An Assignment

On
Methods of Sterilization

Course No: Chem-4109

Course Title: Pharmaceutical Chemistry

Submitted to:
Jannatul Naime
Assistant Professor
Chemistry Discipline
Khulna University
Khulna.

Submitted by:
Md. Al-Amin Mia Anik
ID No: 181826
4th Year, 1st Term
Chemistry Discipline
Khulna University

Khulna.
 CONTENT
Topic No. Topic’s Name Page No.

01. Microbiological assay 03-07

Definition
Objectives of microbiological assay
Principles of microbiological assay
Methods of microbiological assay
Importance of microbiological assay in
pharmaceutical products

02 Sterilization 08-19
Definition
Types of sterilization
Types of methods

03 Sterility 20-26
Definition
Which are sterile products?
What is sterility test?
Why sterility test is important?
Which methods are followed to perform sterility
test?
How sterility test is performed?
Importance of sterility test

04 Pyrogen 27-31
Definition

1
 Which are Pyrogen products?
What is Pyrogen test?
Why Pyrogen test is important?
Which methods are followed to perform Pyrogen
test?
How Pyrogen test is performed?
Importance of Pyrogen test

Microbiological assay
Definition:

2
A microbiological assay defined as qualitative or quantitative determination of chemical
compound from a simple or even complex material with the use of microorganisms.

Objectives of microbiological assay:

 Determining the concentration of certain compounds (e.g., amino-acids, vitamins and

some antibiotics) in complex chemical mixtures or in body fluids.
 Diagnosing certain diseases.
 Testing chemicals for potential mutagenicity or carcinogenicity.
 Monitoring purposes involving the use of immobilized enzymes.
 Sterility testing of antibiotics.
 Microbiological assays are used during production to determine the potency and quality
control.
 These are used to determine the pharmacokinetics of drugs in animal and human.
 In antimicrobial chemotherapy to monitor, in managing, controlling the chemotherapeutic
agents.

Reference: https://www.onlinebiologynotes.com/microbiological-assay-and-methods/
#:~:text=Microbial%20assay&text=Determining%20the%20concentration%20of%20certain,for
%20po

Principles of microbiological assay:

3
The microbiological assay is the technique in which the potency or concentration of a compound
is assessed by determines its effect on the microorganism. It is a legal quality control
requirement for the assay of several antibiotics in both the British Pharmacopoeia and United
States Pharmacopoeia. Many therapeutic agents that are either inhibit the growth of
microorganisms (antibiotics) or essential for their growth (vitamins and amino acids) are
standardized by microbiological assays.

The microbiological assay of an antibiotic is based upon a comparison of the inhibition of

growth of microorganisms by measured concentrations of the antibiotics under examination with
that produced by the known concentration of a standard preparation of the antibiotic having a
known activity.

Microbiological assay of vitamins is a type of biological assay performed with the help of
microorganisms.

Reference: https://solutionpharmacy.in/microbiological-assay/

Methods of microbiological assay:

Method 1: Cylinder/Cup-plate method

Principle: This method depends on the diffusion of an antibiotic from a vertical-cavity through
the solidified agar layer in the Petri plate. The growth of test microorganisms is inhibited entirely

in a zone around the cylinder containing antibiotic solution.

Procedure:

 The nutrient agar is melted, cooled, and poured into a petri dish.
 0.2 ml of known concentration of inoculum is spread on the surface of solidified agar by
spread plate technique.
 Holes or cavities are made by a sterile borer.

4
  0.2, 0.4, 0.6, 0.8, and 1 ml of antibiotic is poured into the cup of the agar plate and then
incubated at 37°C for 24 hours.
 A zone of inhibition is observed for an antibiotic that has antimicrobial activity.
 The zone of inhibition is measured by scale from the backside of the plate from the center
of the cavity or hole.

Fig: Cup-plate method for microbial assay

Reference: https://solutionpharmacy.in/microbiological-assay/

Method 2: Tube assay method or Turbidity Method:

Principle: This method depends on the inhibition of the growth of microbial culture in a uniform
solution of the antibiotic in the fluid medium that is favorable to its rapid growth in the absence
of antibiotics.

