13 Validation of Terminal Sterilization Thomas J. Berger and Kevin D. Trupp Department of Microbiology and Global Process Sterilization Engineering, Hospira, Inc., Lake Forest, Illinois, U.S.A.
INTRODUCTION INTRODUCTION TO PARENTERAL PARENTERAL PRODUCT STERILIZATION
The previous chapter discussed the steam sterilization approach for the processing of hard goods or porous loads. loads. This chapter will discuss discuss the sterili sterilizati zation on validation approach that can be used in the processing of parenteral products by terminal sterilization using moist heat. The underlying principles of steam sterilization are applicable applicable to both hard goods and terminal terminal sterilizat sterilization ion of parenter parenteral al products, products, but both have their unique characteristics. An organized sequential flow of activities must occur as new parenteral formulations are developed, and subsequent subsequently ly processed processed in the manufactur manufacturing ing facility facility. The moist heat sterilizat sterilization ion of pharmaceut pharmaceutical ical solutions solutions is established and verified through a series of activities that confirm the product has received a defined thermal exposure that renders the product sterile. R&D activities can include sterilization developmental engineering studies consisting of sterilization cycle development; container thermal mapping; microbial closure validation, D- and z and z-value -value analysis; container–closure integrity validations as well as final formulation stability studies. The subsequent production phase activities must include include initial initial sterilizat sterilization ion vessel vessel qualification qualification which which demonstr demonstrates ates that the vessel vessel will deliver deliver the defined defined steriliza sterilization tion process process in a consiste consistent nt and reproduci reproducible ble manner. Also, solution and container–closure microbial validat validation ion studies studies must be conduct conducted ed at subproc subprocess ess production production sterilizati sterilization on conditions conditions employing employing heatresis resistan tantt microor microorgan ganism isms. s. Equipme Equipment nt validat validation ion,, filtrat filtration ion studie studiess and assess assessmen mentt of the biobur bioburden den on Abbrev iation s used in this chapte r : AAMI, AAMI, Associa Association tion for the Advanceme Advancement nt of Medical Medical Instrumen Instrumentatio tation; n; APE, antimicr antimicrobia obiall preservative efficacy; efficacy; API, active pharmaceutical pharmaceutical ingredient; ASME, American Society of Mechanical Engineers; BET, bacterial endotoxin endotoxin testing; testing; BI, biologic biological al indicato indicator; r; BIER, BIER, biologica biologicall indicator evaluator resistometer; DP, direct plate; EMA, European Medicinal Medicinal Agency; Agency; F/N, fraction/nega fraction/negative; tive; GAMP, GAMP, good automated manufact manufacturing uring practice; practice; HMI, human–ma human–machine chine interface; interface; I/O, input/output; ICH, International Conference on Harmonization; ISPE, ISPE, Internatio International nal Society Society for Pharmace Pharmaceutic utical al Engineerin Engineering; g; LAL, limulus limulus amebocyt amebocytee lysate; lysate; LVP, VP, large volume parentera parenterals; ls; MES, manufac manufacturing turing execution system; MOS, maintenan maintenance ce of sterility; sterility; P&D, penetratio penetration n and distribution distribution;; PLC, programm programmable able logic controller; PSLR, predicted spore logarithmic reduction; R&D, research and development; RTD, resistance temperature detector; SCADA, SCADA, supervis supervisory ory control control and data acquisit acquisition ion;; SLR, SLR, spore spore logarithm logarithmic ic reduction reduction;; SVP, SVP, small small volume volume parenter parenterals; als; TC, thermocouple.
component parts, as well as the environment, must also be ascertained. The developmental and production phases of sterilization technology activities are then drafted into documents that are submitted as part of a new drug application for the particular parenteral formulation. These reports must follow applicable regulatory requirements for products that are terminally sterilized. Such stud studie iess allow allow one one to esta establ blis ish, h, with with a high high leve levell of sterilization assurance, the correct sterilization cycle (F0, temperature, product time above 100 C, etc.) to be used for the steam sterilization of a specific parenteral formulation in a particular container–closure system. 8
STERILIZER DESIGN
The validation of a steam sterilization cycle is dependent on the equipment chosen. The sterilizer and its support systems must be designed and constructed to deliver the effective cycles repeatedly and consistently. Qualification of the sterilizer consists consists of proper proper design, design, installation installation according to design, operational testing to ensure that design criteria and operational requirements are met and performance qualification to confirm that the product is sterilized per specification. Sterilizer design is geared to the type of product or materials/equipment to be sterilized. All steam sterilization cycles are based on contact with saturated steam, steam–air mixtures or superheated superheated water. water. Saturated Saturated steam is water vapor in equilibrium with liquid water. The values of temperature and pressure at which pure satura saturated ted steam can exist are shown by the phase diagram in Chapter 12, Figure 3. Saturated steam can exist only along the phase boundary for liquid and gaseous water; that is, the relation between its temperature and pressure is fixed. An increase or reduction in the temperature of saturated steam must result in a corresponding increase or decrease in its pressure and vice versa. versa. Steam– Steam–air air mixtur mixtures es can be used used when when overpr overpress essure ure is required required to maintain maintain product product shape shape or containe containerr integrity. Superheated water cycles require air overpressure and the water is either heated by direct injection of steam or indirectly via a heat exchanger. Parts and hard goods are typically steam sterilized using a saturated steam process whereas the trend for product sterilization is towards the use of superheated water or steam–air mixture processes. These processes are needed as a majority of the new products require air overpressure during the sterilization process to maintain desired container characteristics and integrity.
