Pharmaceutical Sterilization

Alaaldin M. Alkilany, PhD

Professor of Pharmaceutics and Nanotechnology

College of Pharmacy
Qatar University 1
Sterilization Methods
• A sterilization process should represent a compromise between
achieving target SAL and maintaining product stability.
• Validation based on the intended application and continuous
monitoring of a sterilization process is always required.
• Sterilization is not an alternative to GMP; it is only the final stage in
a program of microbiological control.
• 5 methods of sterilization are recognized in Eur Ph:
1. Steam sterilization (heating in an autoclave)
2. Dry heat
3. Gas sterilization
4. Ionizing radiation
5. Filtration
Heat Sterilization
• Heat is the most reliable and widely used means of sterilization
• Heat sterilization is limited to thermostable products.
• For moisture-resistant products: moist heat (121-134 °C) sterilization
is used
• For moisture-sensitive products: dry heat (160-180 °C) sterilization is
used.

https://www.youtube.com/watch?v=0dlDf8HiiQw
https://www.youtube.com/watch?v=UbzIcjA72xs&t=2s
Heat Sterilization Processes
• Heat sterilization cycle contains a heating-up stage, a holding stage and a cooling
stage.
• Holding stage is the main stage for killing. However, during both the heating-up and
cooling-down stages, the product is held at an elevated temperature and these
stages contribute to the overall biocidal potential of the process.

The most commonly employed standard temperature/time cycles for bottled fluids and
porous loads (e.g. surgical dressings) are 121 °C for 15 minutes and 134°C for 3 minutes,
respectively.
Moist Heat Sterilization
Applications:
• culture media
• containers and closures
• aqueous injections
• ophthalmic preparations
• irrigation fluids
• surgical and diagnostic equipment
• dressings, sheets
• processing (decontamination) of soiled
and contaminated items
Moist Heat Sterilization
Pressure-temperature relationships and antimicrobial efficacies of alternative steam
sterilization cycles
Tem Holding time (min) steam pressure (psi) IF
115 30 10 2.5
121 15 15 10 bottled fluids
126 10 20 21
134 3 30 40 surgical dressings
 Dry Heat Sterilization
• The instrument used is a hot air oven with perforated
shelves to allow for heat & air flow
• Higher temperatures in the range 160-180 °C are used
at normal air pressure
• Its application is generally restricted to glassware and
metal surgical instruments, non-aqueous
thermostable liquids and thermostable powders
• The major industrial application is in the sterilization
of glass bottles which are to be filled aseptically, and
here there is another advantage which is destroying
bacterial endotoxins (pyrogens) which are difficult to
be eliminated by other means.
• For Depyrogenation of glass, temperatures of
approximately 250 °C are used.
 Gaseous Sterilization
• Performed using the chemically reactive gases
(e.g.ethylene oxide and formaldehyde), which possess
broad-spectrum biocidal activity.
• Application: sterilization of re-usable surgical
instruments, certain medical, diagnostic and electrical
equipment, and the surface sterilization of powders.
Ethylene oxide treatment is also considered as an
alternative to radiation sterilization in the commercial
production of disposable medical devices.
(plastics, optics and electrics)!
• These techniques do not offer the same degree of
sterility assurance as heat methods and are reserved
for temperature-sensitive items.
 Gaseous Sterilization
• Disadvantages:
- lengthy process
- Need to remove toxic residues
- Gases are potentially mutagenic and carcinogenic (They
produce symptoms of acute toxicity including irritation
of the skin, conjunctiva and nasal mucosa;
consequently, strict control of their atmospheric
concentrations is necessary and safe working protocols
are required to protect personnel)
- Can be hazardous and explosive!
 Ethylene Oxide
• Commonly used (more than formaldehyde).
• Good penetrating powers (diffuses into many packaging
materials including rubber, plastics, fabric and paper)
• Need humidity to kill: Organisms are more resistant to
ethylene oxide in a dried state, like those protected from the
gas by inclusion in crystalline or dried organic deposits. Thus, a
further condition to be satisfied in ethylene oxide sterilization
is attainment of a minimum level of moisture in the
immediate product environment. This requires a sterilizer
humidity of 30-70% and frequently a pre-conditioning of the
load at relative humidity levels of greater than 50%.
QA in Gas Sterilization

