Sterilization Procedures & Sterility Assurance

Sterilization Procedures & Sterility Assurance Chapter 21 Introduction • Sterilization is killing or removing all forms of microbial life from a product through the application of a biocidal agent or a certain physical process • Sterilization is required for: - Parenteral and ophthalmic products - Products in contact with broken skin & mucosal surfaces or internal organs - Minimizing the health hazard associated with contaminated items such as microbiological materials and soiled dressings • Sterilization is usually achieved by elevated temperature (moist or dry), reactive gas, irradiation, filtration or other new methods. 1 Introduction • In sterilization, there is a potential risk of product damage, which for a pharmaceutical preparation may result in reduced therapeutic efficacy, stability or patient acceptability • Therefore, there should be a balance between - the maximum acceptable risk of failing to achieve sterility and - the maximum acceptable level of product damage i.e. we need to ensure maximum microbial kill/removal with minimum product deterioration • This is best determined from a knowledge of - the properties of the sterilizing agent - the properties of the product to be sterilized - the nature of the likely contaminants Sensitivity of Microorganisms • There is a general pattern of sensitivity, regardless of the utilized method, to sterilization processes : Vegetative bacteria or fungi and larger viruses > small viruses and fungal spores > bacterial spores • Therefore most durable bacterial spores are used as reference organisms (biological indicators) for testing the efficiency of sterilization processes • These are usually represented by: - Bacillus stearothermophilus for moist heat - B. subtilis for dry heat and gaseous sterilization - B. pumilus for ionizing radiation • Pharmacopoeial methods were developed by careful analysis of experimental data on the survival of these bacterial spores following treatment with heat, ionizing radiation or gas. However, prions still form problems & doubts surround the adequacy of the suggested processes against them 2 Survivor curves • When exposed to a killing process, populations of microorganisms generally lose their viability in an exponential fashion, independent of the initial number of organisms • This can be represented by the ‘survivor curve’, which is a plot between the log of the fraction of survivors against the exposure time or dose • Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally be applied to any biocidal process. Survivor curves • Generally, all survivor curves have a linear portion which may be A. B. Continuous Modified by an initial shoulder C. Modified by reduced rate of kill at low survivor levels N.B. a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores 3 Expressions of Resistance • D-value - It is the time (in heat treatment) or dose (in radiation treatment) required at certain conditions to achieve 1-log cycle ( i.e. 90%) reduction in survivors - The calculation of the D-value assumes a linear survivor curve (type A), and must be corrected to allow for any deviation from linearity with type B or C curves Expressions of Resistance • D-value calculation 4 Expressions of Resistance • Z-value - Used in order to assess the influence of temperature changes on thermal resistance to heat treatment - It represents the increase in temperature needed to reduce the D-value of an organism by 90% (i.e. 1-log reduction) - For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean Zvalues are given as 10°C and 22°C, respectively Expressions of Resistance • Z-value calculation 5 Sterility Assurance • The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever - Thus, there are no degrees of sterility; an item is either sterile or it is not • Survivor curves showed that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. • Survivor curves also show that true sterility, i.e zero survivors, can only be achieved after an infinite exposure period or radiation dose. - Then, it is illogical to claim, or expect, that a sterilization procedure will guarantee true sterility. Sterility Assurance • Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process - i.e. there is no practical absolute 'sterile' term • According to this, the concept of sterility assurance level (SAL) or a microbial safety index has arisen which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. • For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a nonsterile unit is 1 in 1 million units processed (i.e. SAL of 10-6). 6 Sterility Assurance • At Y, there is 10-1 bacterium in one bottle, i.e. in 10 loads of single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z, there is 10-6 bacterium in one bottle, i.e. in 1 million (106) loads of single containers, there is one chance in 1 million that one load would be positive. Sterility Assurance • The sterilization protocol necessary to achieve such sterility assurance with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: 𝑡 𝐼𝐹 = 10𝐷 where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed The needed IF is the ratio between the initial bioburden and the required sterility assurance i.e. 