STERILIZATION METHODS

1. STERILIZATION METHODS The term sterilization for pharmaceutical preparations, means the complete destruction of all living organisms and their spores or their complete removal from the preparation. Five sterilization processes are described in the USP: a. STEAM b. DRY-HEAT, c. FILTRATION, d. GAS, e. IONIZING RADIATION.

2. Sterilization processes are commonly used for parenteral products, except gas and ionizing radiation, which are widely used for devices and surgical materials. The selection of the sterilization method depend on: a. The nature and amount of product, b. Whether the product and container-closure system will have a predominately moist or dry environment during sterilization. Both of these factors are of great importance in determining the conditions (time and temperature ) of any sterilization method chosen.

3. For sterilization purposes, microorganisms can be categorized into three general categories: A. Easy to kill with either dry or moist heat; B. Susceptible to moist heat, but resistant to dry heat (Bacillus subtilis); C. Resistant to moist heat but susceptible to dry heat (Clostridium sporogenes).

4. A. STEAM STERILIZATION (Autoclave) • Sterilization of product and equipment by saturated steam (moist heat ) is one of the most widely used treatment in the parenteral drug industry. • Is conducted in an autoclave and employs steam under pressure. • ADVANTAGES OF STEAM STERLIZATION • a. Very efficient procedure, • b. The method of choice for products and articles that • can withstand the treatment, • c. Quick and inexpensive.

5. The mechanism of microbial destruction in moist heat is by: • Denaturation and coagulation of some of the organism's essential protein. • b. The presence of the hot moisture within the microbial cell permits destruction at relatively low temperature. • "Important factors should be controlled : • Application of pressure • Time of application • c. The velocity of the steam entering the autoclave • d. The efficiency of water separation from incoming steam • e. The size of the drain, • f. The penetration time of the moist heat

6. a. Application of pressure: Because it is not possible to raise the temperature of the steam above 100C. under atmospheric conditions, pressure is employed to achieve higher temperature (it should be recognized that the temperature, not the pressure is destructive to the microorganisms and that the application of pressure only for the purpose of increasing the temperature of the system).

7. b. Time of application: Time is an important factor in the destruction of microorganisms by heat. The usual conditions (time/pressure/temperature), are as follow: 10 pounds pressure (115.50C) for 30 minutes 15 pounds pressure (121.50C) for 20 minutes 20 pounds pressure (126.50C) for 15 minutes As can seen, the greater the pressure applied, the higher the temperature obtainable and the less the time required for sterilization. The temperature at which most autoclaves are routinely operated is usually 1210C.

8. c. The velocity of the steam entering the autoclave d. The efficiency of water separation from incoming steam e. The size of the drain, f. The penetration time of the moist heat into the load may vary with the nature of the load, and the exposure time must be adjusted to account for this latent peroid (an estimate of these latent period must be added to the total time in order to ensure adequate exposure times).

9. A. Applicable for pharmaceutical preparations and materials that can withstand the required temperature and are penetrated by, 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 containers as ampuls, are readily sterilized by this method • C. Also applicable to bulk solutions, glassware and • instruments. APPLICATION OF AUTOCLAVE

10. AUTOCLAVE NOT APPLICABLE FOR: A. The sterilization of oils, fats, oleaginous preparations B. Other preparations not penetrated by the moisture C. Sterilization of exposed powders that may be damaged by condensed moisture.

11. B. DR Y -HEA T STERILIZA TION • Dry heat sterilization is widely used to sterilize glassware and equipment parts in manufacturing areas for parenteral products. • Is usually carried out in sterilizing ovens specifically designed for this purpose. • Because dry heat is less effective in killing microorganisms than is moist heat, higher temperature and longer period of exposure are required. Depending on the size and type of product and on the container and its heat distribution characteristics.

