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PHA425
STERILISATION METHODS
IN PHARMACEUTICAL
MANUFACTURING

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 2

At the end of these lectures students should be able

to –
• Discuss the principles and practices of various
LEARNING sterilisation methods used for sterile
OBJECTIVES manufacturing
• Compare advantages and limitations of
sterilisation methods
• Explain factors affecting heat, radiation, chemical
and filtration sterilization methods
• Define heat sterilization parameters and perform
calculations to estimate parameters such as
heating time and sterility assurance level
• Discuss how to plan and perform the end
product sterility testing

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REFERENCES

Russell, Hugo & Ayliffe Gardner & Peel

Disinfection, Preservation and Sterilisation, Disinfection and
Sterilisation Infection Control

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STERILISATION
A process intended to kill (or) remove all types of micro-
organisms with an acceptably low probability of one
organism surviving

• one in a million chance of one non-sterile item

• sterility assurance level (SAL) of 10-6

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 METHODS OF STERILISATION

• Heat
o Moist
o Dry
Chemical
• Ethylene
oxide
• Aldehydes

5
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 STERILISATION BY HEAT

• Heat  thermal energy  lethal to Micro-organisms

• Damage to cytoplasmic membrane, cellular enzymes, DNA,
RNA
Mechanism
• Moist heat  Protein coagulation
• Dry heat  Oxidation

6
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 HEAT INACTIVATION DEPENDS ON

Heat
• Heating temp  sufficient quantity of heat to produce lethal effect
Organisms
• microbial population (initial bio-burden)
• heat resistance of organisms
Time
• sufficient heating and holding time to destroy all organisms
Heat, Organisms, Time - HOT!
Efficiency of heat inactivation (or) thermal death rate increases with
•  heating temperature
•  heating time
•  initial population
•  heat resistance of micro-organism

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 THERMAL DEATH KINETICS

At constant temperature -
• the same percentage of microbial population will be destroyed in
each unit of time at the rate proportional to the number of surviving
organisms
• Example - If a heat sterilisation process kills 90% of m.o population
per minute -
• for initial population of 1000 m.o -
• 90% of 1000 will be killed in the first min (100 remaining)
• 90% of 100 will be killed in the second min (10 remaining)
• 90% of 10 will be killed in the third min (1 remaining)

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 THERMAL DEATH KINETIC

Log10 Nt = Log10 N0 – kT t
steeper slope =  kT = faster rate of kill

Log10 No. of Survivors (Nt)

flatter slope =  kT = slower rate of kill
• Plot Log10Nt vs. t (time)

• Slope = kT = thermal death rate constant

at temperature T kT
• kT = 1/t log10 N0/Nt

Nt= the number of organisms surviving at

time t
N0 = the initial number of micro-
organisms
k = rate constant Time of heating t (min)
t = time of heating (min)

9
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 DECIMAL REDUCTION TIME (D)
Log10 Number of Survivors

4  Heating time (in minutes) required for

10 fold reduction (or) 1 log cycle
Slope = kT = 0.5 decrease in the number of surviving
3
organisms
2 ▪ Relationship between D and kT

1 Dtemp = 1/kT

0
D 2 4 6
-1 Time of heating (min)  D value defines the rate of thermal
-2
inactivation at a specific temperature
 e.g. D160 = 2 mins
10
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 EXAMPLE CALCULATION

• You have 1000 x 1 mL injection vials, each estimated to have

contaminated with 10 organisms per 1 mL solution.
• If D160= 3 min for a heat steriliser, how long would you need
to heat these vials to get a probability contamination level of
10-6 for the whole batch?
Thermal death kinetic equations
• kT = 1/t log10 N0/Nt
• D = 1/kT

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 ANSWER

N0= 1000vials x 1 mL x 10 = 104 • kT = 1/t log10 N0/Nt

Nt = 10-6
D160= 3 min • 1/3 = 1/t log10 104 /10-6

t =?
• 1/3 = 1/t log10 1010

kT = 1/D • 1/3 = 10/t

kT = 1/3

• t = 30 mins

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HOW DO WE KNOW WHETHER ORGANISMS
ARE HEAT SENSITIVE OR HEAT RESISTANT?