𝑛𝑒𝑒𝑑𝑒𝑑 𝐼𝐹 = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑏𝑖𝑜𝑏𝑢𝑟𝑑𝑒𝑛 𝑠𝑡𝑒𝑟𝑖𝑙𝑖𝑡𝑦 𝑎𝑠𝑠𝑢𝑟𝑎𝑛𝑐𝑒 7 Sterility Assurance • Example: - A product with initial bioburden of 104 spores is to be sterilized by moist heat. Given that the Dvalue is 1.5 minutes, calculate the inactivation factor (IF) and treatment time (t) needed to achieve a sterility assurance of 10-6! - Answer: IF = 1010, t = 15 min. Sterilization Methods • A sterilization process should represent a compromise between achieving good antimicrobial activity and maintaining product stability. • Validation based on the intended application and continuous monitoring of a sterilization process is always required. • Sterilization is not an alternative to GMP; it is only the final stage in a program of microbiological control. • 5 methods of sterilization are recognized in Eur Ph: • 1. Steam sterilization (heating in an autoclave) 2. Dry heat 3. Ionizing radiation 4. Gas sterilization 5. Filtration Other methods of sterilization also exist such us UV light and steam/formaldehyde. 8 Heat Sterilization • Heat is the most reliable and widely used means of sterilization. It acts through destruction of enzymes and other essential cell constituents. • Mechanism of action: the lethal effects of heat proceed most rapidly in a fully hydrated state, thus requiring a lower heat input (temperature and time). In this case denaturation and hydrolysis reactions predominate. While in dry heat oxidative changes take place. • Heat sterilization is limited to thermostable products. • For moisture-resistant products moist (121-134 °C) heat sterilization is used, while for moisture-sensitive products dry heat sterilization (160-180 °C) is applied. • If thermal degradation of a product might possibly occur, it can be minimized by selecting the higher temperature range; since the shorter exposure times employed generally result in a lower fractional degradation. Heat Sterilization Processes • In heat sterilization, the articles to be treated must first be raised to sterilization temperature which involves a heating-up stage, then timing for the process at a certain temp. (The holding stage) begins and proceeds until a cooling-down stage. • However, during both the heating-up and cooling-down stages of a sterilization cycle, the product is held at an elevated temperature and these stages contribute to the overall biocidal potential of the process. An approach has been set to convert these stages to the equivalent time at fixed temp! 9 F-value in Heat Sterilization • During the heating, sterilizing and cooling stages of a moist heat (steam) sterilization cycle temperature-time combinations can be converted to the equivalent time at 121 °C. • Using this approach the overall lethality of any process is defined as the F-value, which represents the heat treatment at any temperature as equal to that of a certain number of minutes at 121 °C. • In other words, if a moist heat sterilization process has an F-value of x, then it has the same lethal effect on a given organism as heating at 121 °C for x minutes, regardless of the actual temperature employed or of any fluctuations in the heating process due to heating and cooling stages. F-value in Heat Sterilization • The F-value of a process will vary according to the moist heat resistance of the reference organism. • When the reference spore is B. stearothermophilus with a Z-value of 10°C, then the F-value is known as the F0-value. • There is a relationship between F- and D-values, leading to an assessment of the probable number of survivors in a load following heat treatment F = D (log N0-log N) D: D-value at 121 °C, and No and N represent, respectively, the initial and final number of viable cells per unit volume. 10 F-value in Heat Sterilization • The F-concept enables a sterilization process to be individually developed for a particular product. This means that adequate sterility assurance can be achieved in autoclaving cycles in which the traditional pharmacopoeial recommendation of 15 min at 121 °C is not achieved. • The holding time may be reduced below 15 min if there is a substantial killing effect during the heating and cooling phases, and an adequate cycle can be achieved even if the 'target' temperature of 121 °C is not reached. • Thus, F-values offer alternative sterilizing cycles and a mechanism by which over-processing of marginally thermolabile products can be reduced without compromising sterility assurance • F-values have application in the sterilization of medical and pharmaceutical products by moist heat, less frequently, a direct parallel is used in dry heat sterilization (FH) F-value in Heat Sterilization • F0 value may be calculated either from integrating the 'area under the curve' of a plot of autoclave temperature against time or by using the following equation: F0 = Δt Σ10(T – 121)/Z Where Δt : time interval between temperature measurements; T = product temperature at time t; Z is (assumed to be) 10 °C. - e.g. temp measurement at 1min interval, temp 115 °C if maintained for 1 min will give F0 =0.25 min • In pharmacopeia, the 'preferred' combination of temperature and time is a minimum of 121 °C maintained for 15 minutes, which equates to an F0 value of 15. 