrigations
 Sterile solutions used for irrigation or flushing of body cavities,
wounds, or surgical sites.
Examples include wound irrigation solutions, bladder irrigations,
and nasal irrigations.

12. Formulation Considerations
API(solute)
Desired features forAPI
 The physical & chemical properties must be determined
 Exceptional purity (special parenteral grade must be
used)
 Compatibility with excipients
 Effect of process on stability
 Should address the Solubility issues
 Pyrogen free
Vehicles
a. Water
Should pass total solid content test (10 ppm) but for sterile
WFI it is 20 to 40 ppm
 Conductivity tests not more than 1micromho (1 megohm,
approximately 0.1 ppm NaCl)
 WFI (USP) may be pyrogenic but sterile WFI is Free from
pyrogen. 13

13. b. Non-aqueous water imiscible solvents
Must not be irritating
 Non toxic
 Non sensitizing
e.g fixed oils like Isopropyl myristate,
oil, Corn oil, Cotton seed oil, Soyabean oil
c. Non-aqueous water miscible
Peanut oil, Seasame
Ethyl alcohol, Propylene glycol PEG 300, 400, 600, Glycerine
Added substances
(1) Antibacterial agents-in bacteriostatic concentration must be
added in multiple dose vial
Non irritant
 Should pass preservative challenge test
hydrophilic surfactants may interact with esters of
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parahydroxybenzoate

14. may interact with the container and closure of the parenteral product,
necessitating an increase in the concentration of preservative required
or, preferably, a change in the type of container closure
 E.g, Phenol has been shown to interact with rubber closures
 Nitrile closures.
Methyl and propyl eater of P-hydroxy benzoic acid from the complex with
polysorbate 80 and hence decrease antibacterial activity
Some examples: Phenyl mercuric nitrate (0.001%), Phenyl mercuric acetate
(0.002%), Methyl paraben (0.01 to 0.18%), Propyl paraben (0.005 to
0.035%), Thiomersal (0.001 to 0.02%), Phenol (0.065 to 0.5%) ,
Chlorobutanol (0.25 to 0.50%).
2. Antioxidants
To slowdown or inhibit oxidative
 Oxidation may be facilitated by
via a chain reaction process.
degradation of therapeutic agents.
free radicals, with breakdown occurring
Radicals are formed due to the action of light, heat or transition metals
(e.g. iron, copper) that are present in the formulation 15

15.  These agents either act to prevent the formation of free radicals
[e.g. butylated hydroxyanisole (0.01 to 0.015%), butylated
hydroxytoluene- (0.01 to 0.015%), Tocopherols]
Strong Reducing agent e.g sodium bisulfate, Sodium metabisulfite,
ascorbic acid (0.1 to 0.15%) (All are used for aqueous system)
Buffers
Should have a capacity to maintain
 Maintain the solubility of the drug
the preparation
3.
the PH
in the vehicle over the shelf-life of
 Enhance the chemical stability of the therapeutic agent by
maintaining the pH of the formulation within the range of optimum
chemical stability of the therapeutic agent
e.g.Acetate, citrates and phosphate buffers
In most case, the biological effectiveness of a drug is maximum at or near
biological fluid pH rather than at the stabilizing pH. 16

16. (4) Tonicity contributors
 Should contribute to isotonicity (about 290 mOsm/L)
 Reduces the pain of injection
 The tonicity of parenteral formulations is an important design criterion
 In the presence of a hypotonic solution, red blood cells will swell (due to
water entry into the cell) and eventually burst (termed haemolysis)
 whereas, in the presence of a hypertonic solution, water will leave the
red blood cells, leading to crenation.
5. Chelating agents
Should avoid degradation due to the presence of heavy metals such as
cupper and iron e.g EDTA(0.01 to 0.075%)
To bind trace amounts of heavy metals
There by reducing their ability to generate free radicals or to be involved in
electron transfer reactions.
6. Solubilizing agents: e.g. Tweens & polysorbates
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17. Components and Container/Closures
 Containers
1. Glass containers
2. Plastic containers
 Containers use considerations
 The size of single dose containers is limited to 1000ml by the USP
and multiple dose containers to 30ml
 Rubber Closures
 Used to seal the opening, providing a material soft and elastic
enough to permit entry and withdrawal of hypodermic needle
1. Glass containers
Glass is commonly used in pharmaceutical packaging because it
possesses superior protective qualities, it is economical, and readily
available in a variety of sizes and shapes
It is essentially chemically inert, impermeable, strong, and rigid,
and has FDA clearance. 18