12. Individual unit to be sterilized should be as small as possible, and the sterilizer should be loaded in such a manner as to permit free circulation of heated air throughout the chamber. • Two principal methods of dry-heat sterilization are infrared and convection hot air) Infrared rays will sterilize only surfaces • Dry heat sterilization is usually conducted at temperature of 160-1700C for 2 hrs or 2600C for 45 min. Higher temperatures permit shorter exposure time. • If a chemical agent melts or decomposed at 170 °C, but is unaffected at 140 °C, the lower temperature must be used and the exposure time would be increased

13. Dry heat kill microorganisms primarily through oxidation. • Dry heat sterilization is generally employed for substances that are not effectively sterilized by moist heat such as: • a. Fixed oils, • b. Glycerin, • c. Various petroleum products such as petrolatum, liquid • petrolatum (mineral oil), • d. Various heat-stable powders such as zinc oxide, kaolin and • sulfur.

14. C. GAS STERILIZATION (ETHYLENE OXIDE) • Some heat-sensitive and moisture-sensitive materials can be sterilized by exposure to ethylene oxide (ETO) or propylene oxide gas than by other means • (ETO) gas is a colorless gas and widely used as a sterilant in hospitals and industry • These gases are highly flammable when mixed with air but can employed safely when properly diluted with an inert gas such as carbon dioxide or a suitable fluorinated hydrocarbon.

15. The mechanism by which ETO kills miroorganisms is by alkylation of various reactive groups (interference with the metabolism) in the spore or vegetative cell. • One of the more resistant organisms to ETO is B. subtilis. Which can be used as USP biological indicator for monitoring the effectiveness of ETO sterilization cycle.

16. Several factors are important in determining whether ETO is effective as a sterilizing gas. • a. Gas concentration (500mL/L), • b. Temperature (50-600C), • c. Humidity (60%), • d. Exposure time (4-16 hrs)

17. Sterilization Process The basis of the lethal action of radiations on microorganisms is the production of ionizations and excitations when radiation traverses the cell. However, lethally irradiated cells remain intact, respiration continues normally, motile cells retain their motility for some time and, under conditions where reproduction is possible, death may not occur until after one or two divisions of a cell.

18. APPLICATION OF GAS STERILIZATION • The great penetration qualities of ETO make it a useful sterilizing agent in special applications: • Sterilization of medical and surgical supplies such as • catheters, needles, and plastic disposable syringes in • their final plastic packaging • b. Sterilize certain heat-labile enzyme preparations • c. Certain antibiotics, and other drugs (with tests to assure • of the absence of chemical reactions)

19. D. STERILIZATION B Y FILTRATION Sterilization by filtration, depends upon the physical removal of microorganisms by adsorption on the filter medium or by sieving mechanisms, is used for sterilization of heat-sensitive solutions Filters have variety of pore-size specifications; one of these filters, the millipore filters

20. Millipore filters are: • Are thin plastic membranes of cellulosic esters with millions of pores/square inch of filter surface • The pores are extremely uniform in size and occupy approximately 80% of the filter membrane's volume • 3) The remaining 20% being the solid filter material • 4) This high degree of porosity permits high flow rates • 5) Millipore filters are made from a variety of polymers to provide membrane characteristics required for the filtration of almost any liquid or gas system

21. 6) Filters are made of various pore size to meet the • selective filtration requirements • 7) They are available in pore size from 14-0.025μm where the smallest bacteria, about 0.2μm, and viruses about 0.025μm

22. Factors that affecting the removal of M.O. A. Pore size of filter B. Electrical charge of the filter and that of the M.O., C. PH of the solution, D. Temperature, E. Pressure or vacuum applied

23. ADVANTAGES OF BACTERIAL FILTERS • a. Its speed in the filtration • b. Its ability to sterilize thermolabile materials • c. Inexpensive equipment required • d. The complete removal of living and dead M.O. as well as other particulate matter from the solution

24. DISADVANTAGES OF BACTERIAL FILTERS • The membrane is fragile thus it is essential to be sure that • the membrane is not ruptured • b. Filtration of large volumes of liquids would require more • time (particularly if the liquids were viscous) • c. Are useful when heat cannot be used and small volumes • of liquids

25. RADIO-STERILIZATION

26. Advantages of Radiosterilization 1) Highly effective. 2) Treatment times is very short. 3) It is a continuous process suitable for long production run. 4) The thermal conductivity of the material is irrelevant. 5) It is a cold process resulting in a temperature rise of only a few degrees and so is suitable for thermolabile materials. 6) Materials may be irradiated in the dry or frozen state 7) Products are processed in the final container after packaging with no risk of subsequent contamination until used. 8) The process may be controlled and monitored accurately.

