King Saud University

College of Pharmacy
Department of Pharmaceutics
PHT 351 Sterile Dosage Forms
Summer Semester of 1423-1424H

PRINCIPLES AND METHODS

OF STERILIZATION

Ibrahim A. Alsarra, Ph.D.

1
Methods of Sterilization

2
 Outlines
u Physical Sterilization
1- Heat
2- Filtration
3- Radiation

u Gas Sterilization

u Sterility Testing and Pyrogen Testing

3
Introduction

USP described five sterilization processes:

Steam (moist) Sterilization
Dry Heat Sterilization
Filtration Sterilization
Gas Sterilization
Ionization Sterilization

4
 Methods of Sterilization

Moist Heat Dry Heat Chemical (cold) Radiation Filtration

Ethylene oxide
Saturated heat autoclaves Batch sterilizers
Vaporized hydrogen peroxide
Superheated water autoclaves Continuous tunnel sterilizers Electromagnetic particulate Membranes
Hydrogen peroxide/steam
Air oven steam autoclaves
Other gases

5
Introduction … cont.

The sterilization processes are commonly used for parenteral products,

except gas and ionizing radiation, which are widely used for devices
and surgical materials.

To assist in the selection of the appropriate sterilization method, certain

basic information must be considered:
1- The nature and amount of product
2- Whether the product and container-closure system will have a
predominately moist or dry environment during sterilization.

6
Introduction … cont.

For sterilization purposes, microorganisms can be categorized into three

general categories:
1- Easy to kill with either dry or moist heat
2- Susceptible to moist heat, but resistant to dry heat (Bacillus subtilis)
3- Resistant to moist heat, but susceptible to dry heat
(Colstridium sporogenes)
Sterilization tests are performed to verify that an adequate sterilization
process has been carried out. Validation of sterilization cycle also gives
assurance of process (including the performance of the equipment and
personnel)
7
 General Facts

The procedure to be used for sterilizing a drug, a pharmaceutical

product, or a medical device is determined to a large extent by the
nature of the product. It is important to remember that the same
sterilization technique cannot be applied universally because the
unique properties of some materials may result in their destruction
or modification.

Methods of inactivating microorganisms may be classified as

either physical or chemical. Physical methods include: moist heat,
dry heat and irradiation. Sterile filtration is another process, but it
only removes, not inactivates, microorganisms, chemical methods
include the use of either gaseous or liquid sterilants
8
 General Facts… (cont.)

Heat stable reusable medical devices that enter the blood stream or
enter normally sterile tissue should always be reprocessed using heat-
based methods of sterilization (e.g., steam autoclave or dry heat oven).

Laparoscopic or arthroscopic telescopes (optic portions of the endoscopic

set) should be subjected to a sterilization procedure before each use; if
this is not feasible, they should receive high-level disinfection. Heat
stable accessories to the endoscopic set (e.g., operative
instruments) should be sterilized by heat-based methods (e.g., steam
autoclave or dry heat oven).

9
 General Facts … (cont.)

Reusable devices or items that touch mucous membranes should, at a

minimum, receive high-level disinfection between patients. These devices
include reusable flexible endoscopes, anesthesia breathing circuits, and
respiratory therapy equipment.

Medical devices that require sterilization or disinfection must be thoroughly

cleaned to reduce organic material or bioburden before being exposed to the
germicide, and the germicide and the device manufacturer's instructions
should be closely followed.

10
 General Facts … (cont.)

Except on rare and special instances (as mentioned previously), items that do
not ordinarily touch the patient or touch only intact skin are not involved in
disease transmission, and generally do not necessitate disinfection between
uses on different patients. These items include crutches, bed boards, blood
pressure cuffs, and a variety of other medical accessories. Consequently,
depending on the particular piece of equipment or item, washing with a
detergent or using a low-level disinfectant may be sufficient when
decontamination is needed.