13
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 Z VALUE
Z =10
Z=3

Defines the heat sensitivity or resistance of micro-organisms

heat resistant organisms have large z values and heat sensitive

organisms have small z values

defined by increase in temperature (C) required to bring about ten-

fold decrease in decimal reduction time (D value)

e.g. D121 = 2 min D124 = 0.2 min Z value = 3 C

D121 = 5 min D131 = 0.5 minZ value = 10C

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 F0 VALUE
15
• F0 value defines lethality in terms of the equivalent heating
time in minutes at 121C, using the reference organism
with a z value of 10 C
• i.e. “standard HOT”= Heat, Organism,Time
• F0 value for a steam sterilizer can be calculated from the
temperature vs. time profile during heating, holding and cooling
processes (BP limit = F0 of 8 for heat treatment).

• The calculation of F0 takes into account the equivalent

lethal effect of lower heating temperatures compared
with 121 C

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 TEMPERATURE VS. TIME PROFILE
OF HEAT STERILISATION PROCESS

• F0 = ∑ t x 10 (T-121)/z
Temp t = time interval
(T) between temp
measurements
Holding time T = product temperature

Heating
Cooling
time time

Time (t)
• F0 value calculated from area under the curve of this plot
• e.g. temp 115° C maintained for 1 minute time slice will give F0 value of
 t = 1 min, T = 115° C
F0 = 1 x 10 (115 -121)/10 = 0.251 min
• Sum up all the F0values for all time slices to get an F0 value for the whole process

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 STERILITY ASSURANCE LEVEL (SAL)
17

• Defines the degree of sterility in the treated batch of product

• for example, SAL of 10-6 means, probability of one un-sterile item in

106 items treated ( i.e. 1 in a million chance of an organism
surviving )

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 EXAMPLE CALCULATION

• A batch of containers with estimated contamination level of 103 is to be heat

sterilised. The D value for the most heat resistant contaminating organism is
2 mins at 121 C.
• What is the total sterilising time at this temperature to give 1 in a million
probability of finding one un-sterile container?
• SAL required = 10-6
• Initial contamination level = 103
• D121 value = 2 mins
• i.e. every 2 mins (90%) or 10 fold reduction in m.o., 10% survived
• 1000  100  10  1 = 3 log cycles = 2 x 3 = 6 mins
• in a million probability of contamination = 10-6  6 more log cycles
reduction = 2x 6 =12 mins
• Total = 6 + 12 = 18 mins (total heating time at 121C)

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 MOIST HEAT STERILISATION
19
• saturated steam at high temp holds load of latent heat which is
available for transfer without drop in temperature when it
condenses onto a cooler surface

9 Kj/mole 40 Kj/mole
Water 0oC Water 115oC Steam 115oC

• Heat transfer from steam to object occurs until object reaches

temp of the steam

• Protein coagulation and denaturation of m.o during this

process

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 AUTOCLAVING

• Moist heat sterilisation

• heating under high steam pressure

in an autoclave oven
Process involves -
1. Removal of air
2. Heat to temperature under high
steam pressure
3. Hold temp for required time
4. Cool
5. Depressurise

20
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 MOIST HEAT

• Used for sterilization of:

• dressings, surgical and diagnostic equipment, containers
and closures
• terminal sterilization of aqueous injections, ophthalmic
preparations, irrigation & haemodialysis solutions
• not suitable for non-aqueous preparations
• preferred method of sterilization

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 MONITORING STEAM
STERILIZATION

Instrumental Chemical Biological

indicators indicators indicators
Thermometers Browne’s tube Spore strips
(red  green) Attest vials
Pressure (>105 viable spores/unit)
gauges Bowie-Dick tape
(light  dark stripes) B. stearothermophilus
Thermocouples (D121 = 2 min)
(temp sensitive tips Steam-Clox strip
connected to temp
(purple  green)
recorder outside
steriliser)
22
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 REQUIREMENTS OF BIOLOGICAL INDICATORS

✓Strain should be stable

✓Non-pathogenic
✓Resistant to method used
✓Reproducible recovery
✓Not less than 105 viable spores/unit
✓Number should match the IF of the process

