King Saud University College of Pharmacy Department of

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King Saud University College of Pharmacy Department of Pharmaceutics PHT 351: Sterile Dosage Forms

King Saud University College of Pharmacy Department of Pharmaceutics PHT 351: Sterile Dosage Forms Summer Semester of 1423 -1424 H PRINCIPLES AND METHODS OF STERILIZATION Ibrahim A. Alsarra, Ph. D.

Methods of Sterilization 2

Methods of Sterilization 2

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

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

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 Saturated heat autoclaves Superheated water autoclaves Air

Methods of Sterilization Moist Heat Dry Heat Saturated heat autoclaves Superheated water autoclaves Air oven steam autoclaves Batch sterilizers Continuous tunnel sterilizers Chemical (cold) Radiation Filtration Ethylene oxide Vaporized hydrogen peroxide Hydrogen peroxide/steam Other gases Electromagnetic particulate Membranes 5

Introduction … cont. The sterilization processes are commonly used for parenteral products, except gas

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:

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,

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

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,

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),

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

STEAM STERILIZATION

Terminology • Bioburden- The degree of contamination with microorganisms and organic parameters • Bioresistance-

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

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

Terminology … cont. u TDT is the time required to kill a known population of microorganisms in a specific suspension at a particular temperature u Increasing temperature decreases TDT u Lowering the temperature increases TDT u Acidic or basic p. Hs decrease TDT u Fats and oils slow penetration and increase TDT 15

Terminology … cont. • Spore- Certain microorganisms which are capable of forming a thick

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 u u Steam contacts a cooler surface, condenses, causing

Action (mechanism) of Steam u u u Steam contacts a cooler surface, condenses, causing a huge decrease in volume and setting up a negative pressure that draws more steam Condensation occurs as long as there is a temperature differential Action of steam ensures: Surface heating, penetration, and protein coagulation 17

Advantages of Steam Sterilization • Stainless steel instruments withstand repeated processing without damage and

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

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: An apparatus for sterilization by steam under pressure, usually at temperatures of 250

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

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

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

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,

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

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)

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

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.

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

Types of steam sterilizers 1. Gravity displacement Definition: Gravity pushes air through the packages and down through the drain. Sterilization begins when steam passes thermometer and reaches the desired temperature. 29

Gravity Displacement Uses 1. Metalware 2. Glassware 3. Thermoplastics 4. Linens 5. Rubber 30

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

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

Pre-vacuum Sterilizer Uses: Metalware Rubber Thermoplastics 32

Types of steam sterilizers … cont. 3. High Pressure (flash) Sterilizer Definition: Another sterilizer

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

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

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

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

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

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.

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 items sterilized in sterilizer for last 24 hour period 39

STERILIZATION by FILTRATION

STERILIZATION by FILTRATION

Filtration - It depends upon the physical removal of microorganisms by adsorption on the

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 heatsensitive 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

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

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 - p. H 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 the solution 43

Filtration …cont. • Disadvantages: 1 - The membrane is fragile and because of that,

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

OTHER TECHNIQUES

Ultrasonic vibrations – High frequency sound waves – Know as Sonicator, untrasonic machines –

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

PYROGENS AND PYROGEN TESTING

PYROGEN - A pyrogen is a material which when injected into a patient will

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

PYROGEN… cont. Two main procedures are used for the detection of pyrogens. A. The traditional method: It requires the administration of the sample to laboratory rabbits whose body temperature is monitored for a period of time thereafter. 49

PYROGEN… cont. Steps: 1 - Render the syringes, needles, and glasswares free form pyrogens

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

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

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

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

PYROGEN… cont. B. Limulus Amoebicyte Lyste Test (LAL) … cont. Proclotting enzyme Endotoxin Activated clotting enzyme Coagulation Clottable protein The Lysate Clotting Mechanism GEL 54

PYROGEN… cont. B. Limulus Amoebicyte Lyste Test (LAL) … cont. In the test procedure,

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