Asst. Prof. Dr. Mehmet İLKTAÇ · 2018. 4. 24. · Air in the sterilizator decreases the temperature and prevents the steam penetrating the products ... STERILIZATION METHODS I

PHAR457

Asst. Prof. Dr. Mehmet İLKTAÇ Lecture 4: 24/04/2017

STERILE PHARMACEUTICALS AND STERILITY

Injections (Parenteral products)

Ophthtalmic preparations

Irrigation fluids

Dialysis solutions

Medical implant devices

Products injected or targeting

internal organs, and broken skin

MUST be

STERILE

STERILE BODY PARTS / BROKEN PHYSİCAL

BARRIER

BODY PARTS CONTAINING

FLORA

STERILE PRODUCTS

NON STERILE PRODUCTS

STERILIZATION Sterile: Object free from all viable forms of microorganisms

Sterilization: Physical or chemical process that kills all forms of microorganisms.

NOT SUBJECTIVE

INCLUDING SPORES !!

Either sterile or not !!!!!

MOST RESISTANT

Sterility assurance level (SAL): the probability of a single unit being non-sterile after it has been subjected to sterilization.

STERILITY

İmpossible to be sure that all pharmaceutical units in a lot are sterile.

Sterility of a pharmaceutical lot is described by STERILITY

ASSURANCE LEVEL

Parenteral pharmaceuticals SAL is 10-6

Sterility assurance: No surviving microorgansims present in a product.

In order to obtain desired low level of SAL,

Use of validated sterilization methods

Use of validated aseptic processing under appropriate GMP (Validation, Documentation, SOPs..) during manufaturing

Environmental monitoring of facilities, personnel and processes

Human with microbial infection/carrier/normal flora

Water and biofilm in the water system

Air

Facility Environment (floor, walls ...)

Stressed environmental microorganisms

penetrating filters

SOURCES OF THE CONTAMINATION OF STERILE PRODUCTS

SENSITIVITY OF MICROORGANISMS TO STERILIZATION

Spor

Enveloped viruses

Mycobacteria

Naked viruses

Fungi

Most resistant

Most

sensitive

Prion

Vegetative bacteria

MICROORGANISMS LOSE THEIR VIABILITY IN EXPONENTIAL FASHION DURING STERILIZATION

The type of microorganism (Fungi, naked virus,

vegetative bacteria, spore forming bacteria ...)

The moisture content of the environment and of the

microorganisms (saturated water vapour, spore ...)

The initial number of microorganism (initial load)

Concentration of sterilization agent (Degree of

temperature, dosage of radiation ..)

Time of exposure

FACTORS THAT DETERMINE THE MICROORGANISMS SURVIVE A STERILIZATION PROCESS

METHODS OF STERILIZATION FOR PHARMACEUTICAL PRODUCTS

Heat Sterilization

Moist Heat (Steam Sterilization)

Dry Heat

Radiation Sterilization

Sterilization by Filtration

Gaseous Sterilization

STERILIZATION METHODS I. Heat Sterilization The most reliable and frequently used method.

Antimicrabial activity Denaturation of the proteins (enzymes, cell membrane etc...)

Heat is much more effective in the presence of water (humidity)

In the presence of humidity, sterility can be obtained at lower level of temperature in a shorter period of time.

Suitable for heat stable products.

STERILIZATION METHODS I. Heat Sterilization

a. Moist Heat Sterilization (Steam Sterilization)

Aqueous injections

Ophthalmic preparations

Irrigation fluids

Containers

Dressing

Sheets

Surgical and diagnostic equipment

Thermostable



Saturated steam at 121-134 °C under pressure (1.5-3 atm) for 15 min and 3 min respectively.



121 °C, 1.5 atm, 15 min.

134 °C, 3 atm, 3 min. Parameters of mosit heat sterilization

Short time Higher level of sterility asurance Less harm to product stability

Most frequently used

Sterilization time should be measured from the moment when all the materials have reached the required temperature.

For dressing

For bottled fluids.