11
STEAM STERILIZATION

12
Terminology

• Bioburden- The degree of contamination with microorganisms and

organic parameters
• Bioresistance- Factors such as heat and/or moisture sensitivities and
product stability
• Biostate- The nutritional, physical, and reproductive phase of
microorganisms
• Bioshielding- Characteristics of the packaging materials
• Bowie Dick test (DART)- A test run daily that validates the vacuum
function of the sterilizer. The test should be run in an empty load at
the same time every day
• Contaminate- To render unfit for use through introduction of a
substance which is harmful or injurious

13
Terminology … cont.

• Contamination- A state of being soiled or infected by contact with

infectious materials
• Culture- A growth of microorganisms on a medium
• Culture Medium- Any substance or preparation used for the growth
and cultivation of microorganisms
• Incubate- To maintain under optimum environmental conditions that
are favorable for growth
• Incubation period- The period between the time infection occurs and
the appearance of the first symptoms
• Incubator- an Apparatus for maintaining a constant and suitable
temperatures for the growth and cultivation of microorganisms
• Nonpathogenic- Not capable of producing disease
• Pathogenic- Capable of producing disease 14
Terminology … cont.

u TDT is the time required to kill a known population of microorganisms

a specific suspension at a particular temperature

u Increasing temperature decreases TDT

u Lowering the temperature increases TDT

u Acidic or basic pHs decrease TDT

u Fats and oils slow penetration and increase TDT

15
Terminology … cont.

• Spore- Certain microorganisms which are capable of forming a thick

wall around themselves enabling them to survive in adverse
conditions. It is highly resistant to heat
• Sterile- Completely devoid of all living microorganisms
• Thermal equilibrium- A condition where all parts of a system have
reached the same temperature; in a steam autoclave, when the
temperature throughout the entire load is the same

16
Action (mechanism) of Steam

u Steam contacts a cooler surface, condenses,

causing a huge decrease in volume and
setting up a negative pressure that draws
more steam
u Condensation occurs as long as there is a
temperature differential
u Action of steam ensures: Surface heating,
penetration, and protein coagulation

17
Advantages of Steam Sterilization

• Stainless steel instruments withstand repeated processing without

damage and no residue
• Steam kills at lower temperatures than dry heat
• Easiest, safest, and surest method of on-site sterilization
• Fastest method, total cycle time is shortest
• Least expensive and most easily supplied agent-piped in from the
facility’s boiler room
• Most sterilizers have automatic controls and recording devices to
eliminate human error from the process

18
Disadvantages of Steam Sterilization

• Preparing, packaging, loading and operating the sterilizer. Also, drying

time
• Instruments need to be free from grease and oil and not be sensitive
with heat
• All items must have direct contact with steam and must be able to
penetrate packaging material
• Timing is adjusted for the differences in load and material and subject to
human error
• The steam may not be pure. Refers to the amount of solid, vapor, or
liquid contamination in the steam

19
Autoclave

Autoclave:
An apparatus for
sterilization by steam under
pressure, usually at
temperatures of 250 degrees
to 270 degrees F

20
Principles

Two important factors should be controlled:

1- Application of pressure:
Because it is not possible to raise the temperature of the steam above
100 °C under atmospheric conditions, pressure is employed to achieve
higher temperature (it should be recognized that the temperature, not the
pressure is destructive to the microorganism and that the application of
pressure solely for the purpose of increasing the temperature of the
system.

21
Principles … cont.

2- Application of time:
Time is another important factor in the destruction of microorganisms by
heat. Most modern autoclaves have gauges to indicate to the operator
the internal conditions of temperature and pressure and timing device to
permit the desired exposure time for the load. The usual conditions
(pressure/temperature/time), are as follows:

Pressure Temperature Time

10 pounds 115.5 °C 30 minutes
15 pounds 121.5 °C 20 minutes
20 Pounds 126.5 °C 15 minutes
22
Standard Temperature and Pressure

250 °F (121 °C) and 15 p.s.i.

23
How Long to Autoclave

Dry goods @ 121 °C Time (min)

u Glassware, empty, inverted 15

u Instruments, wrapped 30

u Utensils, wrapped 30
24
How Long to Autoclave … cont.