Procedure:
 Five different concentrations of the standard solutions are prepared from the stock
solution of standard as per step-wise increased ratio 4:5.
 The concentration of media is selected and unknown substance solution is diluted to that
concentration.

5
  1 ml of each concentration of the standard solution as well as test sample solution in each
of the tubes is placed in duplicate.
 9 ml of nutrient medium is added to each of the tubes.
 Side by side three control samples are prepared. One contained the inoculated culture
medium, another is blank (0.5 ml of dilute formaldehyde solution) and the third one
contains an uninoculated culture medium.
 All the tubes are placed in the incubator for 3-4 hours at 37°C and after incubation 0.5 ml
of dilute formaldehyde is added in each tube.
 The growth of the test microorganism is measured by determining the absorbance at
about 530 nm of each of the solutions in the tubes against the blank.

Fig: Turbidity method of microbial assay

Reference: https://solutionpharmacy.in/microbiological-assay/

Importance of microbiological assay in pharmaceutical products:

 Microbial assays or microbiological assays could be a sort of bioassays designed to
analyze the compounds or substances as a pharmaceutical product that have impact on
micro-organisms.
 They help to estimate concentration and efficiency of antibiotics. Also facilitate in
determination of the simplest anti-biotic as pharmaceutical products appropriate for
patient recovery. This determination is feasible by immune assays for few diseases.

6
  Microbiological assay was found to be convenient, rapid, cost-effective, precise and
accurate in demonstrating pharmaceutical equivalence of antibiotics in different dosage
forms.
 This technique can be used as an alternative method for testing generic antibiotics prior to
their use in animal and human.

Reference: https://www.walshmedicalmedia.com/scholarly/microbial-assay-journals-articles-
ppts-list-828.html

7
 Sterilization
Definition:

Sterilization is defined as a process of complete elimination or destruction of all forms of

microbial life (i.e., both vegetative and spore forms), which is carried out by various physical
and chemical methods.

Types of sterilization:

Reference: http://vin840225ceu.blogspot.com/2015/04/common-types-of-sterilization-
in.html

8
Types of methods:
Physical methods of sterilization:
Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays
in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the
sunlight is more effective in killing germs. due to combination of ultraviolet rays and heat. By
killing bacteria suspended in water, sunlight provides natural method of disinfection of water
bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.

Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand
heat. Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those
articles that cannot withstand high temperatures can still be sterilized at lower temperature by
prolonging the duration of exposure.

Factors affecting sterilization by heat are:

 Nature of heat: Moist heat is more effective than dry heat o

 Temperature and time: temperature and time are inversely proportional. As temperature
increases the time taken decreases.
 Number of microorganisms: More the number of microorganisms, higher the
temperature or longer the duration required.
 Nature of microorganism: Depends on species and strain of microorganism, sensitivity to
heat may vary. Spores are highly resistant to heat.
 Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure. Certain heat sensitive articles must be sterilized at lower
temperature.
 Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.

Action of heat: Dry heat acts by protein denaturation, oxidative damage and toxic effects of
elevated levels of electrolytes. The moist heat acts by coagulation and denaturation of
proteins. Moist heat is superior to dry heat in action. Temperature required to kill microbe by
dry heat is more than the moist heat. Thermal death time is the minimum time required to
kill a suspension of organisms at a predetermined temperature in a specified environment.

9
 1. Dry heat:

Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame.

Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure. This method too is limited to those
articles that can be exposed to flame. Cracking of the glassware may occur.

Incineration: This is a method of destroying contaminated material by burning them in

incinerator. Articles such as soiled dressings; animal carcasses, pathological material and
bedding etc. should be subjected to incineration. This technique results in the loss of the article,
hence is suitable only for those articles that have to be disposed. Burning of polystyrene
materials emits dense smoke, and hence they should not be incinerated.

Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed
to high temperature (160o C) for duration of one hour in an electrically heated oven. Since air is
poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan.
The heat is transferred to the article by radiation, conduction and convection. The oven should be
fitted with a thermostat control, temperature indicator, meshed shelves and must have adequate
insulation.