ACTION OF MECHANISM

Contact of saturated steam with a cooler surface causes condensation leading to the immediate release of the heat energy resulting denaturation of proteins.

At the beginning, efficient air removal from the device is very important.

Air in the sterilizator decreases the temperature and prevents the steam penetrating the products.



AUTOCLAVE



AUTOCLAVE

I. Air removal and steam admission

II. Heating up and exposure

III. Cooling down and drying

Stages of Operation

Vertical autoclave

Horizontal autoclave

Digitalized autoclave

Sterilizing In Autoclave


b. Dry Heat Sterilization ACTION OF MECHANISM

Oxidation of proteins rather than denaturation (steam).

Less efficient than steam sterilization

Higher temperature and higher exposure time

160-180 °C 1.5-2 hours

Parameters


b. Dry Heat Sterilization

Glassware Metallic surgical instruments Thermostable powders

The major application is in sterilization of glass bottles.

Not only removes microorganisms but also ENDOTOXIN

ENDOTOXIN=PYROGEN

For depyrogenation of glass, dry heat temperatures of 250 °C should be used.


b. Dry Heat Sterilization

Sterilization Hold time

Cooling time Heating

Temperature

t

STERILIZATION METHODS II. Radiation Sterilization

a. Ionizing irradition

b. Non-Ionizing irradition

Accelerated electrons Gamma rays

UV rays DNA damage by excitation

DNA damage by free radical

production and ionizing

Non-ionization irradiation is less efective than ionizing irradition.

Spores, viruses, moulds, yeasts, vegetative bacteria

Increasing sensitivity

ACTION OF MECHANISM

Absorbed radiation 25 kGy


Presence of moisture Presence of dissolved oxygen Elavated temperature

Increases the efficacy of radiation

Radiation damages the products in aqueous solution

Unit dose oinment Dry pharmaceutical products Surgical instruments Prostheses Sutures


UV irradition

Less DNA damage (lower energy) Poorly penetrates the packaging material Active for relatively short distance

Unsutiable for pharmaceutical dosage forms

Major applications of UV

Sterilization of air Surface sterilization of aseptic work areas (walls, air flow cabinet) Treatment of manufacturing - grade water

STERILIZATION METHODS III. Sterilization by Filtration

Doesnot destroy; only removes the microorganisms

Prevents flow of both viable and nonviable particles in air

Used for sterilization of liquid solutions and gases

Heat sensitive injections Heat sensitive ophthalmic solutions, Heat sensitive biological products (interferon, serum) Air and other gases for supply to aseptic areas

STERILIZATION METHODS III. Sterilization by Filtration

3 MAJOR MECHANISMS

Sieving Adsorbtion Trapping

Exlusion of particles above a defined size (pore size) Physical interaction

between microbe and filter depends on the tortuosity

of the filter matrix

Only sieving can be regarded as absolute because it ensures the exclusion of all particles above a defined size

For sterilization of solutions, membrane filters of 0.22 or 0.45 µm are used.

STERILIZATION METHODS III. Sterilization by Filtration Filtration sterilizitation of gases is used to provide sterile air to aseptic manufacturing suites and some operating theatres.

Filtration of air is carried out by HEPA filters.

HEPA filters can remove up to 99.9 % of particles more than 0.3 µm.

STERILIZATION METHODS IV. Gaseous Sterilization

Ethylene oxide Formaldehyde

Action of mechanisms

Alkylation of amino, carboxyl, hydroxyl and sulphydryl groups of proteins and imino groups of nucleic acids

Concentration, temprature and moisture increases the efficacy.

Formaldehyde

Ethylene oxide 800-1200 mg/lt; 45-63 °C

15-100 mg/lt; 70-75 °C

Less effecttive than heat sterilization. Reserved for heat sensitive products.