Biohazardous waste bags, @ 121 °C, loosely

tied
Time (min)
– 2 or more bags 100 +
– 1 bag, full 90
– 1 bag, half full 60

Testing available for individual situations

25
How Long to Autoclave … cont.

Liquids, in bottles with vented caps

Size (ml) Time (minutes)
75 25
250 30
500 40
1000 45
1500 50
2000 55
26
Autoclave applicable:

A. Applicable for pharmaceuticals preparations and materials that can

withstand the required temperature and are penetrated but not adversely
affected by, moisture.
B. In sterilizing aqueous solutions, the moisture is already present, and all
that is required is the elevation of the temperature of the solution for the
prescribed period of time. Thus solutions packaged in sealed container as
ampoules, are readily sterilized by this method.
C. Also applicable to bulk solutions, glassware, surgical dressings, and
instruments.

27
Autoclave not applicable:

A. The sterilization of oils, fats, or any oleaginous preparations.

B. Other preparations not penetrated by the moisture.
C. Sterilization of exposed powders that may be damaged by
condensed moisture.

28
Types of steam sterilizers

1. Gravity displacement

Definition: Gravity pushes air through

the packages and down through the
drain. Sterilization begins when steam
passes the thermometer and reaches
the desired temperature.

29
Gravity Displacement Uses

1. Metalware
2. Glassware
3. Thermoplastics
4. Linens
5. Rubber

30
Types of steam sterilizers … cont.

2. Pre-vacuum (high temperature)

Sterilizer

Definition: Air is completely evacuated

from the chamber by a vacuum. The
steam-injector helps eliminate the air
out of packages. Steam then penetrates
the packages on all surfaces.

31
Pre-vacuum Sterilizer Uses:

Metalware
Rubber
Thermoplastics

32
Types of steam sterilizers … cont.

3. High Pressure (flash) Sterilizer

Definition: Another sterilizer that can be

used in either gravity displacement or
pre-vacuum. Gravity displacement is the
most common used. Steam in the jacket
should be maintained at all times.

33
High Pressure Flash Uses

Urgent items: (dropped items for which

no method of sterilization exists) Items
that are dropped, or forgotten

34
Types of steam sterilizers … cont.

4. Washer-sterilizer

Definition: Wash and sterilize

instruments. The water floods the
chamber with cold water to prevent
proteins from setting on the instrument.
The water is then heated with steam to
agitate and clean the instruments.

35
Washer-sterilizer Uses:

to wash and terminally sterilize items

which are non-heat sensitive,
immediately after operative procedures.

NO GLASS, SHARPS OR DELICATE

INSTRUMENTS GO THROUGH THIS
STERILIZER. BREAKAGE COULD OCCUR!!!

36
Biological Indicator Controls/
Spore Tests
Bacillus stearothermophilus: is used strictly as biological
indicator of effective heat sterilization (steam and dry heat
sterilizers) by including filter paper strips carrying
a saturated number of spores into the autoclave cycle.
The strips are then incubated to attempt to recover viable
organism. The usual autoclave cycle of 121 °C for
15 minutes is adequate to kill Bacillus stearothermophilus
with a high margin of safety.
37
Biological Indicator Control/
Spore Tests…cont.
When Used:
Upon installation of new system
After major repairs
Routine quality assurance.
With all implants

38
Biological Indicator Controls/
Spore Tests
• Record results:
– Negative- no color change from original.

– Positive- color changed is usually amber in

color.
– Take sterilizer out of service
– Report to supervisor
– Recall all items sterilized in sterilizer
for last 24 hour period

39
STERILIZATION by FILTRATION

40
Filtration
- It depends upon the physical removal
of microorganisms by adsorption on
the filter medium or by sieving
mechanisms.
- It is used for sterilization of heat-
sensitive solutions.
- Medicinal preparations sterilized by
this method are required to undergo
severe validation and monitoring since
the effectiveness of the filtered
product can be greatly influenced by
the microbial load in the solution being
filtered.
- It removes, but does not destroy
M.O.
- Commercially available filters are
produced with a variety of pore-size
specifications (e.g. Millipore filters). 41
Filtration …cont.
• Most filters consist of:
1- diatomaceous earth
2- cellulose acetate or
3- nitrocellulose