11 Moist Heat Sterilization • Usually performed in an autoclave and involves the use of steam at temperatures in the range 121-134°C. • Moist heat sterilization methods can only be achieved by the generation of steam under pressure. • Application: sterilization of culture media, dressings, sheets, surgical and diagnostic equipment, containers and closures, aqueous injections, ophthalmic preparations, irrigation fluids in addition to the processing (decontamination) of soiled and contaminated items • The most commonly employed standard temperature/time cycles for bottled fluids and porous loads (e.g. surgical dressings) are 121 °C for 15 minutes and 134°C for 3 minutes, respectively. Moist Heat Sterilization 12 Moist Heat Sterilization • Advantage of high temperature-short time cycles: lower fractional degradation & achieving higher levels of sterility assurance due to greater inactivation factors Moist Heat Sterilization • To act as sterilizing agent, steam should provide moisture and heat efficiently to the article to be sterilized. • This is most effectively done using saturated steam, which is steam in thermal equilibrium with the water from which it is derived (i.e. steam on the phase boundary). • Under these circumstances, contact with a cooler surface causes condensation and contraction drawing in fresh steam and leading to the immediate release of the latent heat, which represents approximately 80% of the heat energy. In this way heat and moisture are imparted rapidly to articles being sterilized and dry porous loads are quickly penetrated by the steam. 13 Moist Heat Sterilization • Two methods of steam generation - Wet steam: generated within the sterilizer, in which case it is constantly in contact with water Usually in portable benchtop or 'instrument and utensil' sterilizers - Dry steam: supplied under pressure (350-400kPa) from a separate boiler, with no entrained water droplets In large scale sterilizers • The killing potential of 'wet' steam is the same as that of 'dry' saturated steam at the same temperature, but it is more likely to soak a porous load creating physical difficulties for further steam penetration. Thus, major industrial and hospital sterilizers are usually supplied with 'dry' saturated steam with attention to remove entrained water droplets within supply line. Moist Heat Sterilization • If the temperature of 'dry' saturated steam is increased, then, in the absence of entrained moisture, the relative humidity or degree of saturation is reduced and the steam becomes superheated. • Superheated steam can arise from: - overheating the steam jacket - evolution of heat of hydration when steaming over-dried cotton fabrics. - using too dry a steam supply - excessive pressure reduction during passage of steam from the boiler to the sterilizer chamber • Superheated steam is an inefficient sterilizing agent, it behaves as hot air, i.e. condensation and release of latent heat will not occur unless the steam is cooled to the phase boundary temperature. • The maximum acceptable level of superheat is 5 °C above phase boundary temperature at that pressure. 14 Moist Heat Sterilization • The relationship between temperature and pressure is true only in the presence of pure steam; presence of air contributes to a partial pressure but not to the temperature of the steam. Thus, in the presence of air the temperature achieved will reflect the contribution made by the steam and will be lower than that normally attributed to the total pressure recorded. • Addition of further steam will raise the temperature but residual air surrounding articles may delay heat penetration or, if a large amount of air is present, it may collect at the bottom of the sterilizer, completely altering the temperature profile of the sterilizer chamber. • Therefore, the major aim in the design & operation of a boiler fed steam sterilizer is efficient air removal. Dry Heat Sterilization • The instrument used is a hot air oven with perforated shelves to allow for heat & air flow • Lethal effects of dry heat are due to oxidative processes which are less effective than hydrolytic damage caused by steam sterilization. Thus, dry heat sterilization usually employs higher temperatures in the range 160-180 °C and requires exposure times of up to 2 hours depending upon the temperature employed. • Since bacterial spores resistance varies according to their degree of dryness, conflicting data resulted with regard to recommended exposure temperature & time. • Its application is generally restricted to glassware and metal surgical instruments (where its good penetrability and