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Products in glass containers can utilize the saturated steam processes as described in Chapter 12, but many products and containers require the use of air overpressure during the sterilization process. This section will discuss some of the key design considerations for terminal steam sterilizers and provide some specifics for the various type steam sterilization processes utilizing air overpressure.
Typical Design Considerations for Steam Sterilizers 1. A pressure vessel constructed according to the ASME or equivalent international code. This must withstand at least 50% in excess of the required internal pressures. 2. A safety door mechanism to prevent opening while the unit is under pressure: the locking device may be actuated directly by internal pressure or indirectly through an automatic switch. The door itself may be of the swing-out or sliding type. Separate entry and exit doors are preferable. 3. Process control system (typically a PLC for controlling and monitoring the process). 4. Process data recorder or data collection system. 5. Product racks designed to hold/support the sealed product containers and to provide adequate heating/cooling media flow throughout the product zone. 6. Pressure safety relief valves for both the chamber and jacket (if equipped with jacket). Note. A microbial retentive vent/air filter would not typically be required for processes used for terminal sterilization as there is no direct contact between the heating/cooling media and the contents of the containers.
SteamAir Mixture Sterilization The primary benefit to the steam–air mixture process over a superheated water process is the product is not subjected to direct contact with water (except as condensate), which in some cases can cause cosmetic issues with the container. Steam–air mixture processes typically utilize large recirculating fans to prevent the formation of cold/hot spots in the sterilizer. The steam–air mixture process typically uses an indirect cooling method such as cooling of the jacket or with cooling coils within the sterilizer. Because of this indirect cooling method, the cooling rate of the product is typically much slower and less efficient than direct exposure of the product containers to cooling water. Some of the specific sterilizer design considerations for a steam–air mixture process include the following: 1. A jacket and insulation: the jacket would utilize steam during heating and exposure phases of the cycles and cooling water can be introduced to the jacket during the cooling phase of the process. 2. A thermostatic steam trap to efficiently remove the condensate from the chamber: this is open when cool (in contact with air or condensate) and closed when in contact with steam. As condensate collects, the trap opens owing to the slight temperature reduction and the condensate is discharged. There is also a similar steam trap to remove steam condensate from the jacket.
3. Fan(s) to continuously recirculate the steam–air mixture during heat-up and exposure and to recirculate the air during cooling. 4. Cooling provisions (e.g., cooling coils) to cool the air/product.
Recirculated Superheated Water Sterilization Sterilization with recirculating superheated water (sometimes referred to as a water cascade or raining process) is more efficient than a steam–air mixture and is therefore more common. There are many types of recirculating superheated water processes, the most common is a process where the bottom portion of the sterilizer (below the product zone) is filled with water and a recirculation pump is used to continuously recirculate water from the bottom of the sterilizer to spray nozzles above the product zone. A slight modification to that process is the use of a water distribution pan in lieu of spray nozzles. Another version of the recirculating superheated water process is to completely submerge the product in water but this process is inefficient from a utilities consumption standpoint. All of these recirculating superheated water processes utilize air overpressure and the overpressure can be controlled during the sterilization process to minimize most types of container deformation. There is no limit to the maximum overpressure used but it would typically be limited by the chamber pressure rating. The minimum overpressure will be driven by the temperature being used, the pressure needed to maintain the desired product characteristics and the required overpressure needed to prevent the recirculation pump from loosing prime. These recirculating processes are typically heated and cooled indirectly with external heat exchangers located in the recirculating water loop but direct injection of steam and cooling water can also be used. In addition to the typical sterilizer design considerations mentioned earlier, a superheated water sterilizer would also include a large recirculating water system (e.g., pump, pipes, heat exchangers, headers, spray nozzles) including specific water level control valves and monitoring devices.