Infra-red rays: Infrared rays bring about sterilization by generation of heat. Articles to be
sterilized are placed in a moving conveyer belt and passed through a tunnel that is heated by
infrared radiators to a temperature of 180o C. The articles are exposed to that temperature for a
period of 7.5 minutes.

10
 2. Moist heat:

Moist heat acts by coagulation and denaturation of proteins.

At temperature below 100o C:

Pasteurization: There are two methods of pasteurization, the holder method (heated at 63 o C for
30 minutes) and flash method (heated at 72o C for 15 seconds) followed by quickly cooling to
13o C. Other pasteurization methods include Ultra-High Temperature (UHT), 140 o C for 15 sec
and 149o C for 0.5 sec. This method is suitable to destroy most milk borne pathogens like
Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may
survive pasteurization.

At temperature 100o C:

Boiling: Boiling water (100o C) kills most vegetative bacteria and viruses immediately. Certain
bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores
are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing
activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not
required, certain metal articles and glasswares can be disinfected by placing them in boiling
water for 10-20 minutes.

At temperature above 100o C:

Autoclave: Sterilization can be effectively achieved at a temperature above 100 oC using an

autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature
at which the water boils also increases. In an autoclave the water is boiled in a closed chamber.
As the pressure rises, the boiling point of water also raises. At a pressure of 15 lbs inside the
autoclave, the temperature is said to be 121o C. Exposure of articles to this temperature for 15
minutes sterilizes them. To destroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times are used; 135 o C or 121o C for at
least one hour are recommended.

11
 Fig: Autoclave

Radiation: Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are
low energy rays with poor penetrative power while ionizing rays are high-energy rays with good
penetrative power. Since radiation does not generate heat, it is termed "cold sterilization". In
some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500
percent.

i. Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being
most effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at
this wavelength that the absorption by the microorganisms is at its maximum, which
results in the germicidal effect. UV rays induce formation of thymine-thymine dimers,
which ultimately inhibits DNA replication. UV readily induces mutations in cells
irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc.
that are exposed to the effective UV radiation are inactivated within seconds. Since UV
rays don’t kill spores, they are considered to be of use in surface disinfection. UV rays
are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors,
etc. Disadvantages of using uv rays include low penetrative power, limited life of the uv

12
 bulb, some bacteria have DNA repair enzymes that can overcome damage caused by uv
rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It
doesn't penetrate glass, paper or plastic.

ii. Ionizing rays: Ionizing rays are of two types, particulate and electromagnetic rays.

 Electron beams are particulate in nature while gamma rays are electromagnetic in
nature. High speed electrons are produced by a linear accelerator from a heated
cathode. Electron beams are employed to sterilize articles like syringes, gloves,
dressing packs, foods and pharmaceuticals. Sterilization is accomplished in few
seconds. Unlike electromagnetic rays, the instruments can be switched off.
Disadvantage includes poor penetrative power and requirement of sophisticated
equipment.
 Electromagnetic rays such as gamma rays emanate from nuclear disintegration of
certain radioactive isotopes (Co60, Cs137). They have more penetrative power
than electron beam but require longer time of exposure. These high-energy
radiations damage the nucleic acid of the microorganism. A dosage of 2.5
megarads kills all bacteria, fungi, viruses and spores. It is used commercially to
sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones,
glasswares and fabrics. Disadvantages include; unlike electron beams, they can’t
be switched off, glasswares tend to become brownish, loss of tensile strength in
fabric. Gamma irradiation impairs the flavour of certain foods. Bacillus pumilus
E601 is used to evaluate sterilization process.

Filtration: Filtration does not kill microbes, it separates them out. Membrane filters with pore
sizes between 0.2-0.45 µm are commonly used to remove particles from solutions that can't be
autoclaved. It is used to remove microbes from heat labile liquids such as serum, antibiotic
solutions, sugar solutions, urea solution. Various applications of filtration include removing
bacteria from ingredients of culture media, preparing suspensions of viruses and phages free of
bacteria, measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria,
clarifying fluids and purifying hydatid fluid. Filtration is aided by using either positive or
13
negative pressure using vacuum pumps. The older filters made of earthenware or asbestos are
called depth filters.