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 MOIST HEAT
STERILISATION
Advantages Disadvantages
• Lower temperature
• Heat labile preparations
• Shorter exposure time
• Aqueous thermostable • Corrosive
preparations • Containers must be
• aqueous injections
• parenteral products penetrable by
• O/W emulsions
• eye-drops
steam
• Sealed in heat stable plastic • Difficult to monitor
containers (e.g.
• Damp load (water inside)
PVC, Polyethylene)
• Bursting of rigid containers

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 DRY HEAT STERILISATION
25

• Heat transfer by convection or radiation

• Oxidative destruction of bacterial cell wall and cytoplasmic
components
• Higher pressure and longer holding time
• rubber, plastic packaging/containers could not withstand
• Suitable to sterilise -
• Metal instruments/containers, glassware
• powders, oils, waxes, glycerol (double holding time)

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 HOT AIR OVEN
CHARACTERISTICS

• Electric heater to heat the oven

• Oven heated to the desired temp before loading
• Fan circulation
• for uniform heat distribution
• maximum variation in chamber temp <10C
• load and shelving should allow circulation of hot air
• Thermostat to maintain temp
• Heat lining of the chamber
• Insulation to prevent heat transfer from inside
• Efficiency Indicator organism for HAO
• B. subtilis var niger (D160 = 5 -10 min)
• Innoculum at least 105 viable spores per unit

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 DRY HEAT

• Lethality due to oxidative processes

• Higher temperatures and longer exposure times required
• Typical cycles:
• 160°C for 120 minutes
• 170°C for 60 minutes
• 180°C for 30 minutes

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 DRY HEAT

• Used for:
• glassware, metal surgical instruments, non aqueous
thermostable powders and liquids (oils)
• also used for depyrogenation of glassware (250°C)
• (Pyrogens - substances found in cell wall of some bacteria
which can cause fever when introduced into the body)

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 DRY HEAT STERILISATION

Advantages Disadvantages
Non-aqueous systems slow heating
Non-corrosive long holding times
Penetration high temperatures
Dry process not for aqueous
Oily thermostable preparations
products
–Oily injection
–Ointments

29
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 MOIST VS. DRY HEAT
STERILISATION
Sterilization Moist Heat Dry Heat
method
Method of heat Condensation Radiation
transfer Convection
Mechanism of m.o Protein coagulation Oxidative destruction
destruction

Parameters for 121 C for 15 mins 160-170 C for 1-2 hr

sterilisation efficiency under high steam
pressure 15 psi
Test organism B. stearothermophilus B. subtilis var niger
D121 = 2 minutes D160 = 5 - 10 minutes

Plus advantages and disadvantages of two methods 30

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 RADIATION

Electromagnetic radiation
• Gamma rays, UV rays

radiation dose can cause:

• Chemical reactions in the nucleic acid
• Cell mutation
• Cell death

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 UV IRRADIATION

• Electromagnetic radiation of
• lower energy and
• poor penetration power

• Non-ionising radiation
• Cause excitation of atoms to higher energy state
• Chemical reactions in the nucleic acid
• Alteration of DNA structure
• Mutation
• Cell death
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 APPLICATIONS OF
UV LIGHT
Air
• To reduce m.o counts in clean rooms via UV lamps fitted ducted air
supply systems
• Indirect irradiation in the occupied rooms (reflectors)
Water
• water supply from UV water steriliser equipment
• water inlet and water outlet fitted with UV lamp and continuously
irradiated)
• Lamp protected from water temp fluctuation

Surfaces
• direct irradiation of sterile cabinets (LFC) surfaces
• operator hazards

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 DISADVANTAGES OF
UV LIGHT

• Hazard (operator irridiated)

• Reflectors (e.g. Aluminium/chromium) can be used to protect the
operator and direct and intensify UV exposure to defined areas

• Poor penetration

• Shielding, reflection
• Turbid liquid and opaque surfaces

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35
GAMMA IRRIDIATION
• powerful and penetrating, high-energy
electromagnetic radiation
• shorter wavelength than that of X-rays and also
called nuclear X-rays
• emitted by a decaying unstable nucleus
• the usual source is Cobalt 60 (unstable
radioactive form of Cobalt 59).