https://sterility.com/product/an1087-eogas-dosimeter/

https://www.youtube.com/watch?v=M3ZICe3Vwj8
Let us watch this:
https://www.youtube.com/watch?v=wNlKflMx8xM
Ethylene Oxide
• Disadvantages:
- Highly explosive (in order to reduce this explosion
hazard it is usually supplied for sterilization purposes
as a 10% mix with carbon dioxide, or as an 8.6%
mixture with HFC 124)
- Cannot be detected by smell at low concentrations
- The level of ethylene oxide in a sterilizer decreases
due to absorption during the process
- Desorption phase is required: The treated articles
must undergo a desorption stage to remove toxic
residues (may take many days in open shelves).
Alternatively, forced aeration cabinets can be used
Ethylene Oxide
 Gaseous Sterilization - Formaldehyde
• Formaldehyde has a similar toxicity to
ethylene oxide
• A major disadvantage of formaldehyde is
low penetrating power and this limits the
packaging materials that can be employed
to paper and cotton fabric.
• Formaldehyde gas for use in sterilization is
produced by heating formalin (37% w/v
aqueous solution of formaldehyde) to a
temperature of 70-75°C with steam, leading
to the process known as LTSF (low-temp
steam and formaldehyde sterilization).
 Advantages and disadvantages and
comparison with EtO
Advantages of formaldehyde steam sterilization

1. Very reactive molecule

2. Faster cycle time compared to EtO
3. Cost per cycle is lower than EtO
4. After sterilization most loads are available for immediate use

Disadvantages of formaldehyde gas sterilization

1. The vapour is extremely irritating to the eyes
2. Weak penetrating power compared to EtO
3. Operates on a higher temperature than EtO
4. A relative humidity of ~ 75% is required in order to be effective
as the gas has to dissolve in a film of moisture surrounding the
bacteria
Radiation Sterilization
• Includes:
- Accelerated electrons (electron beams)
- Gamma-rays
- Ultraviolet (UV) light
• Mechanism of action: microbial DNA damage
• UV irradiation is not as efficient as electron beams or gamma-rays.
• In ionizing radiations (gamma-ray and accelerated electrons), activity
increases with the presence of moisture or dissolved oxygen (as a result of
increased free radical production) and also with elevated temperatures.

Gamma-rays UV
e-beams
Radiation Sterilization
Radiation Sterilization - Ionizing Radiation

• Sterilization of heat-sensitive items such as syringes,

needles, cannulas, IV sets, surgical instruments,
sutures, prostheses, unit-dose ointments.
• Cation: certain glass or plastic (e.g. polypropylene,
PTFE) materials used for packaging or for medical
devices can suffer damage upon irradiation.
• Gamma radiation is the most common method
(Electron beams are less penetrating than gamma-
rays)

https://www.youtube.com/watch?v=Tv4KreXY_RU
Radiation Sterilization – UV Light
• Has much lower energy & causes
less damage to microbial DNA.
• Does not penetrate metal at all,
nor glass to any useful degree
This, coupled with its poor
penetrability of normal packaging
materials (can penetrate only some
polymers), renders UV light unsuitable
for sterilization of pharmaceutical
dosage forms.
• Applications: air and surface
sterilization of aseptic work areas
(microbiological safety cabinets),
and for the treatment of
manufacturing-grade water.
 Filtration Sterilization
• The process of filtration is unique amongst
sterilization techniques in that
- it removes, rather than destroys, microorganisms
- It eliminates both viable and non-viable particles
thus it can be used for both the clarification and
sterilization of liquids and gases
• Applications :
- Sterilization of heat-sensitive injections and
ophthalmic solutions and biological products
- Treatment of air and other gases for supply to
aseptic areas
- They may act as part of venting systems on
industrial machines such as fermenters,
centrifuges, autoclaves and freeze-dryers
Note: Membrane filters are used in sterility testing
where they can be employed to trap and concentrate
contaminating organisms from solutions under test.
These filters are then placed on the surface of a solid
nutrient medium and incubated to encourage colony
development
Filtration Sterilization
• The major mechanisms of
filtration are sieving,
adsorption and trapping
Cellulose acetate filters
within the matrix of the filter
material.
• Synthetic membrane filters
(derived from cellulose esters
or other polymeric materials),
Fibrous pads, sintered glass Sintered glass filters
and sintered ceramic products
can be used.

