The major objective of a sterilization process is to destroy all microorganisms present in a given sample. Microbial metabolism is based upon the utilization of inorganic and organic compounds to drive cell growth, division, and
Product Reason for recall
Serum Bacterial contamination
Medical device Microbial contamination
Medical device Mold contamination
Medical device Lack of sterility assurance
Medical device Mold contamination
Medical device Mold contamination
Ceftazidine injection Lack of sterility assurance Ceftazidine injection/cefazolin injection Lack of sterility assurance Lidocaine HCl/epinephrine injection Lack of sterility assurance Lidocaine HCl/epinephrine injection Microbial contamination Oxfloxacin otic solution Lack of sterility assurance Ticacillin disodium/clavulanate Lack of sterility assurance Potassium injection
Various injectables Microbial contamination Glycyrrhizinic acid injection Mold contamination Sodium chloride respiratory therapy Ralstonia pickettii
maintenance [17]. Enzymatic reactions are essential to microbial growth, re- production, survival, and distribution in the environment. All sterilization processes inactivate or interfere with these enzymatic reactions that support microbial metabolism. When exposing a microbial population to a sterilizing agent, the microbial inactivation follows an exponential death curve [16]. The probability of a population of microorganisms to survive a sterilization process is determined by their number, types, and resistance to the steriliza- tion process. Furthermore, other factors such as moisture content, thermal energy, and time of exposure also affect microorganisms’ survival. After the completion of a given sterilization cycle, for a pharmaceutical product, ste- rility means that the product has been sterilized where individual units have a probability of being nonsterile or have a SAL equal to 1 10 6or more (terminally sterilized injectables). This indicates that there is a probability of one in a million that a microorganism can survive the sterilization process. 3.1. Steam Sterilization
When a sample is placed in an autoclave that employs saturated steam and pressure, that sample is sterilized using the most common method of steri- lization. This method is called steam sterilization. The basic principle of op- eration is that the air in the chamber is displaced by the saturated steam, achieved by employing vents and traps. To displace the air more effectively from the chamber and from within articles, the sterilization cycle may include air and steam evacuation stages. The cycles for different products are based upon the heat penetration, distribution, and resistance of test articles. Tem- peratures of 121jC and pressures of 15–21 psi are always used. However, the time required for complete sterilization must be determined during the vali- dation process of different load configurations. These configurations are based upon the different types and numbers of materials treated by any particular sterilization process. During the validation, two parameters are measured. The first one is the mapping of the heat distribution inside the chamber to determine the ‘‘cold’’ spots. This will determine the uniformity and variability of the temperature inside the chamber. The second parameter is the heat penetration with real load configurations. These loads represent the types of material sterilized on a daily basis such as growth media, laboratory instrumentation, glassware, plastic containers, and biological waste. The placement of biological indicators (BIs) inside the autoclave near or inside the loads will allow the determination of the amount of temperature and pressure reaching into the different loads. It is important that the right temperature and pressure reach all materials inside the chamber for complete microbial kill. After incubating the different BIs, the absence of growth indicates the complete sterilization of all articles.
3.2. Dry Heat Sterilization
Dry heat sterilization utilizes a drying oven with heated filtered air. The air is distributed throughout the chamber by convection or radiation, and by em- ployment of a blower system with devices for sensing, monitoring, and con- trolling physical parameters. Acceptable range for temperature in the empty chamber is +15jC when the unit is operating at not less than 250jC.
A continuous process is employed for the sterilization and depyroge- nation of glassware. Because dry heat is frequently used to eliminate pyro- genic substances from glassware and containers, a challenge with a given concentration of pyrogen must be part of the validation system. Standard methods require the inoculation of 1000 or more Unites States Pharmaco- poeia (USP) units of bacterial endotoxin. The bacterial endotoxin test (BET) is used to demonstrate a 3-log cycle reduction [18]. Pyrogenic substances are bacterial components that cause fever and other pathogenic conditions in humans. Therefore, it is important to eliminate any of these substances from materials and equipment.
