Pharmaceutical Microbiology Lecture 1 Introduction to Sterilization PDF

Sally A. Mohamed, PhD Department of Microbiology and Immunology Faculty of Pharmacy, Cairo University, Egypt [email protected] Pharmaceutical Microbiology Lecture1 Introduction to Sterilization  Microbiological Quality The incidence and level of m...

Sally A. Mohamed, PhD Department of Microbiology and Immunology Faculty of Pharmacy, Cairo University, Egypt [email protected] Pharmaceutical Microbiology Lecture1 Introduction to Sterilization  Microbiological Quality The incidence and level of microbial contamination in a pharmaceutical product. Microbiological quality in pharmaceuticals: Why ? Humans live in an environment, which is rich in unseen microorganisms that contaminate everything they handle. The level of microbial contamination of a product is in principle inversely proportional to the level of hygiene applied during manufacture.  Specifications Various compendia describe minimum standards that each pharmaceutical must meet in order to assure its efficacy and safety to the patient. Products with better specification are encouraged, but those with lower specification are not permitted. Compendia The concepts referred below are not exclusively pharmaceutical concepts: it applies to all industries and services, including agricultural and animal production systems.  Good Manufacturing Practice (GMP) Provision of suitable premises, equipment, and environment with trained personnel to operate approved procedures for production.  In Process Control (IPC) In-Process Control is commonly understood as checks being performed during a production process for the purpose of monitoring and if necessary, to adjust the process to assure that the product conforms to its specifications.  Microbiological Quality Control (QC) Analytical procedures performed on a pharmaceutical product in order to test for conformity to specifications of microbiological quality. "destructive tests" Applied to a "sample" of a “batch”  Good Analytical Practice (GAP) This is equivalent to GMP, as applied to analytical procedures. It is also called Good Laboratory Practice (GLP).  Microbiological Quality Assurance (QA) Measures taken during manufacture which ensure that the product will in fact comply with specifications for microbiological quality.  Total Quality Assurance (TQA) The net result of GMP, GAP, and QC which "builds quality into the product" assuring the highest possible level of "error-free" product. The concept of TQA has now been expanded by the International Standard Organization in the form of the "ISO" system for certifying not just total quality but also the capability of TQA. ISO-17025 (LABS). Pharmaceutical preparations Criteria and standards for the microbiological quality of medicines depend upon the route of administration of the medicine in question. Sterile: parenteral, ophthalmic products Nonsterile: tablets, syrup, skin ointment and suppository  Microbial Quality of Parenteral Preparations Since such products are to be applied to sterile human tissues. Such products require: testing for sterility as a QC procedure. Microbiological Quality of Ophthalmic Preparations Compendia requirements for ophthalmic includes : I. Be sterile upon dispensing II. Be filled in containers which reduce the chances of microbial contamination during storage and/or use by the patient (the use of single- dose containers proved impractical) in small volumes (mostly not exceeding 5 ml). III. Contain a preservative which is especially effective against Ps. aeruginosa, sufficiently powerful to “more or less re-sterilize” the preparation within a few hours of re- contamination.  Microbial quality of Nonsterile products ▪ Total microbial count ▪ Absence of specified pathogens The sources of contamination  Considerations that limit contamination a) Appropriate design of premises and layout of equipment. b) Careful design of Standard Operating Procedures (SOP) for production and for maintenance of premises and of equipment. c) Calibration of equipment. d) Validation (retrospective, concurrent and prospective) of the performance of equipment, procedures and personnel. e) Monitoring of microbial contribution of various elements of the process as part of In Process Control (IPC) and documentation of the batch records. f) Use of preservatives g) Elimination of the microorganisms through Sterilization and disinfection Microbial Death!  Kinetics of microbial death and death (survivor) curve In bacteria, death could be defined as the potential inability to multiply, not the lack of active metabolism. t= 1/K log a/b 𝒕 = extinction time 𝑲 = constant depending on the nature of the reaction 𝒂 = initial number of viable cells (bioburden) 𝒃 = number of remaining viable cells The rate of killing process is proportional to the initial number of viable cells (𝒂) 17  Kinetics of microbial death and death (survivor) curve Plotting log 𝑏 against t, a straight line is observed with negative slope representing the exponential or logarithmic phase of death (the longer the contact time with the killing agent, the greater number of dead cells). 18 How to decrease the Bioburden? Physical: heat, radiation …... etc Chemical: Antibiotics, Disinfectants.. etc Sterilization!  What is sterilization? Sterilization is the process of freeing of an article from all living organisms including spores, either through killing or removing. The product may contain dead microorganisms, except when filtration is used where the final product will not contain living or dead organisms. Sterility is an absolute term either sterile or non-sterile. An article should never be described as partially or relatively sterile. Sterility is uncertain and so it is more useful to describe the product subjected to a sterilization process as sterilized rather than sterile. 