noncorrosive nature are of benefit), non-aqueous thermostable liquids and thermostable powders 15 Dry Heat Sterilization • The major industrial application is in the sterilization of glass bottles which are to be filled aseptically, and here there is another advantage which is destroying bacterial endotoxins (pyrogens) which are difficult to be eliminated by other means. • For Depyrogenation of glass, temperatures of approximately 250 °C are used. • The F-value which was developed for steam sterilization has equivalent in dry heat sterilization although with less common application. The FH describes the lethality of dry heat in terms of equivalent no. of minutes exposure at 170°C, with assumed Z- value of 20°C Gaseous Sterilization • Performed using the chemically reactive gases ethylene oxide, and formaldehyde which possess broad-spectrum biocidal activity. • Application: sterilization of re-usable surgical instruments, certain medical, diagnostic and electrical equipment, and the surface sterilization of powders. Ethylene oxide treatment is also considered as an alternative to radiation sterilization in the commercial production of disposable medical devices. (plastics, optics and electrics)! • These techniques do not offer the same degree of sterility assurance as heat methods and are reserved for temperature-sensitive items. • Mechanism of action of the two gases: alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids. • At the concentrations employed in sterilization protocols, type A survivor curves are produced. 16 Gaseous Sterilization • The lethality of these gases increases in a nonuniform manner with increasing concentration, exposure temperature and humidity. That’s why, sterilization protocols have been established using a standard product load containing suitable biological indicator test strips. • Concentration ranges (weight of gas per unit chamber volume) are usually 800-1200 mg/L for ethylene oxide and 15-100 mg/L for formaldehyde, with operating temperatures in the region of 45-63 °C and 70-75 °C, respectively. Gaseous Sterilization • Disadvantages: - The sterilization processes are lengthy (3-4 hrs) and therefore unsuitable for the re-sterilization of high-turnover articles, even at the higher concentrations and temperatures. - Further delays occur because of the need to remove toxic residues of the gases before release of the items for use. - As alkylating agents, both gases are potentially mutagenic and carcinogenic (also the ethylene chlorohydrin which results from ethylene oxide reaction with chlorine) - They produce symptoms of acute toxicity including irritation of the skin, conjunctiva and nasal mucosa; consequently, strict control of their atmospheric concentrations is necessary and safe working protocols are required to protect personnel. - Ethylene oxide can not be detected by smell at low concentration while formaldehyde can normally be detected by smell at concentrations lower than those permitted in the atmosphere 17 Gaseous Sterilization - Ethylene Oxide • EtO is more commonly used than formaldehyde. • Ethylene oxide gas is highly explosive in mixtures of >3.6% v/v in air; in order to reduce this explosion hazard it is usually supplied for sterilization purposes as a 10% mix with carbon dioxide, or as an 8.6% mixture with HFC 124 • Alternatively, pure ethylene oxide gas can be used at below atmospheric pressure in sterilizer chambers from which all air has been removed. • The efficacy of ethylene oxide treatment depends upon achieving a suitable concentration in each article and this is assisted by the good penetrating powers of this gas, which diffuses into many packaging materials including rubber, plastics, fabric and paper. • Organisms are more resistant to ethylene oxide in a dried state, like those protected from the gas by inclusion in crystalline or dried organic deposits. Thus, a further condition to be satisfied in ethylene oxide sterilization is attainment of a minimum level of moisture in the immediate product environment. This requires a sterilizer humidity of 30-70% and frequently a pre-conditioning of the load at relative humidity levels of greater than 50%. Gaseous Sterilization - Ethylene Oxide • Disadvantages: - Explosive! - Cannot be detected by smell at low concentrations - The level of ethylene oxide in a sterilizer decreases due to absorption during the process - The treated articles must undergo a desorption stage to remove toxic residues. Desorption can be allowed to occur naturally on open shelves, in which case complete desorption may take many days, e.g. for materials like PVC, or it may be encouraged by special forced aeration cabinets where flowing, heated air assists gas removal, reducing desorption times to between 2 and 24 hours. 