Rotary and Shaker Sterilization In some cases, certain products (i.e., suspensions and emulsions) require agitation during the sterilization process. For those types of products, it is typical to use a rotating rack within the sterilizer but other agitation methods such as an internal shaking device are available. Refer to Figure 1 for the typical design of a rotary sterilizer and Figure 2 for the typical design of a sterilizer using a shaking mechanism. It is possible to use any of the sterilization processes listed above with product agitation.
Continuous Sterilization For this version of the superheated water process, containers are terminally heat sterilized in a continuous sterilizerby a process where the containers movethrough a constantly controlled environment in carriers with individual compartments. The time, temperature, and pressure requirements are set to predetermined values and are automatically and continuously controlled, monitored, and recorded. Refer to Figure 3 which depicts the pattern
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Figure 1 Photo of sterilizer with a rotary mechanism. Source : Photo Provided by Fedegari Autoclavi SpA, Albuzzano, Italy.
that containers (e.g., parenteral flexibleproduct containers) follow as they move automatically through the continuous sterilizer. The water lock of the pressure vessel is used to provide product entrance into and out of the overpressure environment. The overpressure environment is constantly maintained within predetermined limits. The sterilizing phase begins as the product enters the hot water environment within the pressure vessel. The hot water environment may be a superheated water
Suspension Rods
Platform Frame
Platform Lock Arm
Lock Screw
Platform Rails
Figure 2
Bridge Plate
Photo of sterilizer with a shaking mechanism.
VALIDATION OF TERMINAL STERILIZATION
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spray, which is circulated over the top of the continuously moving carriers. The residence time of the product within this sterilizing environment and the water temperature are controlled within predetermined limits to assure the required heat input. Cooling begins as the product transfers from the sterilizing environment and enters the cooling environment, which is also within the pressure vessel. The cooling water environment is a cool water spray that is circulated over the top of the continuously moving carriers. The temperature of this water is controlled within predetermined limits to assure that the required degree of cooling is achieved before the product leaves the cooling environment. A system of fixed temperature sensors located in the entering and exiting recirculating water for both heating and cooling continuously monitors, records, and controls the temperature of the process water. Air overpressure is required to protect the container from stress while exposed to the high sterilizing temperatures. STERILIZER CONTROL SYSTEMS
A key to effective sterilizer operation lies in the automated process control system. By eliminating the dependence on operator intervention and data recording, automatic temperature and sequential control provides assurance that the “validated” sterilization cycle is consistently and repeatedly delivered. A typical control system for a new sterilizer includes the following hardware components: & PLC & Operator interface panel(s) & Data recorder/data collection system & Process Variable Sensors & I/O devices The PLC is most commonly used as the primary component of the automated process control system as it provides sequential control of the process, provides control of all proportional valves, controls all devices, receives operator input via the operator interface panels and provides process information (such as process variable information and alarms) to the operator via displays and/or operator interface panels. The PLC typically contains specific recipe information for the various cycles to be utilized. In some cases the PLC can be used for data collection, but it is much more common to use a separate data recorder/data collection system. The operator interface panel can be as simple as switches and displays or as complex as a stand-alone PC running a SCADA with a HMI software package. These devices are typically used to select the recipe, start the cycle and display process information during the cycle. The higher level PC based SCADA type operator interface panels can provide detailed cycle reports and trending information. The data recorder/data collection system can range from a simple strip chart recorder to a full-blown MES type data collection system. In many cases the PLC can also provides batch data logging functionality. The minimum variables to record for steam sterilization processes are typically time, temperature, and pressure.
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Steam
Flowrate Sensor
Heaters Surge Tank Pump Indexer Sterilizing Tank Entry Lock Take-Up Shuffle Temperature Sensor
Exit Lock
Cooling Tank Tower Water Heat Exchangers
Surge Tank Pump
Temperature Sensor Mixing Tank
Tower Water
Surge Tank Pump Ozone
Figure 3
Unloading
Pump
Schematic of a sterilizer for “continuous” processing of flexible containers.