Surface sterilization of powders Surgical instruments


Alternative to sterilization by radiation

Ethylene oxide Formaldehyde

Both are TOXIC, CANCEROGENIC, MUTAGENIC

Need to remove toxic residues of the gases before release of the items for use


Ethylene oxide

Highly explosive Supplied as a 10% mixture with CO2 (inert gas)

Good penetration into packaging material

Heated filtered air is pumped for 24 h to remove toxic residues

STAGES OF ETHYLENE OXIDE STERILIZATION

I II III IV


Formaldehyde

İnflammable

Heating formalin at 70-80 °C with steam

Toxic. Need desorbtion

Low penetration to packing material. Only used for paper and cotton packaging material

STAGES OF FORMALDEHYDE STERILIZATION

I II III IV

VALIDATION AND IN-PROCESS MONITORING OF STERILIZATION PROCEDURES

I. Calibration of all the physical instruments (thermocouples, pressure gauges and timers)

II. Production of evidence that the steam is of the desired quality (e.g. chamber temperature is that expected for pure steam at the measured pressure)

III. Leak tests and steam penetration tests

IV.Biological indicators alone or in combination with bioburden organisms to demonstrate that the sterilization cycle is capable of producing an acceptable level of sterility assurance

STAGES

V. To demonstrate repeatability of the above (typically for three runs)


VI. Documentation of all of these aspects.

I. Calibration of all the physical instruments (thermocouples, pressure gauges and timers)

II. Production of evidence that the steam is of the desired quality (e.g. chamber temperature is that expected for pure steam at the measured pressure)

III. Leak tests and steam penetration tests

IV. Biological indicators alone or in combination with bioburden organisms to demonstrate that the sterilization cycle is capable of producing an acceptable level of sterility assurance

V. To demonstrate repeatability of the above (typically for three runs)

VI. Documentation of all of these aspects.


ALL OF THESE PARAMETRES ARE

MONITORED BY

INDICATORS

INDICATORS FOR VALIDATION AND IN-PROCESS MONITORING OF STERILIZATION PROCEDURES 1. Physical Indicators

Heat treatment Temperature is recorded by thermocouples

Moist Heat Temprature by thermocouples, Pressure is recorded by gauges

Gaseous ster. Temperature, gas concentration, humidity and

pressure is recorded

Radiation Radiation dose is recorded by dosimeter

Sterilization parameters are measured by a device

1. Physical Indicators

INDICATORS FOR VALIDATION AND IN-PROCESS MONITORING OF STERILIZATION

PROCEDURES

PHYSICAL INDICATORS REFLECTING INADEQUATE EXPOSURE TO STERILIZATION PARAMETERS

MEANS

THE STERILIZATION PROCESS IS NOT UNDER CONTROL

CORRECTIVE ACTIONS!!!


PROCEDURES 1. Physical Indicators

PHYSICAL INDICATORS BEING UNDER THE APPROPRIATE RANGES

DO NOT

PROOF THE EFFICACY OF STERILIZATION PROCESS DEFINITELY.

2. Chemical Indicators


PROCEDURES

Sterilization agents change the chemical or physical characteristics of a chemical substance

Color change Solid chemical melts

One parameter More than one parameter

Heat Steam Radiation Gases

More Reliable

2. Chemical Indicators


PROCEDURES

Failed chemical indicator indicates failure in the sterilization process.

Appropriate change in chemical indicator doesnot rule out sterilization failure.

CORRECTIVE ACTIONS!!!

NOT DEFINITE RESULT

Should not be used as a substitute for biological indicators


PROCEDURES 2. Chemical Indicators

AIR REMOVAL TEST (STEAM PENETRATION TEST)

Unless air is removed efficiently, air trapped in the sterilizer will prevent the steam penetrating to the load.

BOWIE DICK TEST Chemical indicator are used

Perform daily at the beginning of every day with empty autclave

BOWIE DICK TEST

3. Biological Indicators


PROCEDURES

Consist of standardized bacterial spore preparations

Located at strategic sites where the sterilization agent is presumed to be the least accesible together with load.