18 Gaseous Sterilization - Formaldehyde • Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v aqueous solution of formaldehyde) to a temperature of 70-75°C with steam, leading to the process known as LTSF (low-temp steam and formaldehyde sterilization). • Formaldehyde has a similar toxicity to ethylene oxide and although absorption to materials appears to be lower similar desorption routines are recommended. • A major disadvantage of formaldehyde is low penetrating power and this limits the packaging materials that can be employed to paper and cotton fabric. Radiation Sterilization • Includes: - Particulate radiation: accelerated electrons (electron beams) - Electromagnetic radiation: gamma-rays and ultraviolet (UV) light • Mechanism of action: The major target for these radiations is microbial DNA, with damage occurring as a consequence of ionization and free radical production by ionizing radiation (gamma-rays and electron beams) or excitation by non-ionizing radiation (UV light). Excitation is less damaging and less lethal than ionization, therefore, UV irradiation is not as efficient as electron beams or gamma-rays. 19 Radiation Sterilization • Vegetative bacteria are the most sensitive to irradiation (with notable exceptions, e.g. Deinococcus [Micrococcus] radiodurans), followed by moulds and yeasts, with bacterial spores and viruses as the most resistant (except in the case of UV light where mould spores prove to be most resistant). • The resistance of the organism to radiation depends on the extent of DNA damage required to produce cell death & the repair capacity of m.o. • In ionizing radiations (gamma-ray and accelerated electrons), activity increases with the presence of moisture or dissolved oxygen (as a result of increased free radical production) and also with elevated temperatures. Radiation Sterilization - Ionizing Radiation • Applications: sterilization of heat-sensitive items such as syringes, needles, cannulas, IV sets, surgical instruments, sutures, prostheses, unit-dose ointments. • However, undesirable changes can occur in irradiated preparations, especially those in aqueous solution where radiolysis of water contributes to the damaging processes. Thus, radiation sterilization is generally applied to articles in the dry state • Also, certain glass or plastic (e.g. polypropylene, PTFE) materials used for packaging or for medical devices can also suffer damage. • With these radiations, destruction of a microbial population follows the classic survivor curves and a D-value, given as a radiation dose, can be established for the reference bacterial spores (e.g. Bacillus pumilus) permitting a suitable sterilizing dose to be calculated. • Electron beams are less penetrating than gamma-rays 20 Radiation Sterilization – UV Light • Has much lower energy & causes less damage to microbial DNA. • Does not penetrate metal at all, nor glass to any useful degree This, coupled with its poor penetrability of normal packaging materials (can penetrate only some polymers), renders UV light unsuitable for sterilization of pharmaceutical dosage forms. • Applications: air and surface sterilization of aseptic work areas (microbiological safety cabinets), and for the treatment of manufacturing-grade water. Filtration Sterilization • The process of filtration is unique amongst sterilization techniques in that - it removes, rather than destroys, microorganisms - It eliminates both viable and non-viable particles thus it can be used for both the clarification and sterilization of liquids and gases • Applications : - Sterilization of heat-sensitive injections and ophthalmic solutions and biological products - Treatment of air and other gases for supply to aseptic areas - They may act as part of venting systems on industrial machines such as fermenters, centrifuges, autoclaves and freeze-dryers - Membrane filters are used in sterility testing where they can be employed to trap and concentrate contaminating organisms from solutions under test. These filters are then placed on the surface of a solid nutrient medium and incubated to encourage colony development 21 Filtration Sterilization • The major mechanisms of filtration are sieving, adsorption and trapping within the matrix of the filter material. • Only sieving can be regarded as absolute since it ensures the exclusion of all particles above a defined size. • Synthetic membrane filters (derived from cellulose esters or other polymeric materials) approximate most closely to sieve filters • Fibrous pads, sintered glass and sintered ceramic products can be regarded as depth filters relying principally on mechanisms of adsorption and entrapment. Filtration Sterilization 22 Filtration Sterilization - Liquids • In order to be used as a method of sterilization, the m.o removal efficiency of filters must be high. • Membrane filters of 0.2-0.22 mm nominal pore diameter are most commonly used. • Sintered filters are used only in restricted circumstances, i.e. for the processing of corrosive liquids, viscous fluids or organic solvents. • Two filters of 0.2 mm pore diameter from different manufacturers will not behave similarly, because, in addition to the sieving effect, trapping, adsorption and charge effects all contribute significantly towards the removal