Different types of filters are:

1.Earthenware filters

2. Asbestos filters

3. Sintered glass filters

4. Membrane filters

5. Air Filters

Chemical methods of sterilization:

ALCOHOLS:

Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol

Application: A 70% aqueous solution is more effective at killing microbes than absolute
alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to
ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers.
Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable.

ALDEHYDES:

Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably
damages nucleic acids. It kills all microorganisms, including spores.

Examples: Formaldehyde, Gluteraldehyde

Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of
rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc.

14
Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with
potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5%
tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure
of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at
40o C for 20 minutes is used to disinfect wool and 0.25% at 60 o C for six hours to disinfect
animal hair and bristles.

Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration,
leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde
requires alkaline pH and only those articles that are wettable can be sterilized.

PHENOL:

Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of

enzymes.

Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene,

chlorhexidine, chloroxylenol (Dettol)

Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar
derivatives. They act as disinfectants at high concentration and as antiseptics at low
concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against
spores and most viruses. They are not readily inactivated by organic matter. The corrosive
phenolics are used for disinfection of ward floors, in discarding jars in laboratories and
disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin
disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand
wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation
and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary
ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg.
Savlon). Chloroxylenols are less irritant and can be used for topical purposes and are more
effective against gram positive bacteria than gram negative bacteria. Hexachlorophene is
chlorinated diphenyl and is much less irritant. It has marked effect over gram positive bacteria

15
but poor effect over gram negative bacteria, mycobacteria, fungi and viruses. Triclosan is an
organic phenyl ether with good activity against gram positive bacteria and effective to some
extent against many gram negative bacteria including Pseudomonas. It also has fair activity on
fungi and viruses.

Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic

soaps. Chloroxylenol is inactivated by hard water.

HALOGENS:

Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl
groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture
iodine, iodophores)

Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be
combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such
as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic.
For hand washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted
in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household
bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In
higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is
used in serology and virology. Used at a dilution of 1:10 in decontamination of spillage of
infectious material. Mercuric chloride is used as a disinfectant.

Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is
corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

HEAVY METALS:

Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are
bacteriostatic.

16
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g.,
mercurochrome, merthiolate)

Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia
neonatorum (Crede’s method). This procedure is no longer followed. Silver sulphadiazine is used
topically to help to prevent colonization and infection of burn tissues. Mercurials are active
against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used
in preservation of serum. Copper salts are used as a fungicide.

Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

SURFACE ACTIVE AGENTS:

Mode of actions: They have the property of concentrating at interfaces between lipid containing
membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain
hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both
of these, they concentrate on the surface of membranes. They disrupt membrane resulting in
leakage of cell constituents.

Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents
containing negatively charged long chain hydrocarbon are called anionic detergents. These
include soaps and bile salts. If the fat-soluble part is made to have a positive charge by
combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents
are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride
act as cationic detergents.

Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They
are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter.
Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source.

17
DYES:

Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic
acids.

Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes
such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only
euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also
germicidal. It intercalates between base pairs in DNA. They are more effective against gram
positive bacteria than gram negative bacteria and are more bacteriostatic in action.

Applications: They may be used topically as antiseptics to treat mild burns. They are used as
paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain
selective media.

HYDROGEN PEROXIDE:

Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen
peroxide produces hydroxyl-free radical that damages proteins and DNA.

Application: It is used at 6% concentration to decontaminate the instruments, equipments such

as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising
wounds and ulcers. Strong solutions are sporicidal.

Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter

drastically reduces its activity.

ETHYLENE OXIDE (EO):

Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and
hydroxyl- groups. Properties: It is a cyclic molecule, which is a colorless liquid at room
temperature. It has a sweet ethereal odor, readily polymerizes and is flammable.

Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is

highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or
18
dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well
absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles,
rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine,
respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

Reference: Microbiology by Michael Joseph Pelczar. (BOOK) & GOOGLE

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 Sterility
Definition:
Sterility can be defined as the freedom from the presence of viable microorganisms. However,
the conditions that guarantee absolute sterility are usually too harsh for active ingredients, and
the definition of sterility for a medicinal product must be defined in functional terms.

