Validation of sterilization - Pharmaceutical Microbiology Third Semester PDF Notes

 Validation of sterilization


       Validation – meaning and significance

       Sterilization indicators

      Physical indicators

      Chemical indicators

      Biological indicators

Intended learning objectives

At the end of this lecture, the student will be able to:

       Explain the significance of validation in sterilization

       List the sterilization indicators

       Describe the working of sterilization indicators

Validation- History

          The concept of validation was first proposed by two Food and Drug Administration (FDA) officials, Ted Byers and Bud Loftus, in the mid-1970s in order to improve the quality of pharmaceuticals

          It was proposed in direct response to several problems in the sterility of large volume parenteral market

           The first validation activities were focused on the processes involved in making sterile products, but later on were applied for  associated processes such as  environmental control, media fill, equipment sanitization and purified water production

Validation of sterilization

       Validation means demonstrating that a process will consistently produce the results that it is intended to

       Validation is a process of establishing documentary evidence demonstrating that a procedure, process, or activity carried out in production or testing maintains the desired level of compliance at all stages. 

       Validation would be necessary for each of the individual aspects of the manufacturing process,

       Environmental monitoring

       Raw materials quality assessment

       Sterilization process itself

       The sterility testing procedure

Sterilization indicators

                Sterilization procedures should be monitored through a combination of physical, chemical, and biological techniques designed to evaluate the sterilizing conditions and the procedure's effectiveness

       Physical indicators

       Chemical indicators

       Biological indicators

Physical indicators

Observing the gauges or displays on the sterilizer for

       Assessing the cycle time


       Pressure of sterilization equipment

       Relative humidity

Heat sterilization

       A temperature record chart is made - part of the batch documentation

       Compared against a master temperature record (MTR)

       Information on heat distribution and penetration within a sterilizer can be gained by the use of thermocouples

Gaseous sterilization

       Measurement of elevated temperatures using temperature probes

       Routine leak tests to ensure gas-tight seals

       Pressure and humidity measurements are recorded.

       Gas concentration is measured, often by reference to weight of gas used

Radiation sterilization

       A plastic (often perspex) dosimeter is used

       It  gradually darkens in proportion to the radiation absorbed

Integrity testing sterilizing filters

       Membrane filters have been used successfully for many years to remove yeast, bacteria and particulate from fluid streams

       To test the integrity of filters, the following tests are done:

a)      Destructive test

b)      Non - destructive integrity test

a) Destructive test- Destructive challenge testing is the best way to determine a sterilizing filter's ability to retain bacteria. During the bacterial retention test, 0.22 µm filter discs and devices are challenged with a solution of culture medium containing bacteria (Brevundimonas diminuta ATCC 19146) at a minimum challenge of 107 per cm2. The effluent is then passed through a second 0.45 µm filter disc that is placed on an agar plate and incubated.

 - The filter cannot be used for filtration purposes again

b) Non - destructive integrity test, may be done on filters before and after filtration process

-This is done to ensure that the filter meets specification, is properly installed and intact during filtration, and to confirm the rating of the filter

- Integrity testing before filtration process monitors filter integrity, preventing use of a non-integral filter. 
- Integrity testing after a batch has been filtered can detect if the integrity of the filter has been compromised during the process

There are 3 major tests used to determine the integrity of a membrane filter: the Bubble Point Test, the Forward Flow Test, and the Pressure Hold Test

        All three tests are based on the same physics, the flow of a gas through a liquid-wetted membrane under applied gas pressures

        They differ in which part of the flow/pressure spectrum they examine

       This is an indirect method for the measurement of pore size of filter

Bubble point pressure test

       Bubble point test  is based on the fact that liquid is held in the pores of the filter by surface tension and capillary forces

        The minimum pressure ( as seen on the pressure dial) required to force liquid out of the pores is a measure of the pore diameter
The bubble point is expressed as :

k = shape correction factor
Ύ = surface tension
cos ɵ =  liquid solid contact angle
d = pore diameter

       Consists of immersing the filter candles in water or filling the funnel with the liquid in the case of a membrane filter or sintered glass filter

       Air or gas is passed from the bottom of the filter

       The pressure of Air/ gas is gradually increased until the first bubble is seen at the filter/liquid interface

Filtration sterilization

       Bubble point pressure test

Chemical indicators

       Chemical indicators are designed to respond to a characteristic change to one or more of the physical conditions within the sterilizing chamber

       The principle is  based on the ability of heat, steam, gases and ionization radiation to alter the chemical and/or physical characteristics of  chemical substances

       Change should take place only when satisfactory conditions for sterilization prevail

       Used for monitoring a sterilization process

       Chemical indicators generally undergo melting or colour changes

       Included in strategically placed containers or packages

       Monitors  the conditions prevailing at the coolest or most inaccessible parts of a sterilizer

Bowie Dick test: Organic chemical in a printing ink base impregnated into a carrier material. A combination of moisture and heat produces a darkening of the ink

Brown’s tube: Sealed tubes partly filled with a solution which changes colour at elevated temperatures; rate of colour change is proportional to temperature

Thermalog S: Consists of a blue dye in a waxy pellet. At autoclaving temperatures, and in the continued presence of steam, the pellet melts and travels along a paper wick forming a blue band the length of which is dependent upon both exposure time and temperature

Thermalog G: Same as Thermalog S. Used for gaseous sterilization

Biological indicators

       Consist of standardized bacterial spore preparations

       Usually in the form either of suspensions in water or culture medium or of spores dried on paper, aluminium or plastic carriers.

       As with chemical indicators, they are usually placed in dummy packs located at strategic sites in the sterilizer

       After the sterilization process, the aqueous suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium, which is then incubated and periodically examined for signs of growth.

       The bacterial species to be used in a BI must be selected carefully, as it must be non-pathogenic and should possess above-average resistance  to the particular sterilization process.

Filtration sterilization

       Measuring the ability of a filter to produce a sterile filtrate from a culture of a suitable organism

       Serratia marcescens  has been used for filters of 0.45mm pore size,

       Brevundimonas diminuta (formerly Pseudomonas diminuta) having a minimum dimension of 0.3mm is applied to filters of 0.22mm pore size.


       Validation means demonstrating that a process will consistently produce the results that it is intended to

       Sterilization indicators - Physical indicators, Chemical indicators, Biological indicators

       Physical indicators validate the temperature, pressure, radiation dose or filter pore size

       Chemical indicator - Based on the ability of heat, steam, sterilant gases and ionizing radiation to alter the chemical and/or physical characteristics of a variety of chemical substances

       Biological indicators - Consist of standardized bacterial spore preparations

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