of particles. Consequently, the depth of the membrane, its charge and the tortuosity of the channels are all factors which can make the performance of one filter superior to that of another. Filtration Sterilization - Liquids • Therefore, the major criterion by which filters should be compared, is their titre reduction values, i.e. the ratio of the number of organisms challenging a filter under defined conditions to the number penetrating it • The filter must be sterilizable, ideally by steam sterilization - Membrane filters may be sterilized once for one use Except industrial filters which can be re-sterilized few times - Sintered filters can be re-sterilized many times • Filtration sterilization is an aseptic process and it requires careful monitoring of filter integrity & final product sterility testing. 23 Filtration Sterilization - Liquids • Membrane filters, in the form of discs, can be assembled into - Pressure-operated filter holders mounting and in-line use or - Vacuum filtration tower devices for syringe • Membrane filters are often used in combination with a coarse-grade fiberglass depth pre-filter to improve their dirt-handling capacity. Filtration Sterilization - Gases • The principal application for filtration sterilization of gases is in the provision of sterile air to  Aseptic manufacturing sites  Hospital isolation units  Some operating theatres • Other applications include  Sterilization of venting air in tissue and microbiological culture  Decontamination of air in mechanical ventilators  Treatment of exhausted air from microbiological safety cabinets  The clarification and sterilization of medical gases 24 Filtration Sterilization - Gases • Filters employed generally consist of pleated sheets of glass microfibres separated and supported by corrugated sheets of Kraft paper or aluminium and thus are acting as depth filters • These are employed in ducts, wall or ceiling panels and laminar airflow cabinets. • Although acting as depth filters, these high-efficiency particulate air (HEPA) filters can remove up to 99.997% (99.97% minimum) of particles greater than 0.3 mm in diameter. • In practice their microorganism removal efficiency is rather better since the majority of bacteria are found associated with dust particles and only the larger fungal spores are found in the free state Filtration Sterilization - Gases 25 Filtration Sterilization - Gases • Air is forced through HEPA filters by blower fans, and pre-filters are used to remove larger particles to extend the lifetime of the HEPA filter. • The operational efficiency and integrity of a HEPA filter is monitored by Pressure differential and airflow rate measurements Dioctylphthalate smoke particle penetration tests. New Sterilization Technologies • For thermolabile products & aqueous drugs that are damaged by radiation, aseptic manufacture is costly option with lower SAL. • 2 examples of new technologies: high intensity light & low temperature plasma but still unsuitable for protein or nucleic acid-containing biotechnology products • High intensity light  It is based on the generation of short flashes of broad wavelength light from xenon lamp that has an intensity of 100,000 that of the sun, almost 25% of the flash is UV  Application: sterilization of water & terminal sterilization of injectables in UV transmitting plastic ampoules (e.g. polyethylene).  But not useful for coloured solutions or those with solutes that has high UV absorbance 26 New Sterilization Technologies • Low Temperature Plasma  Plasma is a gas or vapour that has been subjected to electrical or magnetic field which causes a substantial proportions of the molecules to become ionized.  Thus it is a cloud of neutral species, free radicals, ions & electrons in which the positive & negatively charged particles are equal.  Two types: low pressure and atmospheric pressure.  Plasma may be generated from many substances: among the established methods, chlorine and hydrogen peroxide plasmas are used which possess excellent antimicrobial activity.  Application: on most items sterilized by ethylene oxide, i.e. medical devices but not drugs. It is not used for powders, liquids & certain fabrics  Advantages: does not need elimination of toxic gases at the end of cycle unlike LTSF & ethylene oxide & there is no significant corrosion or reduction of sharpness of exposed surgical instruments. Sterilization Control & Sterility Assurance • Currently, awareness of the limitation of sterility testing in terms of their ability to detect low microbial count resulted in relying on satisfactory quality standards during the whole manufacturing process. i.e. the quality is assured by process monitoring & performance criteria, which are considered under four subjects: 1. Bioburden determination 2. Environmental monitoring 3. Validation & in process monitoring of sterilization procedures 4. Sterility test 27 1. Bioburden Determination • Bioburden is the total no. of m.o /ml or g regardless of type (yeast, bacteria, anaerobic,etc..). • There