Ceramic filters
Filtration Sterilization - Liquids
• Membrane filters of (0.2-0.22 µm)
nominal pore diameter are most
commonly used.
• Two filters of 0.2 mm pore diameter
from different manufacturers will not
behave similarly, because, in addition to
the sieving effect, trapping, adsorption
and charge effects all contribute
significantly towards the removal of
particles.
• Consequently, the depth of the
membrane, its charge and the
tortuosity of the channels are all factors
which can make the performance of
one filter superior to that of another.
Filtration Sterilization - Liquids
• Membrane filters, in the form of discs, can be assembled into
- Pressure-operated filter holders for syringe mounting and in-line use
or
- Vacuum filtration tower devices
• Membrane filters are often used in combination with a coarse-
grade fiberglass depth pre-filter to improve their dirt-handling
capacity.
Filtration Sterilization - Gases
• The principal application for filtration sterilization of gases
is in the provision of sterile air to
ØAseptic manufacturing sites
ØHospital isolation units
ØSterilization of venting air in tissue and microbiological culture
ØDecontamination of air in mechanical ventilators
ØTreatment of exhausted air from microbiological safety cabinets
ØThe clarification and sterilization of medical gases

HEPA
filter can remove 99.97% min of particles
greater than 0.3 mm
 New Sterilization Technologies
• Low Temperature Plasma
ØPlasma is a gas or vapour that has been subjected to
electrical or magnetic field which causes a substantial
proportions of the molecules to become ionized.
ØThus it is a cloud of neutral species, free radicals, ions &
electrons in which the positive & negatively charged
particles are equal.
ØTwo types: low pressure and atmospheric pressure.
ØPlasma may be generated from many substances: among Cold plasma
the established methods, chlorine and hydrogen peroxide
plasmas are used which possess excellent antimicrobial
activity.
ØApplication: on most items sterilized by ethylene oxide,
i.e. medical devices but not drugs.
It is not used for powders, liquids & certain fabrics
ØAdvantages: does not need elimination of toxic gases at
the end of cycle unlike LTSF & ethylene oxide & there is no
significant corrosion or reduction of sharpness of exposed
surgical instruments.
https://www.youtube.com/watch?v=HPcsTav95nc
 New Sterilization Technologies
• high intensity light!
• unsuitable for protein or nucleic acid-containing
biotechnology products
ØIt is based on the generation of short flashes of broad
wavelength light from xenon lamp that has an intensity of
100,000 that of the sun, almost 25% of the flash is UV
ØApplication: sterilization of water & terminal sterilization of
injectables in UV transmitting plastic ampoules (e.g.
polyethylene).
ØBut not useful for coloured solutions or those with solutes
that has high UV absorbance
Sterilization Control & Sterility Assurance
• Currently, awareness of the limitation of sterility
testing in terms of their ability to detect low
microbial count resulted in relying on satisfactory
quality standards/practices during the whole
manufacturing process.
• i.e. the quality is assured by process monitoring &
performance criteria, which are considered under
four subjects:
1. Bioburden determination (API, excipients and
packaging)
2. Environmental monitoring (manufacturing area and
regular check!)
3. Validation & in process monitoring of sterilization
procedures (chemical and biological indicators)
4. Sterility test
1. Bioburden Determination
• It is important to have a low pre-sterilization
bioburden by:
• having high quality of raw materials,
• the environment does not encourage microbial growth
of the m.o already present in raw material,
• low microbial contamination during manufacture.
• Therefore, manufacturing process may utilize
adverse temp, extreme pH, organic solvent in order
to prevent increase in the microbial load.
2. Environmental Monitoring
• The level of microbial contamination in manufacturing
areas is monitored regularly so as not to exceed certain
levels.
• M.O. in atmosphere are monitored by settle plates &
air sampler.
• Contamination on surfaces or manufacturing
equipment is measured by swabs or contact plates
(Rodac-replicate organism detection & counting-plates)
which are petri dishes overfilled with agar media
• Operators in manufacturing areas are monitored.
samples from clothes (face mask, gloves & fingerprint)
are taken