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36
MECHANISM OF EFFECT

• High energy, highly penetrating electromagnetic rays

• Ionization of vital cell components and cell death
• Ionisation of DNA
• Ionisation of cellular water and production of free radicals reacting
with DNA

• Indicator Organism B. Pumilus

(D value = 0.3 MRad)

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 FACTORS AFFECTING
RADIATION
STERILIZATION
37

• Amount of energy absorbed per kg of material depends on

• Power of the source (energy out put)
• time of exposure
• density of the material treated
• Oxygen
• ↑ effect of oxidation
• Oxidising agents sensitize spores in anaerobic condition
• Temperature
• Synergistic effect
• Resistance at freezing temp
• Organic substances
• Protective effect

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ADVANTAGES DISADVANTAGES

• Materials are not wetted or heated • Hazardous

• Operated at ambient temp • Chemical changes in drug
• Packed and sealed container in • Container deterioration
packaging
• Small volume, low density
• Continuous and automated
• Expensive
• Plant

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 PHARMACEUTICAL
APPLICATIONS

• Drugs in dry state

• Freeze dried antibiotics, vaccines, vitamin
• anaesthetics, hormones, albumin, enzymes
• Oil, creams, ointments, waxes, talc, powders, capsule shells
• Detergents and emulsifiers
• Heat sensitive medical equipments
• Disposable syringes/needles
• Bandages and surgical dressings
• Sutures
• Catheters
• Gloves
• Eye droppers
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 METHODS OF
STERILISATION
Heat
✓Moist
✓Dry
Chemical
• Ethylene
oxide
• Aldehydes

40
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 CHEMICAL STERILISATION

• Ethylene Oxide (C2H4O )

• Formaldehyde (H. CHO)

CH2CHO

CH2
• Glutaldehyde
CH2CHO

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 MECHANISM OF
CHEMICAL
STERILISATION

• Alkylating agents
• Site of action – Cell DNA during replication
• biocidal, mutagenic, carcinogenic

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 ETHYLENE OXIDE
CH2 CH2
• Flammable
• highly explosive at concentration
> 3.6% v/v in air
O
• Used as a non-flammable mixture
• Highly diffusible through
with inert diluent gases (e.g. CO2,)
packaging materials
• Freely soluble in water
• Pure gas may be used in chamber
• Dissolve in rubber, plastic,
without air at sub atmospheric
silicones and oil
pressure

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 FACTORS AFFECTING ETHYLENE
OXIDE STERILIZATION

Concentration
• 400mg/L – 700mg/L
• Allowance for absorption by packaging materials
• Items rinsed and cleaned and dried first
• Packaging must allow permeability of steam and EtO vapour

Temperature
• ↑ effect at lower concentrations
• ↑ temp will decrease exposure time
• 2.8h @ 55°C
• 4.8h @ 37°C
Moisture
• moisture dissolves gas to form concentrated solution for the effect
• Optimal effect at 33%RH
• activity decreases at lower or higher RH

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 GAS CHAMBER CONDITIONS

Exposure
Vacuum

Pre-conditioning Air Washes

Humidification

Cycle Time
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 MONITORING OF
ETHYLENE OXIDE
STERILISATION

• Thermocouples
• Humidity sensors
• Weight of the EtO released from the gas cartridge for
concentration estimation
• Chemical indicators
• Strips change colours on exposure to EtO
• Biological indicators (B. subtilis var niger)
• Attest vials
• Spore strips
• Indicator Organism =B. subtilis var niger

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Limitations Applications
• Toxicity
• Carcinogenic • Dressings
• Allowable exposure 50 ppm • Containers
• Minimum detectable by • Medical equipment
odour 700 ppm
• endotracheal tubes
• Difficulty in Monitoring • anaesthetic masks
• Removal of Residues • Implants
• Time consuming • cardiac valves
• breast implants

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 CHEMICAL STERILIZATION
Formaldehyde
Glutaldehyde
• Alkylating reaction with Protein, DNA, & RNA of
organisms
• Formaldehyde Fumigation
• Conc 3.5 mg/L @ 70-75C temp @ 60-90% RH
• Glutaldehyde solution for soaking and rinsing of heat
sensitive instruments
• HIV inactivated within 2 min in 2% solution
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 LIMITATIONS

• Poor penetration
• Absorption by wrapping material
• Inactivation
• Toxicity
• E.g. HCHO 2-5 ppm causes immediate respiratory, eyes
and skin irritation)
• Contact dermatitis
• Difficult to monitor