• 0.45 µm filters removes most

bacteria

• 0.20 µm more inclusive,

viruses need 0.01 µm

• Useful for sterilizing liquids

(enzymes, vaccines) that are
destroyed by heat

42
Filtration …cont.
• Factors affecting removal of M.O.:
1- Pore size of filter
2- Electrical charge of the filter
and that of the M.O.
3- pH of the solution
4- T, P, and Vacuum applied

• Advantages:
1- Speed
2- Good for thermolabile
materials
3- Inexpensive
4- The complete removal of living
and dead M.O. as well as
other particulate matter from
43
the solution
Filtration …cont.
• Disadvantages:

1- The membrane is fragile and

because of that, it is essential to
determine that the assembly was
properly made and the membrane was
not ruptured

2- Filtration of large volumes of liquids

would require more time (particularly
if the liquids were viscous)

3- Useful when heat cannot be used

and small volumes of liquids.

44
OTHER TECHNIQUES

45
Ultrasonic vibrations

– High frequency sound waves

– Know as Sonicator, untrasonic machines
– Killing by –shock waves that disintegrates cell
wall
and membranes. 46
PYROGENS AND PYROGEN TESTING

47
PYROGEN

- A pyrogen is a material which when injected into

a patient will cause a rise in body temperature
(pyrexia).
- The lipopolysacchride that comprise a a major
part of the cell wall of gram-negative bacteria are
called endotoxins, and it is these that are the
most commonly encountered pyrogens.
- Bacterial cells may be pyrogenic even when they
are dead and when they are fragmented, and so a
solution or material that passes a test for sterility
will not necessarily pass a pyrogen test.

48
PYROGEN… cont.
Two main procedures are used for the detection of pyrogens.

A. The traditional method:

B. It requires the administration of the sample to laboratory rabbits
whose
C. body temperature is monitored for a period of time thereafter.

49
PYROGEN… cont.

Steps:
1- Render the syringes, needles, and glasswares free form pyrogens by
heating at 250 °C for not less than 30 minutes.
2- Warm the product to be tested to 37 °C ± 2 °C
3- Inject into an ear vein of each of three rabbits 10 ml of the product per
kg of body weight.
4- Record the temperature at 30-minute intervals between 1 and 3 hours
subsequent to the injection
50
PYROGEN… cont.

Steps:
5- If no rabbit shows an individual rise in temperature of 0.5 °C or more
above its respective control temperature, the product meets the
requirements for the absence of pyrogens.
6- If any rabbit shows an individual temperature rise of 0.5 °C or more,
continue the test using five other rabbits.

51
PYROGEN… cont.

Steps:
7- If no more than three of the eight rabbits show individual rise in
temperature of 0.5 °C or more and if the sum of the eight individual
maximum temperatures rises does not exceed 3.3°, the material under
examination meets the requirements for the absence of pyrogens.

52
PYROGEN… cont.

B. Limulus Amoebicyte Lyste Test (LAL)

In recent years, it has been shown that an extract from the blood cells of
the horseshoe crab (Limulus polyphemus) contains an enzyme and
protein system that coagulates in the presence of low levels of
lipopolysaccharides.

The discovery has led to the development of the Limulus Amoebicyte

Lyste Test (LAL) for the presence of bacterial endotoxins.

53
PYROGEN… cont.

B. Limulus Amoebicyte Lyste Test (LAL) … cont.

Proclotting enzyme

Endotoxin

Activated clotting enzyme

Coagulation Clottable protein GEL

The Lysate Clotting Mechanism 54
PYROGEN… cont.
B. Limulus Amoebicyte Lyste Test (LAL) … cont.

In the test procedure, the lysate is mixed with equal volume of the test
solution in a depyrogenated container, such as a glass tube. The tube is
then incubated undisturbed at 37 °C for a period of about 60 minutes.
The test is a pass or fail test. The end point is identified by gently inverting
the glass tube. A positive result is indicated by the formation of a solid
clot. The clot doesn’t disintegrate when the tube is inverted. A negative
result is indicated if no gel clot has been formed.

55