A. Sterilization

1. King Saud University College of Pharmacy Department of Pharmaceutics PHT 351: Sterile Dosage Forms Summer Semester of 1423-1424H A. Sterilization Ibrahim A. Alsarra, Ph.D.

2. Outlines • Principles of Sterilization: Introduction • Methods of Sterilization: An introduction • Sterilization Criteria • Sterilization Validation and Monitoring PHT 351: Sterile Dosage Forms

3. I. Principles of Sterilization PHT 351: Sterile Dosage Forms

4. Introduction Definitions and Terminologies • Sterility: The total absence of viable microorganisms and it is an absolute state • Sterilization: The inactivation or elimination of all viable microorganisms and their spores, based on a probability function. The sterilization process is usually the final stage in the preparation of the product. • Aseptic Processing: Those operations performed between the sterilization of an object or preparation and the final sealing of its package. These operations are carried out in the complete absence of microorganisms. PHT 351: Sterile Dosage Forms

5. Introduction …(cont.) • Disinfection: A process which aims to reduce the number of harmful (pathogenic) microorganisms in a particular situation. It is not an absolute process i.e. will eradicate infective vegetative organisms but not spores. • Antiseptics: Chemical substances applied to living tissues in humans or animals in order to arrest or prevent the growth of microorganisms by inhibiting their activity without necessarily destroying them. • Bactericide: Any agent that destroys microorganisms PHT 351: Sterile Dosage Forms

6. Introduction …(cont.) • Bacteriostat: Any agent that arrests or retards the growth of microorganisms. • Germicide: Any agent that destroys microorganisms, but not necessarily bacterial spores. • Sterility Assurance Level (SAL): A term related to the probability of finding a nonsterile unit following a sterilization step. It usually is expressed in terms of the negative power of 10 (i.e. 1 in 1 million = 10-6). • Bioburden: The number of viable microorganisms in or on an object or population entering a sterilization step (usually expressed in colony forming units per unit time). PHT 351: Sterile Dosage Forms

7. Introduction …(cont.) The aim of sterilization process: Is to destroy or eliminate microorganisms that are present on or in an object or preparation, to make sure that this has been achieved with an extremely high level of probability and to ensure that the object or preparation is free from infection hazards. The currently accepted performance target for a sterilization process: Is that it provide a probability of finding a nonsterile unit of less than 1 in 1 million. That is, the process (including production, storage, and shipment) will provide a Sterility Assurance Level (SAL) equal to or less than 10-6. PHT 351: Sterile Dosage Forms

8. Contamination: General Facts Some microbes (bacteria, molds, etc) multiply in the refrigerator, others at temperatures as high as 60o. Microbes vary in their oxygen requirements from strict anaerobes that cannot tolerates oxygen to aerobes that demand it. Slightly alkaline growth media will support the multiplication of many organisms while others flourish in acidic environments. Some microorganisms have the ability to use nitrogen and carbon dioxide from the air and thus can actually multiply in distilled water. In general, however, most pathogenic bacteria have rather selective cultural requirements, with optimum temperatures of 30 to 37 and a pH of 7.0 PHT 351: Sterile Dosage Forms

9. II. Methods of Sterilization PHT 351: Sterile Dosage Forms

10. PHT 351: Sterile Dosage Forms

11. III. Sterilization Criteria PHT 351: Sterile Dosage Forms

12. 1. Death Rate or Survival Rate Each sterilization method can be evaluated using experimentally derived values (death and survival rates) representing the general inactivating rates of the process. Plotting the logarithm of surviving organisms against time of exposure to the sterilization method. In most instances, these data show a linear relationship, typical first order kinetics, and suggest that a constant proportion of contaminant population is inactivated in any given time interval. Based on such inactivation curves, it is possible to drive values that represent the general inactivation rates of the process. For example, based on such data, it has become common to drive a decimal reduction time or D value, which represents the time under a stated set of sterilization exposure conditions required to reduce a surviving microbial population by a factor of 90%. PHT 351: Sterile Dosage Forms