Typical sensors include temperature measurement devices (RTDs or TCs), pressure measurement devices, and where applicable level measurement devices and flow measurement devices. It is customary that the temperature sensor used to control the process temperature not be used to provide the batch record process data. An independent/secondary temperature sensor for batch reporting provides a high degree of insurance that the cycle actually ran within its defined limits. Heavy wall thermowells should not be used, as this will affect the time response of the measurement. Thin-walled thermowells or temperature elements with stainless steel sheaths should be used for temperature measurement. The pressure sensor should be equipped with a sanitary-type diaphragm and connected to the sterilizer using a sanitary fitting. A sanitary diaphragm can introduce errors to the pressure measurement due to the stiffness of the diaphragm. This stiffness is related to the size of the diaphragm. The impact is negligible for diaphragms above 3 inches diameter. This should be considered when sizing the connection to the sterilizer. Sterilizers that maintain a specific water level (i.e., recirculated water process) should be equipped with liquid level sensors. These sensors may be in the form of singlepoint-level-typeprobe or a continuous levelsensor. Regardless of what type sensor is used a separate high-level sensor must also be provided. The separate high-level sensor provides greater assurance that the collected water at the bottom of the vessel remains below the product level.
Sterilizers that rely on recirculated water as part of the sterilization process can include a flow sensor. The flow sensor may be a direct measurement such as a flow meter (i.e., coriolis, ultrasonic, magnetic, etc.) or an indirect measurement such as differential pressure sensor across the recirculation pump. Direct measurements are always preferred. For I/O devices, there are analog types and discrete types. The analog inputs are typically from process sensors and the analog outputs are typically for control of proportional valves. The discrete inputs are typically from switch type (operator and process) devices and the discrete outputs are typically for activating hardware such as valves, pumps, lights, etc. The design and development of the sterilizer control system software should follow the principles of ISPE GAMP 4 Guide for Validation of Automated Systems (1). This guideline details a software life cycle from conception thru decommissioning. STERILIZATION CYCLES
The type of steam sterilization cycle to be utilized is dependent on product needs and equipment availability. As discussed in Chapter 12, the sterilization of hard goods or porous loads typically require the use of a pulsed prevacuum cycle as it is preferable to remove the air from the porous materials being sterilized whereas in the terminal sterilization of aqueous solutions in sealed containers,
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the major concern is to provide rapid heat transfer to the wall of the filled product containers and air removal is not required (nor even desirable as the hydrating moisture is contained within each container). Parenteral products may be filled into rigid or flexible containers. In either there is typically airor nitrogen present in the headspace above the liquid. As the solution is heated, this gasexpandsand adds to the internal pressure increase resulting from the evolution of water vapor from the aqueous vehicle within the heated container. Thus, the pressure within the container will exceed the chamber pressure during steam process for sealed containers. Glass vials can be sealed with special closures to withstand this pressure. As long as the pressure differential between the chamber and the containers does not become too great during the steam exhaust portion of the cycle, the vials will not burst. If rapid cooling of the load is desired, the pressure differential might become significant enough to cause closure integrity to be lost. Plastic bags, semi-rigid containers and syringes present a greater problem because they do not have the inherent strength of glass and may burst or deform as the pressure differential increases. To prevent this, air must be injected into the chamber to raise the pressure above the saturation pressure of the steam. This is particularly important during the cooling cycle, when the chamber pressure is reduced at a much faster rate than that within the container. The following section provides a description of the various steam sterilization cycles used for parenteral products in sealed containers.
Saturated SteamPre-Vacuum Cycle For a saturated steam process, the most common (and perhaps most effective) method to remove the entrapped air from the sterilizer is to remove it mechanically before the actual sterilization begins. This is done by means of a mechanical vacuum pump or steam eductor. This cycle can be used for products in glass containers. A sketch of a typical pre-vacuum cycle is shown in Chapter 12, Figure 7.
Saturated SteamGravity Displacement or Steam Purge Cycle Other means for eliminating air without a vacuum source include the use of a gravity displacement cycle or a steam purge cycle. For the gravity displacement cycle, steam is introduced on the side or top of the vessel and the cold air is forced out via the drain. The steam purge cycle uses large quantities of steam distributed via headers under the entire product zone with numerous large vents located at the top of the vessel. For these types of cycles, the appropriate vents or drains should be fully open and the large steam supply valve fully open for an extended time and temperature to ensure that the air is adequately removed from the sterilizer. Once the vents and drains close, the process runs like a traditional saturated steam process. It is important to determine that the measured temperature and pressure are consistent with the steam saturation curve in Chapter 12, Figure 3. This process can be used with glass containers.