20 Sterilization is required in the following fields Microbiology: media, containers, suspending fluids and contaminated items. Biotechnology: fermentation media and vessels. Microbiology Biotechnology Pharmacy: injectables, ophthalmic, drugs for contact with broken skin or internal organs. Surgery: dressing, sutures, tools and equipment. Injectables Medical devices: syringes, gloves, canules, catheters..etc. Computer and space industry. Computer Surgery What are the methods for sterilization? A. Physical (does not remain in the final preparation) Dry and moist heat Radiation (gamma rays, accelerated electrons or β-particles and to a less extent, ultraviolet rays) B. Filtration (removes rather than destroys microorganisms) C. Chemical (remains in the final preparation except for gases) Gases (ethylene oxide, formaldehyde, steam) Heating with bactericide 22  How to determine the basis for a successful sterilization process? Knowing the Knowing the properties of the Knowing the nature properties of the product to be of possible sterilizing agent sterilized contaminants  Expression of resistance to Sterilization Thermal death point (TDP): lowest temperature required to kill all M.O. in suspension within certain time (usually 10 min). Thermal death time (TDT)(Extinction Time) (t): shortest time required to kill all M.O. in a suspension at certain temperature. Decimal reduction time (D-value): Time at fixed temp /or/ Radiation dose required to reduce the number of viable cells by 90% or one log cycle, assuming linear curve and must be corrected in cases where there is variation from linearity. -It could be used as a measure for the degree of resistance of an organism at particular temperature. 25  D value refers to resistance at a specific temperature (heat sterilization). To assess the relationship of influence of temperature change on thermal resistance, Z value is used: Z-value: The increase in temp required to reduce the D-value of an organism by 90% or one log cycle. It is a measure for the change in resistance upon changing the temperature. Smaller Z-Values indicate sensitivity to heat. 26 Inactivation Factor (IF): It is a measure of the destructive power of a sterilization process. Significance of sterilization The efficacy of a sterilization process can be controlled by its inactivation factor "IF" or its total destructive power There is always a limit to the maximum IF applied otherwise, treatment will be too harsh and product is destroyed. The standard IF applied is 108 , meaning a reduction in the count of microorganisms by 108. (as a numeric example: 108 to 100) (8D values), this is the most commonly used IF. 27 Sterility Assurance Level (SAL): a numerical value denoting the probability of finding a single surviving organism in a product. (practically, the probability of finding a non- sterile container in the batch). It is dependent on the initial count "a" or bioburden SAL = a/IF If the original bioburden (a) is 100 and If the sterilization process has an "IF" of 108 SAL= 102/108 =10-6 (1:106) This means that among 106 containers, there exists one nonsterile container (1 in 1 million units). -If the bioburden is originally high (e.g 108)and the sterilization process has an "IF" of 108 then the SAL = 108/108 = 1(all containers are nonsterile), (1:1).  Official methods for sterilization Heat Cold Moist Heat Dry Heat Gaseous Radiation Filtration (autoclaving) 29  Sterilization by heat Each microorganism (M.O.) has an optimum temperature for growth , below its growth decreases reversibly and above it decreases irreversibly due to degradation of vital molecules. The equation describing the effect of heat: log t = 0.219 E + K T t = extinction time T = absolute temp (T , t ) K = constant depends on the No. and type of the most resistant M.O. E = heat of activation energy required to kill the most resistant M.O. 30  Mode of action of heat Dry heat (no water) Moist heat (water) Death is due to slow protein Death is through protein and nucleic acid denaturation through oxidation of denaturation due to coagulation (the loss certain groups in the protein molecule of the tertiary structure of proteins). requires high E and longer t depends on the presence of water (steam). requires low E and shorter t ✓ The main difference between the two forms of sterilization is how heat is transferred from the sterilization environment to the product in need of sterilization. ✓ For dry heat sterilization, heated air is in direct contact with the load items and the transfer of its thermal energy onto the load items. ✓ Moist heat steam works by transferring the energy contained within heated and pressurized steam (saturated steam) onto the load items. 31 Mode of action of heat (continued) At constant temperature, bacteria are killed by moist heat in shorter time than dry heat. (T/F Question) When applying heat in presence of a bactericide chemical agent, death is due to the chemical agent and heat only enhances the rate of reaction or decreases the resistance of the microorganism to the chemical agent. 32  Factors affecting heat sterilization 1. Time and temperature of exposure. 2. Nature of the article to be sterilized. 3. Number of microorganisms (bioburden). 4. Type of microorganisms (species, strain, spore formation). Sensitivity of microorganisms to sterilization Generally, microbial sensitivity to heat sterilization follows the following pattern: a. The most sensitive forms are vegetative bacteria, fungi, yeast and large viruses. Significant response to moist heat at 60-70 °C. b. Less sensitive forms are thermophilic bacteria, small viruses, resistant fungal spores. Significant response to moist heat at 70-90 °C. c. The most resistant forms are bacterial endospores which must be subjected to temperature in the range of 90-115 °C with full hydration before significant reduction in viability occurs. 34  Why are endospores resistant to heat? 1. Contain the least amount of water (10% unbound water). 2. Spore wall contains calcium and dipicolinic acid which prevent the penetration of moisture but does not prevent the penetration of heat. 3. SH groups of proteins are protected through the formation of disulfide bond. 35