Which are sterile products?

There are many types of sterile products:

1) Injections
2) Non-injectable sterile fluids
3) Ophthalmic preparations
4) Dressings

Injection:
Injections may be aqueous solutions, oily solutions (because of poor aqueous solubility or the
necessity for a prolongation of drug activity), aqueous suspensions or oily suspensions. They
may be aseptically produced or terminally sterilized in their final containers.

a) Formulation philosophy: Many injections are formulated as aqueous solutions, with Water
for Injections as the vehicle. Their formulation depends on several factors including the
aqueous solubility of the active ingredient, the dose, its thermal stability, the route of
administration, and whether the product is to be offered as a multiple-dose product (i.e. with
doses removed on different occasions) or as a single-dose form (as the term suggests, only
one dose per container). Some types of injections must be isotonic with blood serum. This
applies particularly to large-volume intravenous infusions if at all possible; hypotonic
solutions may cause lysis of red blood corpuscles and thus must not be used for this purpose.
b) Intravenous infusions: Intravenous infusions consist of large-volume injections or, drips
(500 ml or more) that are infused at various rates (50–500 ml/h) into the venous system.
They are generally sterilized in an autoclave.
c) Intravenous additives: A common hospital practice is to add drugs to infusions immediately
before administration. Regularly used additives include potassium chloride, lidocaine
(lignocaine), heparin, certain vitamins and antibiotics. Potentially this can be a hazardous

20
 practice. For instance, the drug may precipitate in the infusion fluid because of the pH (e.g.
amphotericin) or the presence of calcium salts (e.g. thiopentone); the drug may degrade
rapidly (e.g. ampicillin in 5% w/v glucose); multiple additions may lead to precipitation of
one or both of the drugs or to accelerated degradation; and finally, drug loss may occur
because of sorption by the container. For instance, insulin is adsorbed by glass or by
polyvinyl chloride (PVC); glyceryl trinitrite and diazepam are absorbed by PVC. Apart from
these problems, if the addition is not carried out under strict aseptic conditions the fluid can
become contaminated with microorganisms during the procedure. Thus any addition should
be made in a laminar-flow workstation or isolator, and the fluid should, ideally, be
administered within 24 hours of preparation.
d) Total parenteral nutrition: Total parenteral nutrition (TPN) is the use of mixtures of amino
acids, vitamins, electrolytes, trace elements and an energy source (glucose and fat) in the
long-term feeding of patients who are unconscious or unable to take food.
e) Small-volume injections: This category includes single-dose injections, usually of 1–2 ml
but as high as 50 ml, dispensed in borosilicate glass ampoules, plastic (polyethylene or
polypropylene) ampoules or, rarely, multiple-dose glass vials of 5–25 ml capacity stoppered
with a rubber closure through which a hypodermic needle can be inserted, e.g. insulins,
vaccines. Preservatives must be added to injections in multiple-dose containers to prevent
contamination during withdrawal of successive doses. However, preservatives may not be
used in injections in which the total volume to be injected at one time exceeds 15 ml. This
may occur if the solubility of a drug is such that a therapeutic dose can only be achieved in
this volume of solvent. There is also an absolute prohibition on the inclusion of preservatives
in intra-arterial, intracardiac, intrathecal or subarachnoid, intracisternal and peridural
injections, and various ophthalmic injections.
f) Small-volume oily injections: Certain small-volume injections are available where the drug
is dissolved in a viscous oil because it is insoluble in water and therefore a non-aqueous
solvent is used. In addition, drugs in non-aqueous solvents provide a depot effect, e.g. for
hormones. The intramuscular route of injection must be used.
g) Freeze-dried products: freeze-drying (lyophilization) consists of preparing the drug
solution (with buffers and cryoprotectants), filtering through a bacteria-proof filter,

21
 dispensing into containers, removing water in a freeze-drier, then capping and closing the
containers. Many biotechnology products are freeze-dried.
Freeze-drying is an aseptic process whereby water is removed from a frozen product mainly
by sublimation, i.e. by the conversion of ice directly into the vapor state without the
intermediary of liquid water. Drugs are reconstituted into solution immediately prior to
injection.

h) Glass containers: Single-dose injections are usually packed in glass ampoules containing 1,
2 or 5 ml of product. To ensure removal of the correct dose volume by syringe and needle, it
is necessary to add an appropriate overage to the ampoule. Thus a 1 ml ampoule will actually
contain 1.1 ml of product and a 2 ml ampoule should contain 2.15 ml of product.