13. 1. Death Rate or Survival Rate… (cont.) Death value (D): is the time required to reduce the population by 90% at specified temperature. D value can be obtained from the death curve (i.e. the straight line relationship between the log viable count of a bacterial population and time when the population is exposed to a lethal temperature. The graph shows three hypothetical population of the same organism exposed to the same killing agent (e.g. heat). Notice that the populations A, B, and C die at the same rate (all three lines are parallel) although population C dies sooner than B and B dies sooner than A because of the initial sizes (ml of microorganism) are smaller. PHT 351: Sterile Dosage Forms

14. 1. Death Rate or Survival Rate…(cont.) New Finding It is sometimes known as Thermal death time was found that complete killing of microorganisms is approached by applications of 6 D values. By extending the process to include 6 D values, most of the remaining population is inactivated, reducing the population of one organism surviving to one in 1 million. PHT 351: Sterile Dosage Forms

15. 2. D Value and Inactivation Factor (IF) From the D value for a particular combination of organism/time/temperature an “inactivation factor” (IF) can be calculated, e.g. if the D value of an organism exposed to a temperature of 121 °C for 15 minutes was 2 minutes: The IF would be 1015/2= 107.5 PHT 351: Sterile Dosage Forms

16. 3. Death Rate Constant (K) • Microbiologist in various industries employee a value known as the death constant (k) to compare susceptibilities of different microorganism to a given control agent. The more rapidly a population can be sterilized, the longer the death rate constant. • The death rate constant is the same and independent of the initial population size. It would only be different if were are comparing different organisms. The death rate constant can be calculated using the following formula: Where: T= Time in minutes of exposure to a killing agent N0= Initial number of microorganisms Nt = Final number of microorganisms after treatment PHT 351: Sterile Dosage Forms

17. 3. Death Rate Constant (K)…(cont.) Example IF at time 0, the size of population is 10,000,000 and after 3 minutes the population is reduced to 10,000; the K value is found to be 1.00 for curve A. Calculate K value for curve B and C? PHT 351: Sterile Dosage Forms

18. 4. Z Value or Thermal Destruction Value Z value relates the heat resistance of a microorganism to changes in temperature. The Z value is the number of degrees in temperature change required to produce a 10-fold change in D value. Bacterial spores have a Z value in the range 10-15 °C while most non sporing organisms have Z values of 4-6 °C. PHT 351: Sterile Dosage Forms

19. 4. Z Value or Thermal Destruction Value … (cont.) Example: If the D value for Bacillus stearothermophilus spores at 110 °C is 20 minutes and they have a Z value of 9 C, this means that at 119 °C the D value would be 2.0 minutes and at 128 °C the value Z value would be 0.20 minutes. PHT 351: Sterile Dosage Forms

20. 5. Q Value or Temperature Coefficient Q value also gives a measure of the relative heat resistance of different microorganisms and describes the change in the death rate over a 10 °Cchange in temperature. It does the same, like Z value, but is less commonly used. PHT 351: Sterile Dosage Forms

21. 6. F Values • F value is a measure of the deadliness or lethality of the total process of sterilization and equates heat treatment at any particular temperature with the time in minutes at a designated reference temperature that would be required to produce the same lethality in an organism of stated Z value. The death rate constant can be calculated using the following formula: Where: Tc= Load temperature at time dt Z= 10 °C PHT 351: Sterile Dosage Forms

22. VI. Sterilization Validation and Monitoring PHT 351: Sterile Dosage Forms

23. Indicators for Validation …(cont.) A. Biological Indicators • It involves incorporating a viable culture of a stated species of microorganisms. The biological indicators are usually used to check or monitor the efficacy of a sterilization cycle. • The efficacy of a biological indicator depends on: • 1- Sensitivity • 2- The viability of the organisms • 3- The storage conditions before use and after the • incubation • 4- The conditions of the culture after sterilization PHT 351: Sterile Dosage Forms

24. Indicators for Validation …(cont.) A. Biological Indicators …(cont.) PHT 351: Sterile Dosage Forms

25. Indicators for Validation …(cont.) B. Chemical Indicators • They are used to indicate whether a particular batch of product has been through a sterilization process; they are not used to indicate whether a specific process of sterilization was suitable or successful. • Disadvantage: • They undergo some physical and chemical changes when exposed to the conditions of the sterilization process. PHT 351: Sterile Dosage Forms

26. Indicators for Validation …(cont.) B. Chemical Indicators … (cont.) PHT 351: Sterile Dosage Forms