27. Applications • Sterilization of materials for which conventional methods are unsatisfactory (catgut, rubber, certain dressings, oils) or to disposable plastic materials which cannot be heat sterilized. • Medical applications as the sterilization of tissue graft materials, such as freeze dried bone or aorta, the production of vaccines and the elimination of the virus of serum hepatitis by the irradiation of freeze-dried plasma. • In food sterilization using low radiation doses to eliminate insect infestations of stored foods.

28. 4.A list of medical products which are known to have been radiosterilized is as following: • Antibiotics of the tetracycline group. • Arterial prostheses. • Cardiac valve prostheses. • Endotracheal tubes. • Cannulae. • Atropine eye drops. • Chloramphenicol eye ointment. • Dialysis units.

29. Plastic catheters, gloves, hypodermic syringes (with • needles), Petri dishes, tubing. • Rubber catheters and gloves. • Surgical blades • Surgical dressings: bandages, gauze, eye pads, swabs. • Sutures: catgut', collagen, silk, polyester, nylon. • Transfusion giving and taking sets.

30. Selection of the Sterilizing Process • The criteria for the selection of the type of radiation • include the following: • The specific ionization produced should be relatively low. A relatively small number of ionizations within a microorganism is sufficient to cause inactivation so radiations which produce a high specific ionization are less efficient and are more likely to damage the product. • The radiation should be capable of adequate penetration of the material and deliver a reasonably uniform dose therein.

31. (3) The radiation source should be capable of delivering high doses economically, at the same order of cost as conventional sterilization techniques. (4) It should be possible to irradiate materials in any physical state. (5) The dose must be accurately reproducible and capable of accurate measurement and control. (6) The safety of operators should be ensured and radioactivity should not be induced in any irradiated material. The only types of radiation to meet the above criteria are γ-rays and electron beams. Other radiations have limited and specialised uses.

32. Determination of the Sterilizing Dose • The selection of the radiation dose required to sterilize various materials depends on several factors: • The nature of the species present and their radiation resistance in the particular environment and under the conditions of processing used, • The margin of safety required.

33. The wide variations in sensitivity between species has already been referred to and the most resistant species likely to occur as a contaminant in the material and the degree of such contamination, are the two most important considerations. Since the principal type of material sterilized by radiation comprises individual solid items, nonuniformity of contamination is the normal situation and results in a higher dose requirement than if contamination were uniform, since all items have to be treated according to the highest contamination level which occurs.

34. The margin of safety required depends on the risk of the occurrence of non-sterile articles which is considered to be acceptable for the intended purpose, thus the sterilization process is a probability function. An increase in the dose, and so in the inactivation factor, gives a lower probability of residual contamination.

35. Thus selection of an appropriate sterilizing dose may be difficult and in the absence of precise information about the initial conditions large doses are used, in the range 2.5 to 4.5 Mrad. The 2.5 Mrad dose has been widely accepted but it has been suggested that this may be inadequate. With these high doses, especially at 4.5 Mrad, the effects of irradiation on the material may be important and each type of product requires careful evaluation as to stability, activity, physical properties, storage life and acute and chronic toxicity.

36. Factors Affecting the Radiation Sensitivity The Species: The variation in radio-sensitivity between species is the most important factor in the determination of the sterilizing effect of a given dose. The radio-resistance of microorganisms generally increases in the order vegetative bacteria, fungi, bacterial spores, viruses.

37. Inoculum level: The higher the initial population the greater the dose required to achieve inactivation. This population effect causes the need to keep the initial degree of contamination as low as possible so that the sterilization of articles is achieved with minimum dose. Gaseous environment: The presence or absence of oxygen during and after irradiation influences the sensitivity to radiation damage.