After sterilization, incubate and check for growth

Near door Near vacuum pump To the lowest shell



PROCEDURES

SPORES MOST RESISTANT

IF YOU KILL SPORES YOU KILL EVERY MICROORGANISM

Bacillus stearothermophilus

Bacillus subtilis

Bacillus subtilis

Bacillus subtilis

Bacillus pumilis

Bacillus stearothermophilus



PROCEDURES

ADVANTAGE

THE MOST DEFINITE METHOD THAT SHOWS THE EFFICACY OF STERILIZATION

DISADVANTAGE

INCUBATION PERIOD TO CHECK THE GROWTH (upto 7 days) CAUSES DELAY IN THE RELEASE OF THE PRODUCT.

3. Biological Indicators (BI)


PROCEDURES

BIs must be used for each cycle of gaseous sterilization

For methods other than gaseous sterilization BIs should be used for validation. NO POCESS MONITORING IS RECOMMENDED


PROCEDURES

For initial validation of membrane filtration method:

Serratia marcescens

Brevindimonas diminuta

For 0.45 µm membrane filter

For 0.22 µm membrane filter

STERILITY TESTING

2 methods: STERILITY TESTING

1. Membrane Filtration (MF) Dilute the product in a

sterile diluent.

Add polysorbate 80/myristate into the diluent for insoluble products.

Filter the suspension using 0.45 µm membrane filters.

Aseptically transfer the membrane filters to Thioglucollate broth and Soybean Casein Digest broth

Wash the filter with diluent.

The Method suggested by

pharmocopeias except for devices

2. Direct Inoculation (DI)

Directly inoculate the products into Thioglucollate broth and Soybean Casein Digest broth.

For insoluble substances add polysorbateto the medium.

STERILITY TESTING

For both methods, antimicrobial activity of the product should be removed by:

Dilution

Membrane Filtration

Neutralizing Substance

Use DI ıf MF method is unsuitable for the product. Devices are tested using DI method

NEUTRALIZATION AGENTS

STERILITY TESTING OBSERVATION AND INTERPREATION OF RESULTS

Thioglucollate broth

Soybean Casein Digest broth

Incubate under ANAEROBIC conditions

at 37 °C for 14 days

Incubate under AEROBIC

conditions at 25 °C for 14 days

FOR ANAEROBIC BACTERIA

FOR AEROBIC BACTERIA, FUNGI AND MOULDS

STERILITY TESTING OBSERVATION AND INTERPREATION OF RESULTS

Check turbidity regularly for 14 days.

No turbidity = No growth of microorganisms

If product renders the medium turbid, transfer a small quantity of medium to fresh medium or to agar plate and incubate for 4 days.

Comply with the test of sterility

Turbidity PRODUCT IS NOT STERILE

Nonsterile products Sterile products

6-8 days after 14 days after

RESULTS Microbial Limit Testing

Sterility Testing

The test should be validated before with test strains:

Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Clostridium sporogenes Candida albicans Aspergillus brasiliensis

SMALL QUANTITY IS APPLİED ONTO THE MEMBRANE FİLTER OR MEDIA AFTER THE PRODUCT İS APPLIED ON FILTER OR MEDIA.

VALIDATION OF STERILITY TESTING

QUANTITY OF THE PRODUCT THAT SHOULD BE TESTED

NUMBER OF ARTICLES TO BE TESTED IN A BATCH

The number of samples tested is very low according to the total number of samples

If a small percentage of container in a batch is contaminated, sterility testing will not detect it.

Then the most important factors to demonstrate the sterility of all units in a lot are:

To monitor and validate the manufacturing process as a whole

Environmental monitoring of manufacturing facility, raw material testing, validated aseptic procedures

To validate and monitor sterilization process

ENVIRONMENTS THAT THE STERILITY TESTING IS CARRIED OUT

Sterility testing should be carried out in class 1000 clean rooms or in a class 100 safety cabinet.

All the reagents and equipments used during the test must be sterile.

Operators should wear gloves, face masks, lab. coats etc...

Environmental monitoring of surfaces, air and personnel during testing must be part ofthe process

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Asst. Prof. Dr. Mehmet İLKTAÇ · 2018. 4. 24. · Air in the sterilizator decreases the temperature and prevents the steam penetrating the products ... STERILIZATION METHODS I