18. have proved useful for a number of reasons including their high
quality, and the freedom of design to which they lend themselves
 are extremely resistant to breakage and thus offer safety to
consumers along with reduction of breakage losses at all levels of
distribution and use.
Primarily made from the following polymers: polyethylene,
polypropylene, polyvinyl chloride, polystyrene, and to lesser extent,
poly-trifluoro-ethylene, and polyamides.
Plastic containers consist of one or more polymers together with
certain additives such as antioxidants,colors, lubricants, plasticizers, and stabilizers.
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Glass does not deteriorate with age, and with a proper closure system, it
provides an excellent barrier against practically every element except light
The major disadvantages of glass as a packaging material are its fragility
and its weight.
(2) Plastic containers

19. Production Considerations
 Includes
 Accumulation and combination of the ingredients
 To enhance the assurance of successful manufacturing
written SOP has to be followed
 Extensive record must be kept to give assurance at
production process.
in all process a
the end of the
Facilities:
The prevention of contamination must be the primary objective in
the design of these facility
Aseptic filling room: perfect cleanliness must be achieved in the
aseptic filling room
Buffer area: The surrounding area should provide a buffer area in
which standard of cleanliness are only slightly lower than those for
the aseptic room
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20. Production considerations
It is crucial for ensuring successful manufacturing processes. Here are some key
points to consider:
1. Accumulation and Combination of Ingredients:
 This involves sourcing raw materials, components, or any other inputs needed for
production.
2. Standard Operating Procedures (SOPs):
 Well-defined and documented SOPs should be established and followed throughout the
production process.
 It provide step-by-step instructions on how to carry out each task, ensuring
consistency, quality, and safety.
 Following SOPs minimizes errors and variations in the production process,
increasing the chances of successful manufacturing.
3. Extensive Record-Keeping:
 Comprehensive record-keeping is essential to track and document all aspects of
the production process.
 This includes recording the quantity and quality of ingredients used, equipment
calibration and maintenance, production timelines, quality control checks, and any
deviations from the standard procedures.
 These records serve as evidence of compliance, traceability, and quality assurance.

21. Parameters to be taken into consideration in
Production Facility:
the Design of a Parenteral
1. Environmental controlling
Effective environmental control, both
essential
Physical & biologic is
The standards of environmental control vary, depending on area
involved (clean-up, packaging, compounding or filling) and the type
of product being produced
Unquestionably, the entire area used for the preparation of product
prepared aseptically (without terminal sterilization) must be
maintained under the most rigid control that technology permits
If product is to be terminal sterilized, somewhat less rigid biological
control of the compounding & filling areas may be acceptable
 but rigid standard cleanliness must be maintained
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22. 2.Traffic control
Carefully designed arrangement to control and minimize traffic,
particularly in and out of the aseptic area, is essential
The only access directly from the outside is to the personnel wash
room, the equipment wash room, the non sterile manufacturing area, &
the capping (packaging )area
Access by personnel to the aseptic corridor and aseptic compounding
& filling rooms is only through an airlock
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23.  All equipment and surrounding work area must be cleaned
thoroughly at the end of the working day
 no contaminating residue from the concluded process may remain
 All cleaned equipment should be selected for its effectiveness and
freedom from lint-producing tendencies
 All cleaned equipment should be reserved for use in aseptic area
4. Surface disinfection
after thorough cleaning ,all surface should
aseptic area
be disinfected, at least in
Irradiation from ultraviolet lamp that are located to provide
adequate radiation intensity on the maximum extent of surface in a
room & that are maintained free from dust and films
 further reduce the viable microorganism present on surface and in
the air
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3. Housekeeping

24. 5. Personnel
They must be inherently neat , orderly, reliable and alert and having
good manual dexterity
All employees should be in good health and should be subjected to
periodic physical examination.
They should report the developing of symptoms of a cold, a sore
throat or other infectious disease
Removing at least street clothing, scrubbing the hand & arms
thoroughly with a disinfectant soap and donning the prescribed uniform
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25. Two categories of manufacturing operations
1. Terminally sterilization
2. Aseptic preparation
1. Terminal sterilization
 It is a process by which product is sterilized in its final
container.
 Containers are filled & sealed under high-quality
environmental conditions to minimize contamination,
 5 to 15% of all sterile products are terminally sterilized.
 Product in its final container is subjected to a
sterilization process such as heat or irradiation or
chemical

27. (2)Aseptic preparation
The drug product, container, and closure are first subjected to
sterilization methods separately, as appropriate, and then brought
together.
• Because there is no process to sterilize the product in its final
container, it is critical that containers be filled and sealed in an
extremely high-quality environment.
Sterilization
Process that eliminates, removes, kills, or deactivates all forms of life.
There are two basic types of sterilization
1. Physical processes of sterilization
a. Thermal methods
 Sterilization by moist heat
 Sterilization by dry heat
b) Non thermal methods
 Ultraviolet light, Sterilization
methods
by radiation, Filtration 33