is max permitted bioburden for each ingredient or product in pharmacopoeia. • It is important to have a low pre-sterilization bioburden by: – having high quality of raw materials, – the environment does not encourage microbial growth of the m.o already present in raw material, – low microbial contamination during manufacture. • Therefore, manufacturing process may utilize adverse temp, extreme pH, organic solvent in order to prevent increase in the microbial load. 2. Environmental Monitoring • The level of microbial contamination in manufacturing areas is monitored regularly so as not to exceed certain levels. • M.o. in atmosphere are monitored by settle plates & air sampler. • Contamination on surfaces or manufacturing equipment is measured by swabs or contact plates (Rodac-replicate organism detection & counting-plates) which are petri dishes overfilled with agar media • Operators in manufacturing areas are monitored (samples from clothes (face mask, gloves & fingerprint) 28 3. Validation & In-Process Monitoring of Sterilization Procedures • Validation: demonstrating that a process will consistently produce the results that it is intended to. • Validation of steam sterilizer: – Calibration of all physical instruments used to monitor the process, thermocouples, pressure gauge, timers – Show evidence that steam is of desired quality (i.e. chamber temp is that for pure steam at certain pressure) – Conduct leak tests & steam penetration test using empty & loaded chamber – Use of process efficiency indicators (alone or with bioburden m.o.) – Show repeatability of the process (at least repeat 3 times) – Documentation for all the previous steps 3. Validation & In-Process Monitoring of Sterilization Procedures • Usually records of temp, pressure, time, humidity are kept for each batch of sterilized product • There are 3 types of sterilization indicators: – Physical indicators – Chemical indicators – Biological (Microbiological) indicators 29 3. Validation & In-Process Monitoring of Sterilization Procedures • Physical indicators: – For heat sterilization (dry & moist): temp record chart for each cycle is compared against master temp record (MTR). Temp is taken from coolest part of loaded sterilizer. Heat distribution studies by placing thermocouples at selected sites of sterilizer or inside the test packs – For gas sterilization: elevated temp are monitored for each cycle, pressure, humidity & gas concentration measurements are recorded. – For radiation sterilization: The use of plastic dosimeter (Perspex) which gradually darkens in proportion to radiation absorbed 3. Validation & In-Process Monitoring of Sterilization Procedures • Physical indicators: – For filtration sterilization: filters are subjected to bubble point pressure test to determine pore size of filters & to ensure integrity of certain types of filters (membrane & sintered glass).  Bubble Point Pressure Test: wetted filter in assembled unit is subjected to increasing air or N2 gas pressure differential. The pressure difference recorded when the 1st bubble of gas breaks away from the filter is related to the max pore size. When gas pressure is further increased slowly, there is a general eruption of bubbles over the entire surface. The pressure difference here is related to the mean pore size. Pressure differential below expected value indicates damaged filter.  Diffusion Rate Test: for membrane filters, measures the diffusion of gas thru a wetted filter at pressures below the bubble point pressure, a faster diffusion rate than expected indicates loss of filter integrity. 30 3. Validation & In-Process Monitoring of Sterilization Procedures • Physical indicators: – Efficiency testing of HEPA filters is by the generation upstream of dioctylphthalate (DOP) or NaCl particles of known dimensions followed by detection in downstream filtered air. Retention efficiency is recorded as % of particles removed under defined test conditions • Chemical indicators: – Are based on the ability of sterilization process to alter the chemical or physical characteristic of chemical substances. E.g. melting or colour change. – However the change recorded does not necessarily correspond to microbiological sterility, so such devices should not be employed as a sole indicator for sterilization. 3. Validation & In-Process Monitoring of Sterilization Procedures • Biological indicators: – Used for thermal, chemical or radiation sterilization; they consist of standardized bacterial spore preparations which are either suspension in water or culture medium or spores dried on paper, Al or plastic carrier. – After sterilization, the spores are aseptically transferred to nutrient medium, incubated & periodically examined for signs of growth. – Selection of BI:  Must be non-pathogenic  Should possess above-average resistance to the particular sterilization 31 3. Validation & In-Process Monitoring of Sterilization Procedures • Biological indicators: – Commonly utilized indicators: Bacillus stearothermophilus: for steam sterilization, hydrogen peroxide & peracetic acid sterilization B. subtilis: for dry heat & ethylene oxide B. pumilus: for ionizing radiation Filtration sterilization:  107 cells/cm2 are filtered & m.o. in filtrate are studied. As inoculum size or pressure increases the extent of their passage through the filter increases  usually using Serratia marcescens: a small G-ve rod shaped bacterium, minimum dimension of 0.5µm, for 0.45µm filters,  more rigorous tests use Brevundimonas diminuta, minimum dimension of 0.3µm, for 0.2-0.22µm filters. 