 Non-injectable sterile fluids:

a) Non-injectable water: This is sterile water, not necessarily of injectable water standards,
which is used widely during surgical procedures for wound irrigation, moistening of
tissues, washing of surgeons’ gloves and instruments during use and, when warmed.
b) Urological (bladder) irrigation solutions: These are used for rinsing of the urinary tract
to aid tissue integrity and cleanliness during or after surgery.
c) Peritoneal dialysis and haemodialysis solutions: Peritoneal dialysis solutions are
admitted into the peritoneal cavity as a means of removing accumulated waste or toxic
products following renal failure or poisoning.
d) Inhaler solutions: In cases of severe asthmatic attacks, bronchodilators and steroids for
direct delivery to the lungs may be needed in large doses. This is achieved by direct
inhalation via a nebulizer device; this converts a liquid into a mist or fine spray.

Ophthalmic preparations:

a) Eye drops:  Eye drops are presented for use in (1) sterile single-dose plastic sachets
(often termed Minims) containing 0.3–0.5 ml of liquid, (2) multiple-dose amber fluted
eye dropper bottles including the rubber teat as part of the closed container or supplied
separately, or (3) plastic bottles with integral dropper.

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 b) Eye lotions: Eye lotions are isotonic solutions used for washing or bathing the eyes.
They are sterilized by autoclaving in relatively large-volume containers (100 ml or
greater) of coloured fluted glass with a rubber closure and screw-cap, or packed in plastic
containers with a screw-cap or tear-off seal.
c) Eye ointments: Eye ointments are prepared in a semisolid base—e.g. Simple Eye
Ointment BP, which consists of yellow soft paraffin (8 parts), liquid paraffin and wool
fat. The base is filtered when molten to remove particles and sterilized at 160°C for 2
hours.
d) Wetting solutions: These are used to hydrate the surfaces of hard lenses after
disinfection. As they must also cope with chance contamination, they must contain a
preservative as well as a wetting agent. They may be isotonic with lachrymal secretions
and be formulated to a pH of about 7.2 for compatibility with normal tears.
e) Soaking solutions: These are solutions for disinfection of lenses but also maintain the
lenses in a hydrated state.

Dressings:

Dressings and surgical materials are used widely in medicine, both as a means of protecting and
providing comfort for wounds and for many associated activities such as cleaning and swabbing.
Methods for their sterilization include autoclaving, dry heat, ethylene oxide and ionizing
radiation. Any other effective method may be used.

Reference: https://basicmedicalkey.com/sterile-pharmaceutical-products/

What is sterility test?

Sterility testing is designed to demonstrate the presence or absence of extraneous viable
contaminating microorganisms in biological parenterals designed for human use. The tests are
based upon the principle that if micro-organisms are placed in a medium which provides
nutritive material and water, and kept at a favourable temperature, the organisms will grow and
their presence can be indicated by a turbidity in the originally clear medium.

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Why sterility test is important?
 Sterility testing is required to ensure viable contaminating microorganisms are not
evident in a product.
 Sterility testing of sterile pharmaceuticals is an important part of GMP microbiology, and
is used to ensure that pharmaceutical and biopharmaceutical therapeutics are actually
sterile and safe for human use.

Which methods are followed to perform sterility test?

Methods for sterility test are:
a) Membrane filtration
b) Direct inoculation

How sterility test is performed?

a) Membrane filtration:
The Membrane Filtration Sterility Test is the method of choice for pharmaceutical products.
An appropriate use of this test is for devices that contain a preservative and are bacteriostatic
and fungistatic under the direct transfer method. With membrane filtration, the concept is that
the microorganisms will collect onto the surface of a sub-micron pore size filter. This filter is
segmented and transferred to appropriate media. The test media are fluid thioglycollate
medium (FTM) and soybean casein digest medium (SCDM). FTM is selected based upon its
ability to support the growth of anaerobic and aerobic microorganisms. SCDM is selected
based upon its ability to support a wide range of aerobic bacteria and fungi (i.e., yeasts and
molds). Incubation time is 14 days.