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 METHODS OF
STERILISATION

Heat
✓Moist
✓Dry
Chemical
✓Ethylene
oxide
✓Aldehydes

50
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 STERILISATION BY
FILTRATION

• Living and non-living particles are removed from liquid and air or
gases by passage through filters
• No killing of micro-organism
• No growth inhibition

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 USES OF FILTERS
Sterilization of air and gases
❑Clean room air supply
❑Laminar flow cabinets
Sterilization of Liquids
❑Heat labile substances
❑Liquid parenterals
❑Administration set
Sterilisation of solid/semisolids
❑Heat labile ointments
❑Solids first dissolved in solvent and filtered sterile solvent
evaporated
Quality Control tests
❑Sterility testing – membrane filtration method
❑Microbial and Particulate level checks
Purification of Water
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 DESIRED PROPERTIES
OF FILTERS
• Inert, non-pyrogenic
• Efficiently remove particles
• Good flow rate
• Resistant to clogging
• Sterilisable
• Reusable
• Do not retain drug and other ingredients
• Do not release filter components

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 DEPTH FILTERS

• Compacted pads of fibres

▪ Polypropylene, cellulose or
glass
• Pores and channels
• variable diameters and
directions

• HEPA filter
• Pre-filters (prior to
sterilzation filter)

54
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PORE SIZE VS. POROSITY

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 DEPTH FILTERS -
LIMITATIONS
• Uniform pressure is important
• High pressure  compression of filter   flow rate
• Liquid remains in the filter must not be driven or sucked or
filter will crack
• Retain liquid, loss of solvent
• Loss of formulation ingredients
• Slow speed of filtration
• May not be suitable for sterilisation (chemical, heat) or reuse
• Filter fibers may shed into the filtrate

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MEMBRANE FILTERS

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 MEMBRANE
FILTERS
Polymeric membranes
hydrophilic, hydrophobic, hydrophobic edge, solvent resistant,
gridded
10 –100 mm thickness
High porosity
Fast flow and speed of filtration
Low fluid retention
Minimal solute adsorption
No shedding of filter components
Narrow pore size range
0.22 , 0.45, 0.8 - 0.12 mm filters
Poor dirt handling capacity
Rapidly clogging
Limited ease of handling

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 MEMBRANE
FILTER -
MECHANISM
• Screening or Sieving out
• Retention
• All particles of size > pore diameter are retained

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 USES OF
MEMBRANE
FILTERS

Pore Size (mm) Use

0.8 Removing particles, glass filters

0.45 Removing particles

Microbial and particulate contamination checks
Sterility testing of sterile pharmaceuticals

0.22 Sterilization by filtration

60
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 MONITORING
61 FILTERS
• Stringent QA checks for the reliability and suitability of
filters used for sterilisation

• Integrity testing of membrane filters

▪ Bubble point test

• Bacterial retention test

• Microbial challenge test

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 BUBBLE POINT
PRESSURE

• Pressure of a test gas (N2) required to push out the liquid in a

wet membrane filter

• at that pressure gas bubbles appear in the liquid

• The gas pressure is a measure of the pore diameter

• Smaller pore diameter requires higher pressure to push the

liquid out

• Larger pore diameter requires lower pressure

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 BUBBLE POINT TEST

N2

Wet Membrane - Higher Pressure Required

to overcome Surface Tension to push water out of
pores (capillaries)

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 BUBBLE POINT
PRESSURE

N2

Pressure to push the liquid out of wet membrane

Gas bubbles appear in the liquid

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 BUBBLE PRESSURE -
APPLICATION

Pore size Bubble Pressure (kPa)

(mm)

0.8 97

0.45 220

0.22 380

• Manufacturer’s recommended bubble pressure

e.g. 0.22 mm = 380 kPa
• Filters pass integrity testing if
Bubble pressure  recommended pressure
i.e. actual pore size  specified size

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 MICROBIAL CHALLENGE
TEST

• Bacteria retention test

• Efficiency of sterilising filter (0.22 mm)

• Challenge organism
Br. diminuta = 0.3 mm diameter
High concentration (107/cm2 area)

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 MICROBIAL CHALLENGE
TEST
• Slow filtration of bacterial suspension
• Entire filtrate is
▪ Cultured in broth medium (or)
▪ Passed through another filter which is then cultured
• Filter passes test if no growth after 72 hr at 30°C

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