29. 2. Chemical processes of sterilization
 Gas sterilization
Thermal methods
The lethal effectiveness of heat on microorganisms depends up on
the degree of heat, the exposure period, and the moisture content
present
Within the range of sterilizing temperatures, the time required to
produce a lethal effect is inversely proportional to the temperature
employed.
e.g. sterilization may be accomplished in 1 hour with dry heat at a
temperature of 170oC,
temperature of 140oC.
Thermal methods of sterilization may be divided
accomplished by dry heat and those by moist heat
but may require as much as 3 hours at a
into those
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30. Sterilization by moist heat (heating in an autoclave)
Water-wettable materials only, and aqueous formulations
Moist heat is more effective than dry heat for thermal sterilization
because moist heat causes the coagulation of cell protein at a much
lower temperature than dry heat, and
Normal moist heat cycles do not destroy Pyrogens
 Temperature, time and pressure monitored
 Quality of the steam – no contamination
Sterilization by dry heat
Used for substances that resist degradation above approximately140oC (284oF)
2 hrs exposure to a temp of 180oC or 45min at 260oC normally can
be expected to kill spores as well as vegetative forms of all MOs.
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31. Radiation Sterilization
No generation of heat except IR radiation. So it is also cold method
sterilization.
Radiations that kill the microorganisms are of two types
of
1. Ionizing radiation:
Have shorter wavelength, higher energy rays with good penetrative power.
Mode of action of ionizing radiation: Ionizing radiation raises the energy
level of atoms sufficient to release electrons resulting in ionization
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Ionizing Radiation are
 Gamma rays: generated by radioactive cobalt (Co60), Sterilization of
vitamins eg. Vit C, antibiotics like Streptomycin , benzylpenicillin
 X –Rays, High speed electrons (beta particles)

32. 2. Non-ionizing rays
Longer wavelength, lower energy, and poor penetration
1. UV RADIATIOS
Source: UV rays are generated using a high-pressure mercury vapour lamp.
Dose: 10-60 microwatts / cm2
 Microbicidal wavelength lie in the range of 200-280 nm
 Raises the energy level of atoms causing only excitation but not ionization.
 This results in interatomic vibration with breakage and induces formation of new
bonds (thymine-thymine
of proteins
dimmers---depolymerization of DNA and denaturation
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33. Sterilization By Filtration
 Cold method of sterilization
 Thermolabile liquids or solutions and air or gases.
 It does not affect the physical or the chemical integrity of the sterilized
material.
 Not used for suspensions.
Types of filters
1. Depth filters: retaining or trapping M.O. within the filter matrix.
Made of sintered glass or ceramic or asbestos or
Disadvantages
Migration of filter material into the filtrate.
Viruses and mycoplasma could pass through
2. Screen filters
cellulosic fibers
Retaining M.O. on the surface of the filter. A. Membrane filters: made up of
Cellulose (rayon), polyvinyl chloride, and a polyvinyl acrylonitrile copolymer
3.Air Filters: HEPAfilter High efficiency particulate air
microns and larger than this size with the efficiency of 99.97%.
HEP
Ais the only means for achieving class 100 clean room.
Particles are mainly trapped (they stick to a fiber)
filter(HEP
A)removes out particles of 0.

34. Gas sterilization (Ethylene oxide ([CH2]2O) Beta-propiolactone (BPL):
 therefore have assumed importance in sterilization
Ethylene oxide ([CH2]2O) is a gas at room temperature and it is highly
flammable and explosive when mixed with air
So, it should be admixed with inert gases like carbon dioxide (10%) or
one or more of the fluorinated hydrocarbons (Freons) in certain
to render nonflammable and safe to handle.
proportions
Mode of action: It is an alkylating agent. Cause alkylation of
amino-, carboxyl- and hydroxyl- groups.
sulfydryl-,
Application:
 It has good penetration and used to sterilize heat labile articles such as rubber,
plastics, syringes, disposable petri dishes, complex apparatus like heart-lung
machine, respiratory and dental Equipments.
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 Sterilization of surfaces capable of killing spores rapidly

35. Disadvantages: It is highly toxic, irritating to eyes, skin, highly
flammable, mutagenic and carcinogenic.
Beta-propiolactone (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl-
and hydroxyl- groups.
Application:
 has broad-spectrum activity and effective sporicidal agent
 0.2% is used to sterilize biological products.
 It is used to sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.
44