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 Fig 2.

b) Direct Inoculation:

This method is the method of choice for medical devices because the device is in direct
contact with test media throughout the incubation period. Viable microorganisms that may
remain in or on a product after sterilization have an ideal environment within which to grow
and proliferate. This is especially true with damaged microorganisms where the damage is
due to a sub-lethal sterilization process. All microorganisms have biological repair
mechanisms that can take advantage of environmental conditions conducive to growth. The
direct transfer method benefits these damaged microorganisms. The entire product should be
immersed in test fluid. With large devices, patient contact areas should be immersed. Nova
understands that appropriate modifications are required due to the size and shape of test
samples. The method requires that the product be transferred to separate containers of both
FTM and SCDM. The product is aseptically cut, or transferred whole, into the media
containers. After being transferred, the samples are incubated for 14 days.

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 Fig: Direct inoculation for sterility test

Reference: https://www.novatx.com/sterility-testing/

Importance of sterility test:

Sterility testing of sterile pharmaceuticals is an important part of GMP microbiology, and is
used to ensure that pharmaceutical and biopharmaceutical therapeutics are actually sterile and
safe for human use.

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 Pyrogen

Definition:
A Pyrogen is a substance i.e. products of the growth of micro-organisms or may be parts of dead
cells or metabolic products which cause febrile reactions like fever, chills, back pain etc.

Which are Pyrogen products?

GNB endotoxin is a high molecular weight complex that contains lipopolysaccharide (LPS),
protein, and phospholipid originating from the outer membrane of Gram-negative bacteria.

Depyrogenation is one of the most important challenges for pharmaceutical manufactures of

parenteral drugs, since fever in a patient depends on the total amount of pyrogen delivered to that
patient. 

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115822/#:~:text=Although
%20%E2%80%9Cpyrogen%E2%80%9D%20is%20a%20general,risks%20are%20lower
%20%5B1%5D.

What is Pyrogen test?

Protein and polysaccharide substances called pyrogens, released either from bacteria or viruses
or from destroyed cells of the body, are capable of raising the thermostat and causing a rise in
body temperature. Fever is a highly significant indicator of disease. Pyrogen testing determines
the presence or absence of pyrogens in parenteral pharmaceutical products and is regulated by
several standards from organizations such as the Food and Drug Administration (FDA), United
States Pharmacopeia (USP), or European Pharmacopeia (EP). The sterility of a product does not
imply that it is free of pyrogens. Therefore, drugs that are purported to be sterile must also be
tested for pyrogens to prevent febrile reactions in patients.

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Why Pyrogen test is important?
Bacterial endotoxins (pyrogens) are polysaccharides from bacterial membranes. They are water
soluble, heat stable, and filterable. If they are present in a preparation and administered to a
patient they can cause fever and also leukopenia in immunosuppressed patients.

Pyrogen contamination can occur during production or the administration of pharmaceuticals,

biotherapeutics, and medical devices, but the presence of pyrogens can also be an inherent
characteristic of the product, such as adjuvants in vaccines or synthetic lipopeptides.

Which methods are followed to perform Pyrogen test?

1. Rabbit pyrogen testing

2. Monocyte activation test
3. Bacterial Endotoxin testing (LAL test)
4. Recombinant factor C (rFC) testing

How Pyrogen test is performed?

Rabbit pyrogen testing:

Principle:

The test involves the measurement of rise in body temperature of rabbits following IV injection
of sterile solution of substance being examined.

Animals used: Select same variety of healthy mature rabbits weighing less than 1.5Kg and
should maintain balanced diet. They should not show any loss of body weight during the
preceding week of test.

Temperature measurement:

Accurate temperature sensing devices such as Clinical Thermometer graduated in 0.1°C /

Thermister / Probes are used to measure the temperature of rabbit. Insert the thermometer into
the rectum of rabbit to a depth of not less than 6 cm and record the temperature.