4. Sterility Test • A test which assesses whether a sterile pharmaceutical or medical product is free from contaminating m.o. or not. • The test is performed on a sample taken from a batch. In order to extend a successful result to a whole batch assurance that every unit of the batch was manufactured in a similar manner to the sample taken is needed. • To be sure that no organism is present; a universal growth medium that supports the growth of all types of m.o. should be used, which is not available. Practically, media capable of supporting non fastidious bacteria, yeast & mould are used • Pharmacopeial tests do not look for viruses, which can pass thru sterilization filters. 32 4. Sterility Test • Disadvantages of Sterility Test: – Destructive test – Questionable suitability for testing large expensive or delicate products – It is a statistical process where part of a batch is randomly sampled & batch is released based on this sample – The procedure intends to demonstrate a negative! i.e. failure to detect m.o. could be a consequence of the use of unsuitable media or inappropriate cultural conditions. Methods for Sterility Testing A. Direct inoculation method • By introducing test sample directly into nutrient media. • Two media recommended by pharmacopeias: 1. Fluid thioglycolate medium which is suitable for anaerobic microorganisms (at 30-35 °C) 2. Soybean casein digest medium (tryptone soya broth) which supports the growth of both aerobic bacteria (at 30-35 °C) &fungi (at 20-25 °C) • Limits are placed upon the ratio of the weight of added sample relative to the culture medium to avoid reducing nutrient properties. 33 Methods for Sterility Testing B. Filtration method: • Recommended by pharmacopeias • It involves the filtration of fluids thru sterile membrane filter (pore size 0.45 mm); the m.o. present will be retained on the surface of the filter, the filter is then washed & divided aseptically into two portions which are transferred to suitable cultural media. • Water soluble solids can be dissolved in a suitable diluent, while oil soluble solids are dissolved in a hydrophobic solvent e.g. isopropyl myristate. Methods for Sterility Testing C. Addition of concentrated culture medium : • The concentrated culture medium is added to the pharmaceutical fluid (e.g. IV infusion) in its original container, so that the resulting mix is equivalent to a single strength culture medium. • Hence, the entire volume is sampled in this method. 34 Methods for Sterility Testing • In all cases the media employed should have been assessed for nutritive (growth supporting) properties & a lack of toxicity using specified m.o. (positive control) • The test should be done under strict aseptic conditions to avoid accidental contamination. • Adequacy of facilities is confirmed by sampling air & surfaces; whereas isolators (sterile cubicles) are used to reduce possibility of contamination from the operator • Negative controls should be done by testing samples that are known to be sterile (e.g. samples subjected to reliable process like radiation or to several cycles of sterilization). Methods for Sterility Testing • If the product has antimicrobial activity (antibiotic or preservative), its activity must be nullified during sterility testing which can be achieved by: 1. Inactivating (neutralizing) agent e.g. enzymes like penicillinase, chloramphenicol acetyltransferase 2. Dilution (for substances with high conc exponent e.g. phenols, cresols, alcohols 3. Membrane filtration thru hydrophobic membrane filter which is washed to remove traces of antibacterials, and then transferred to the culture medium • Sterility test provides no guarantee to the sterility of the batch, it is an additional check & gives confidence to the efficacy of a sterilization or aseptic process. 35 Parametric Release • Parametric release allows for releasing terminally sterilized batches without being tested for sterility • Many authorities rely on the validation & reliable performance of sterilizers rather than on test for sterility • The sterilization cycle will be validated to have a sterility assurance of 10-6 or less as minimum safety factor. • Validation includes investigating heat distribution, heat penetration, bioburden, container closure & cycle lethality studies. • To subject a product for parametric release, presterilization testing of bioburden on each batch and the inclusion of suitable chemical or biological indicator are also required. 36