36. Cleaning equipment and containers
Equipment and container to be used in the processing of sterile
production must be thoroughly clean
Equipment cleaning procedures are required to prevent cross-
contamination or adulteration of drug products
 Purpose: to eliminate any sort of contamination
 Goal: to produce safe, efficacious and quality product
Serial cleaning: between batches of the same product
 Rigorous cleaning: between different products
1.
2.
Equipment disassembly (if required)
Prewash/inspection:
Most important step
 Potable water is sufficient for this step
 Removes all visible material
Wash
Actual washing of the parts or equipment
 Specified detergent (type and amount) or concentration
3.
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37. Temperature should be specified
 Multiple wash steps
 Dissolves most of the residual material
4. Initial rinse
Removes residual liquid (iii, which contains detergents)
 Quality of water should increase as stages of rinsing
increase (potable water→ purified water→ distilled water
WFI)
or
5. Final Rinse
Using highest-quality water (Purified Water, or WFI)
 Removes already dissolved residues (at a very low level)
hence temp not important
6. Reassemble (if required) 48

38. Rinsing new containers
 Deteregent treatment is usually eliminated
To loosen debris by rinsing ,alternating hot(clean steam) and
cold treatment should be used.
The final rinse should be done by WFI
Cleaning of rubber and plastic components
Rubber usually wash by mechanical agitation in a tank of hot
deteregent solution followed by a series of thorough water
rinsing, and finally rinse by WFI
 Objective to remove surface debris accumulated from molding
operation and from handling and leachable component at or near
surface
Sterilization of equipments
In general , equipment , container, closures and all other
components should be sterilized after cleaning and prior to use
49

39. Compounding the product
Should be done under clean environmental condition
Aseptic conditions usually are not required since it may not be
possible of feasible to sterilize some of the ingredient or the
equipment e.g. large tanks
Order of mixing ingredients may become highly significant.
Filtration of solutions
Objective clarification or sterilization
 Removal of particulate mater, to approximately 0.3 micron result in
sterilization , removes all viable microorganism and spores
After filtration, the solution must be protected from environmental
contamination until it is sealed in the final container
During filtration the solution must pass from a clean environment
to an aseptic area, particularly if it has been sterilized by filtration
process
50

40. Large volume of sterile solution( up to one liter) can be filled by
a. Gravity –slow , but is accomplished in a simple manner
b. Pressure- may be semi automated ,
c. Vacuum- more adaptable to automation
Slight excess is required in each container to provide for the lose that

occur during administration due to adherence to the wall of a
container and retention in the syringe
Emulsion and suspension often require specially designed
equipment due to high viscosity
High pressure must be applied
filling

reservoir
Need continuous agitation in the during filling, to keep the
52

product homogeneous
Filling procedure
A liquid may be subdivided from a bulk container to individual
dose container to uniformly

41. Filling of powders
 More difficult to subdivide accurately and precisely in to
individual dose container than liquid
Can be subdivide in to container by individual weighing
when the powder is free flowing machine filling may be employed
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42. Sealing
Container should be sealed in aseptic area immediately adjacent to
filling machine
Retains the contains of sterile product and assure the user that it is
not opened
Tamperproof sealing is important
Sealing ampoules
Ampoules: They are intended for single use only.
 Tip-seals are made by melting sufficient glass at the tip of the
ampoule neck to form a bead of glass and close the opening
Pull-seal are made by heating the neck of rotating ampoule below
the tip then pulling the tip away to form small, twisted capillary just
prior to being melted close
54

43. Pull-sealing is slower process, but the seal are more reliable than
those from tip-sealing
Powder ampoule or other types having a wide
sealed by pull-sealing
Fracture of the neck often occur during sealing
opening must be
Sealing vial
 Vials can be designed to hold multiple doses.
Rubber closures must fit the opening of the
enough to produce a seal
container snugly
They may be inserted by hand ,using sterile forceps
A faster hand method involves picking up the closure and inserting
it into a vial by means of a tool connected to a vacuum line
Aluminum caps are used to hold rubber closure in place
55

44. Classification of rooms
Divide or defined areas of operation in an aseptic processing facility
should be appropriately controlled to attain
different degrees of air quality depending on the nature of the
operation
satisfying microbiological and particle
GradeA
High risk operations, e.g. filling, aseptic preparation
 usually UDAF(Unit Directional (Laminar) air flow systems used
Grade B
 background environment to Grade A (in case of aseptic preparation
and filling)
Grade C : Preparation of Solution to be filtered
Grade D: Handling of components after washing
56