3.3. Gas Sterilization by Ethylene Oxide
The common agent used in gas sterilization is ethylene oxide. This kind of sterilization process is carried out when a sample cannot withstand the tem- peratures used in steam and dry heat sterilization procedures. Ethylene oxide is highly flammable, mutagenic, and levels the possibility of toxic residues in treated materials. The process is carried in a pressurized chamber similar to steam sterilization but with modifications unique to gas sterilizers. After sterilization is completed, the chamber must be degassed to enable microbial monitoring. Parameters such as gas diffusion, concentration, moisture con- tent, holding time, and temperature are very important factors during the validation of gas sterilization processes. Moisture and gas concentration are also critical factors. Package design and chamber loading patterns must en- hance gas diffusion to optimize gas penetration and microbial death.
3.4. Ionizing Radiation Sterilization
This kind of sterilization process is widely used with medical devices. Fur- thermore, several drugs have also been treated using this procedure. The advantages of ionizing radiation are as follows:
Low chemical reactivity Low measurable residues
The process is controlled by adsorbed radiation dose. Irradiation increases temperature minimally but can affect plastic and glass materials. The two types of irradiation used are radioisotopic decay (gamma) and electron beam radiation. The dose to yield the sterility assurance level re- quired must be determined during process validation. For gamma irradiation, validation procedures include material compatibility, loading patterns, identification of minimum and maximum doses, and timer setting. An effec- tive sterilization dose tolerated without damaging the article must be selected. Specific product loading patterns must be determined with the minimum and maximum dosage distribution. Absorbed dose is determined by employing inoculated products with Bacillus pumilus. Other dosages are based upon the radiation resistance of the natural microbial population contained in the article to be sterilized.
3.5. Filtration
Filtration through microbial retentive materials is frequently used for the sterilization of heat-labile solutions by physical removal of the contained microorganisms [19]. A filter assembly generally consists of a porous mem- brane sealed or clamped into an impermeable housing. The effectiveness of a filter medium or substrate depends upon the filter’s pore size and may depend upon adsorption of bacteria on or in the filter.
Rating the pore size of the filter membranes is based upon using microorganisms of the size represented by ascertaining the capability to re- tain the microbes. For instance, sterilizing filter membranes are capable of retaining 100% of a culture of 107Brevundimonas diminutaATCC 19146 per square centimeter of membrane surface under a pressure of not less than 30 psi (2 bar). These membranes are rated 0.22 or 0.2 Am, depending on the manufacturer’s practice. This rating also applies to reagents and media. However, studies have demonstrated that 0.22-Am filters do not remove all microorganisms under all conditions [20–22]. Environmental bacterial iso- lates have been able to penetrate these filters more effectively than B. diminuta. These studies recommend the use of 0.1-Am filters. However, reg- ulatory agencies and industrial practices are still based upon using 0.22-Am filters.
Filter membranes that are capable of retaining only larger micro- organisms are labeled with a nominal rating of 0.45 um. There is no rating for these kinds of filters. However, they are able to retain B. diminuta and Serratia marcescens ATCC 14756. Test pressures vary from 5 psi, 0.33 bar for S. marcescensto 0.5 psi, 0.34 bar for B. diminuta to high 50 psi, 3.4 bar. Filter membranes rated 0.1 Am are tested using Mycoplasma strains at a pressure of 7 psi, 0.7 bar.
Other important parameters in the validation of a filtration process are as follows:
Product compatibility Sorption
Preservatives and other additives
Effluent endotoxin content.
Microbial bioburden (BB) of the solution to be processed by filtration is a very important parameter to evaluate the effectiveness of a filtration process [23]. Determining the numbers of microorganisms in the sample prior and after the filtration step will indicate the efficiency of a given process. Fur- thermore, pressure, flow rate, and filter characteristics are also important. Membrane filters are based upon materials such as:
Cellulose acetate Cellulose esters Cellulose nitrate Fluorocarbonate Acrylic polymers Polycarbonate Polyester Polyvinyl chloride Vinyl Nylon Polytef Metal.
A filter assemble must be tested for integrity prior to use and also after the filtration process is completed to demonstrate the integrity of the system. Some of the tests are as follows:
Bubble point test Diffusive test Airflow test Pressure hold test Forward flow test.
There should be a correlation between these tests and microorganism reten- tion for the process to be validated.