https://www.youtube.com/watch?v=KdndbLKHxmw
3. Validation & In-Process Monitoring of
Sterilization Procedures

• Validation: demonstrating that a process will

consistently produce the results that it is intended to.
• Validation of steam sterilizer include various elements
such as:
• Calibration of all physical instruments used to monitor the
process (thermocouples, pressure gauge, timers..etc)
• Use of process efficiency indicators (alone or with bioburden
m.o.)
• Show repeatability of the process (at least repeat 3 times)
• Documentation for all the previous steps
• Usually records of temp, pressure, time, humidity are kept for
each batch of sterilized product
 3. Validation & In-Process Monitoring of
Sterilization Procedures
• Biological indicators:
• Used for thermal, chemical or radiation sterilization
• they consist of standardized bacterial spore preparations
which are either suspension in water or culture medium
or spores dried on paper, Al or plastic carrier.
• After sterilization, the spores are aseptically transferred
to nutrient medium, incubated & periodically examined
for signs of growth.
• Selection of BI:
Ø Must be non-pathogenic
Ø Should possess above-average resistance
to the particular sterilization
4. Sterility Test
• A test which assesses whether a sterile
pharmaceutical or medical product is free from
contaminating m.o. or not.
• To be sure that no organism is present; a universal
growth medium that supports the growth of all
types of m.o. should be used, which is not available.
Practically, media capable of supporting non
fastidious bacteria, yeast & mould are used
• Pharmacopeial tests do not look for viruses, which
can pass thru sterilization filters.
4. Sterility Test
• Disadvantages of Sterility Test:
• Destructive test
• Questionable suitability for testing large
expensive or delicate products
• It is a statistical process where part of a batch is
randomly sampled & batch is released based on
this sample
• The procedure intends to demonstrate a
negative! i.e. failure to detect m.o. could be a
consequence of the use of unsuitable media or
inappropriate cultural conditions.
Methods for Sterility Testing
A. Direct inoculation method: By introducing test sample directly
into nutrient media.
B. Filtration method:
• Recommended by pharmacopeias
• It involves the filtration of fluids thru sterile membrane filter (pore size
0.45 mm); the m.o. present will be retained on the surface of the filter, the
filter is then washed & divided aseptically into two portions which are
transferred to suitable cultural media.

C. Addition of concentrated culture medium : The concentrated

culture medium is added to the pharmaceutical fluid (e.g. IV
infusion) in its original container, so that the resulting mix is
equivalent to a single strength culture medium.
https://www.sigmaaldrich.com/QA/en/applications/microbiological-
testing/sterility-testing
Methods for Sterility Testing
• Negative controls should be done by testing samples
that are known to be sterile (e.g. samples subjected to
reliable process like radiation or to several cycles of
sterilization).
• positive control should be used. In all cases the media
employed should have been assessed for nutritive
(growth supporting) properties & a lack of toxicity using
specified m.o.
• The test should be done under strict aseptic conditions
to avoid accidental contamination.
• Sterility test provides no guarantee to the sterility of the
batch, it is an additional check & gives confidence to the
efficacy of a sterilization or aseptic process.
Sterile Manufacturing: Essential
requirements to minimize contamination
• Separated from other units
• Special construction
requirement (material and
design to prevent
contamination and allow
ease of cleaning and
disinfection and
maintenance).
• Entry through airlocks (for
both people/ goods)
• Clean rooms (A, B, C and D)
(HEPA filters)
• cGMP and adherence to
regulatory guidelines
https://www.youtube.com/watch?v=jwi1GElNa7M
Regulatory guidelines
 Sterile Manufacturing unit: Essential
requirements to minimize contamination
• HEPA filters on the ceiling
• Exhaust vents on the floor
• Laminar air flow
 Sterile Manufacturing unit: Essential
requirements to minimize contamination
• Seamless and rounded floor to wall junctions
• Readily accessible corners Floors, walls and ceilings constructed of
smooth hard surfaces that can be easily cleaned
• Limited equipment, personnel
• Layout of equipment to optimize movement of operators
• Airlock and interlocking doors to control air balance and avoid
contamination
Airlock systems
• Goal: Preventing the classified area from the
contamination that may occur during the entry and
exit of personnel and material
• Minimize contamination of dust and particulates
• The airlock consists of a relatively small chamber
with two airtight doors, which do not open
simultaneously (interlock system with alarm if both
are open)
• Doors open to higher pressure side
• High air change (min 20 changes per hour)
 Types of airlock