45. Airborne particulate classification according to different bodies
International Organization of Standards, (ISO), Technical Specification (TC)
ISO classify the rooms form ISO1 to ISO 9
WHO/GMP US 209 E US Consultancy ISO /TC
GradeA M 3.5 Class 100 ISO 5
Grade B M 3.5 Class 1000 ISO 5
Grade C M 5.5 Class 10000 ISO 7
Grade D M 6.5 Class 100000 ISO 8

46. Quality Evaluation of Sterile Products
The three general areas quality control are:
Incoming stock
Manufacturing (processing) and
The finished product
For sterile products, incoming stock control encompasses routine
tests on all ingredients as well as special evaluations such as Pyrogen
tests, glass test on containers, and identity tests on rubber closures
It also may be necessary to perform microbial load (bioburden) tests
to determine the number and types of microorganisms present
Process quality controls include
Testing the water for injection daily,
Ensure the entire environment and personnel are suitable for the
production process
60

47. The finished product evaluation
1. Leaker test (Packaging
types
a) visual inspection
b) bubble test
c) dye test
d) vacuum ionization test
Leaker tests are 4
or sealing integrity test):
This test is carried out to ensure hermetic seal of the ampoules
Method
Leakers usually are detected by providing a negative pressure ( – 27
mmHg/30 mins) within an incompletely sealed ampule, usually in a
vacuum chamber, while an ampule is entirely submerged in a deeply
color dye solution (usually 0.5 to 0.1% methylene blue)
61

48. Subsequent atmospheric pressure then causes the dye to
penetrate an opening, being visible after the ampoule has been
washed externally to clear it of dye
Vials and bottles are not subjected to such a leaker test, because
the rubber closure is not rigid.
2. Clarity test (Particulate matter test)
If the particle size of foreign matter is larger than the size of
R.B.C.. It can block the blood vessel.
USP states that all products contains are visually inspected and
that any with visible particles be discarded
In addition , for large volume infusions the USP established a
limit of 50 particles of 10µm and larger and 5 particles of 25µm and
larger per milliliter
Visual inspection can be done on 100% of the product units
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49. Instrumental method of evaluation for particulate matter in liquids
use the principles of light scattering (Nephelometer), light absorption
and Electrical resistance (Coulter counter) have been used to obtain
particle count and size distribution.
3. Pyrogen Test
 Detected by the fever response of rabbit (Invivo method)
Housing conditions and handling are critical to obtain consistent
results
Because of this rectal thermometer has been largely replaced by
rectal thermocouples, w/h remains in place through out the test,
eliminate handling of rabbit for individual temp reading.
63

50. 1. The rabbit test Recommended procedure
Prior to the test, determine the temperature of each animal by
taking 2 measurements at an interval of 30 min.
 Warm the solution to be tested to approximately 38 °C Inject into a marginal
vein of the ear of each of 3 rabbits 10 ml/kg of body weight
 Record the temperature of the animal during a period of 3 hrs, every 30 min

51. Procedure
 Sham test is performed within 7 days of actual test
 Inject into an ear vein of each of three rabbits 10ml of the
product per kg of body weight, completing each injection within 10
minutes after start of administration
 Record the temperature at 30 minute intervals between 1 and 3
hours subsequent to the injection.
 If no rabbits show an individual rise in temperature of 0.5oC or
more, if it is so, continue the test using five other rabbits
 If not more than 3 of the 8 rabbits show individual rises in temp.
of 0.5oC or more and if the sum of the 8 individual maximum temp.
exceed 3.3oC, the
rises does not material under test meets the
requirements for the absence of Pyrogens.
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52. LALtest for Pyrogen (Endotoxins)
Limulus amoebocyte test or bacterial Endotoxin test for the validation of
depyrogenation process.
Reagent - LAL reagent (Limulus amoebocyte lysate/ limulus
Polyphemus)
Reaction - In presence of Endotoxin a firm gel is formed within 60 min
when incubated at 37 ºC.
Advantage: 5 to 10 times more sensitive than the rabbit test
4. Sterility Test
All products which are labeled as ‗sterile‘ must undergo sterility
test
Sterility testing attempts to reveal the presence or absence of viable
microorganisms in a sample number of containers taken from a batch of
product
The BP (2001) states: the test may be carried out using the technique
of membrane filtration or by direct inoculation of the culture media with
the product being examined
Appropriate negative controls are included in either case using
65
preparations known to be sterile

53. Medium used –
a.
b.
c.
Nutrient broth forAerobic
Fluid thioglycolate (FTM)forAerobic andAnaerobic
Soyabean casein digest (SCD) for FungiAerobic
Incubation–for 2 weeks at 30 to 35°C (For FTM) and 20 to 25°C
(For SCD)
All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood
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54. 69