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Materials used:
Glass ware and syringes must be washed with water for injection and heated to 250°C for 30
minutes / 200°C for 1 hour in hot air oven.
Procedure:
It includes
1. Preliminary test.
2. Main test.
Preliminary test:

 Select fresh animals / Animals not been used during 2 previous weeks.
 Conditioned them for 1 to 3 days.
 With hold the food from animal before 2hours of starting the test and access to water may be
allowed. Record the temperature of animals using thermometer.
 After 90 min, give IV injection 10mL/Kg (Pyrogen free saline solution)
 Record the temperature of animals after IV injection at an interval of 30min and continued
for 3 hrs after injection.
 Animals show temperature variance of 0.6°C should not be used for main test.

Main test:
Preparation of Sample: Dissolve test substances in Pyrogen free saline water and warm the
liquid to 38°C before giving injection.

 3 groups of selected rabbits (preliminary test passed rabbits)

 With held food for 2 hrs before experiment and during the experiment
 Record initial temperatures (mean of two measurements at an interval of 30 minutes)
 Rabbits showing a temperature variance ≥0.2 °C between two successive readings should
not be used. Use only those rabbits that do not deviate a temperature variance 1°C between
two successive readings.
 Inject sample into the marginal vein of the ear of 3 rabbits Not less than 0.5mL/ Kg and not
greater than 10mL/Kg body wt.
 Record the temperature of animals during a period of 3 hours at intervals of 30 minutes.

Interpretation of Results.
Case: 1

 No rabbit shows individual raise in temperature of 0.6 °C (or) Sum of three rabbits raise in
temperature does not exceed 1.4 °C
 Absence of Pyrogens in the test sample.

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Case: 2

 If 2 or 3 rabbits show increase in temperature ≥0.6 °C or Sum of three rabbits raise in

temperature exceeds 1.4 °C
 Continue or Repeat the experiment using additional 5 rabbits.
 Not more than 3 rabbits shows individual raise in temperature of 0.6 °C (or) Sum of eight
rabbits raise in temperature does not exceed 3.7 °C
 Absence of Pyrogens in the test sample.

Monocyte activation test:

The Monocyte Activation Test (MAT) is an alternative to animal-based methods for the

detection of both endotoxin and non-endotoxin pyrogens. The monocyte activation test mimics
the human immune reaction by incubating monocytes with the tested sample. If pyrogens are
present, monocytes are activated and produce inflammatory molecules, cytokines, responsible
for the febrile reaction. The cytokines are then detected using an immunological assay (ELISA)
involving specific antibodies and an enzymatic color reaction.

Bacterial Endotoxin testing (LAL test):

In Vitro assay used to detect the presence and concentration of bacterial endotoxins in drugs and
biological products. Limulus Amoebocyte Lysate (LAL) is an aqueous extract of blood cells
(amoebocytes) from the horseshoe crab, Limulus polyphemus.

LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane

component of Gram-negative bacteria and forms gel which is then used for the detection and
quantification of bacterial endotoxins.

Limitations of LAL Test:

1. Disturbed by endotoxin binding components like lipids, blood components etc.

2. Difficult to correlate with rabbit test.

3. False positive for cellulose and many herbal preparations.

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Recombinant factor C (rFC) testing:

The recombinant Factor C is a genetically engineered protein normally found in the Limulus
amebocyte lysate cascade. In this test, Factor C reacts with endotoxin and couples with a marker
to produce a quantifiable, fluorescent end product. The recombinant Factor C test uses the same
principle as the LAL test, but without the need for animal-derived material.

Reference: https://www.sigmaaldrich.com/BD/en/applications/microbiological-testing/pyrogen-
testing

Importance of Pyrogen test:

Pyrogen detection tests are becoming increasingly important for the analysis of fever-inducing
substances in parenteral drugs.

Testing for pyrogens is a critical step in ensuring parenteral pharmaceutical product and medical
device safety. It is part of the mandatory release tests to avoid life-threatening fever reactions
induced by pyrogenic substances.

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