15 Pa 25 Pa 30 Pa 15 Pa 30 Pa 15 Pa
+ ++ +++ + +++ +

Cascade airlock bubble airlock

30 Pa 15 Pa 30 Pa
+++ + +++

sink airlock
More essentials…
Air showers

Gowning rooms
Cleanroom zoning

A:
aseptic preparation and filling, for B:
operations that affords high risk, the background environment for
e.g. filling, closing, ampoule and the Grade A zone
bottle opening zones

C and D:
clean zones for less responsible
stages of manufacturing sterilized
products (carrying out activities
during which the product is not
directly exposed or operations in a
closed system).
 Cleanroom specs

• Classification is based on the “air quality” in accordance to required

characteristics to minimize the risks of particulate or microbial contamination of
the product or materials being handled
• Note that there are different standards for air classification and different terms
for the same air quality by different agencies (the above is one example)
Types of sterile manufacturing
• Sterile manufacturing with terminal sterilization
• Aseptic manufacturing
vProduction in a controlled environment in order to
prevent any microbiological contamination
vMaterials used in the process are previously
sterilized and introduced in the manufacturing area
as sterile
vThis approach is designed to avoid any
contamination during the production and filling
processes for products which cannot be produced
by terminal sterilization (sterilized at the end of
production process using available sterilization
techniques due to instability)
Types of sterile manufacturing
References and further readings
• Hugo and Russell's Pharmaceutical Microbiology, 8th Edition. ISBN: 978-1-444-33063-2
• Allen LV, Popovich NG, Ansel HC (Editors). Liquid dosage forms in Ansel's Pharmaceutical
Dosage Forms and Drug Delivery Systems. 9 th Ed. Baltimore: Lippincott Williams & Wilkins;
2011. . http://0- pharmacy.lwwhealthlibrary.com.mylibrary.qu.edu.qa/book.aspx?bookid=2266
• Aulton, M.E. Pharmaceutics- Solutions in The Science of Dosage Form Design. 4th Edition.
Churchill Livingstone, 2013.
• Allen LV, Jr. Remington: the science & practice of pharmacy, 22nd ed. Baltimore: Pharmaceutical
Press; 2012.
• Thompson JE, Davidow L (Editors). A Practical Guide to Contemporary Pharmacy Practice. 3rd Ed.
Baltimore: Lippincott Williams & Wilkins; 2009
• Bouman-Boer Y, Fenton-May V, Le Brun P. Practical Pharmaceutics. Springer, 2009.
• Marriott JF, Wilson KA, Langley CA, Belcher A. Pharmaceutical compounding and dispensing.,
Pharmaceutical press, 2010.
• https://www.fda.gov/media/71026/download
• https://www.fda.gov/media/71461/download
• https://www.fda.gov/files/Guidance-for-Industry-for-the-Submission-Documentation-for-
Sterilization-Process-Validation-in-Applications-for-Human-and-Veterinary-Drug-Products.pdf
• https://www.fda.gov/media/75174/download
• https://www.fda.gov/media/81734/download