38. Degree of Hydration: Microorganisms are less radiation sensitive under dry conditions than in the presence of water due to the absence of indirect effects arising from the radiolysis of water. There are a 30 % change in the inactivation rate over a range of about 20 to 40 per cent relative humidity.

39. Temperature: • The radiation sensitivity of microorganisms is reduced at, • or below, the freezing point of water, due to the • immobilization of free radicals and reactive species • produced in the water, thus reducing indirect effects. • At temperatures used for sterilization there is a synergistic effect of temperature and irradiation that sterility may be achieved by a combination of milder treatments of both sterilizing agents. This phenomenon is of potential practical importance in the sterilization of products such as foodstuffs which are both heat and radiation labile.

40. Stage of Cell Division: The sensitivity of vegetative bacteria varies during the growth cycle that the growth phase affect the dose required if there is the possibility of growth in the material to be sterilized. Since bacterial spores are more resistant than vegetative cells of the same species there is an sudden increase in radiosensitivity as spore germination occurs.

41. Protective and Sensitizing Agents: • A large variety of compounds affect the radiation sensitivity of microorganisms. • Protective agents include reducing agents (cysteine, thiourea, cysteamine, β-mercaptoethanol), alcohols and polyhydric compounds (ethanol, glycerol, mannitol) and proteins or complex foodstuffs. • The effect of free radical scavengers abolish part of the oxygen effect. • Sensitization has been reported with oxidizing agents (nitrate, nitrite), water soluble stable free radicals. • Vitamin K5 has been reported both as a sensitizer and as a protective agent with different bacterial species.

42. Testing of Sterilization Process Two types are performed to test the efficiency of sterilization: • Testing the sterility of the final product • Testing the sterilization process (by physical, chemical and biological methods) to confirm that the equipment is working satisfactory.

43. Indicator Tests For Indication Of The Efficiency Of The Sterilization Process Physical indicator tests • The performance of steam and gas sterilizers can be tested by observing the reading of temperature, pressure, vacuum gauges and stage timers throughout an automatically controlled sterilization cycle. • Recorded charts should be examined carefully. • 2) Thermocouples test: is used to measure the temperature at selected sites in the chamber or within the load of a dry heat, steam or gas sterilizer.

44. 3) In case of sterilization by radiation: A measurement of radiation dose i.e. the amount of energy absorbed by the material tested. • In case of sterilization by filtration: • A bubble pressure test is used to determine the pore size of filters.

45. Chemical indicator tests Types that cannot indicate time of exposure: A) Klintex papers: These are paper strips or stickers attach to each object to be sterilized. The word (sterile) is written on the strip (colorless) but after exposure to the sterilizing agent as steam the word (sterile) will be cleared.

46. B) Klintex test tablets: These contain 75% lactose, 24% starch and 1% magnesium trisilicate. They are hard and white but after steam sterilization they become brown and gelatinous. They should be examined soon after removal from the sterilizer.

47. C) Autoclave test tape (Bowie – Dick test): This is a valuable test for confirming that the steam has displaced all the air from a porous load (i.e. air removal test). The tape carries heat sensitive bars which become colored if steam has full penetrated the pack. If air remains, the bars in the centre are lighter in color.

48. Types that indicate time of exposure: Browne’ tubes: Each tube consists of a sealed glass tube which contains a red fluid (an ester and acid - base indicator) that changes to yellow, brown and finally green on heating (the ester undergoes heat hydrolysis to form an acid + alcohol. The acid will change the color of the indicator).

49. There are four types of Browne’s tubes: • Browne’s tube type I:Suitable for ordinary steam sterilizers • Browne’s tube type II:Suitable for high vacuum sterilizers • Browne’s tube type III:Suitable for hot air oven • Browne’s tube type IV:Suitable for I.R conveyer oven.

50. Biological indicator tests Biological indicators consist of bacterial cultures which are usually used in the form of impregnated strips e.g. paper and metal foil and are placed in different sites in the sterilizer. At the end of the process, the bacteria are transferred to a nutrient medium which is incubated and the presence or absence of growth is noted.