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SteamAir Mixture Cycle It is important to understand the physical principle involved in a mixture of steam and air. The fixed relationship between temperature and pressure seen in Chapter 12, Figure 3 no longer applies. Dalton’s law states that the pressure of an ideal mixture of gases is equal to the sum of the partial pressure of the gases, or P
Z
PA C PB C PC :
Raoult’s law further states that, for ideal mixtures, the partial pressureof the gas is equal to its vapor pressure multiplied by the mole fraction in the liquid. For steam in equilibrium with pure condensate, this reduces to PA
pA
Z
where PA is the partial pressure of steam and pA is the vapor pressure of the condensate. The difference between the observed chamber pressure P and PA is the partial pressure of air. The presence of air, although necessary for the maintenance of container integrity, can reduce the heat transfer efficiency. The objective of the design in the “air overpressure” cycle is to maintain a well-mixed chamber. This assures that the heat transfer to the load will be uniform regardless of the presence of air. Mixing may be accomplished in several ways. The air may be injected directly into the incoming steam. Usually, though, some mechanical means is selected. Most steam–air sterilizers use a fan built into the top or end of the chamber, which circulates and mixes the air and the steam (Chapter 12, Fig. 9). Some steam–air sterilizers are capable of using water during the cooldown process to cool the containers more rapidly. This rapid cooling may also be necessary for product stability. Various methods (i.e., direct injection, recirculation through a heat exchanger, etc.) for introducing the cooling media can be utilized.
Recirculating Superheated Water Cycle The typical recirculating superheated water process (sometimes referred to as a water cascade or raining process) begins by the addition of water to the sterilizer to a predefined level (below the product zone). Then a water recirculation pump is started to continuously recirculate water from the bottom of the sterilizer to spray nozzles or a water distribution pan above the product zone. The recirculation pump is on throughout the heat-up exposure and cool-down phases. During heat-up, the water is heated at a pre-defined rate via a heat exchanger in the recirculation loop or with the direct injection of steam. Also during heat-up, compressed air is added to the chamber to attain the desired overpressure levels. Once the temperature set point is achieved, the controller steps into the hold portion of the cycle and the temperature and pressures are maintained at the desired levels. For cooling, the steam supply is shut off and the recirculating water is cooled at a controlled rate by introducing cooling media to a heat exchanger installed in the water recirculation loop or by the direct injection of cooling water into the recirculating loop. This type of process does not require the use of a jacket but does require specific water level controls.
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The recirculating superheated water process is very efficient and the temperatures and pressures can be tightly controlled during the entire process, thus minimizing container stresses.
Container integrity
STERILIZATION CYCLE DEVELOPMENT This section will address sterilization and associated microbiological activities that occur in R&D areas as well as the production environment when using the BI/bioburden approach in support of a parenteral product. The list below depicts some of the sterilization engineering and microbiological activities associated with a parenteral product as it moves through development. These studies or similar ones are ordinarily conducted in developmental sterilizers or may occur as investigative engineering studies in a production sterilizer as appropriate. The overkill method can be used for some of the more stable parenteral formulations, and its validation is accomplished as described in Chapter 12. Sterilization development activity
Cycle development
Container thermal mapping
Formulation development
Parenteral solution microbiological evaluations: Moist heat D - and z -value analysis
Activity statement
Develop preliminary container sterilization specifications with engineering parameters such as temperature, time and F 0 Determine cold spot and assess heat penetration within finished container Perform analytical feasibility studies prior to product finalization with method’s validations
Perform triplicate D -value analysis on each parenteral formulation at three temperatures, e.g., 112 C, 118 C and 121 C and then calculate the z -value Perform on final product if it contains a preservative or if there is a multidose claim for the container Perform studies with a panel of microorganisms to validate 70% recovery for the filtration process Inoculate parenteral product with bioburden and growth promotion compendia organisms to evaluate the product’s ability to support microbial growth 8
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APE
In-process bioburden analysis
Spike hold time studies
Container closure evaluations: Microbial closure inactivation
Perform kill curve kinetics using bioburden and BI (spores) inoculated onto the worse case closure site
Stability runs
BET
Perform dye ingress, microbial challenge or physical integrity tests following exposure to maximum sterilization conditions that stress the container Perform analytical chemistry and microbiological evaluations at various temperatures and times per ICH and or compendia requirements Perform test method validation of API, excipients and final product per compendia requirements
Sterilization engineering personnel primarily focus their efforts in determining whether a parenteral formulation packaged in a particular container configuration can be sterilized in a current cycle or whether a new cycle must be developed. The referenced EMEA (2) decision tree is followed when evaluating a new parenteral product in an LVP or SVP container. Sterilization feasibility studies are conducted in a sterilizer to ascertain the physical effects of the cycle on the product in question. Product attributes that can be affected by a cycle are closure integrity, product potency, pH, color, shelf-life stability, visible, and subvisible particulates as well as final product sterility. Once the basic engineering parameters (e.g., temperature, time and F0) are established, then engineering thermal container mapping studies can be performed (3,4).
Container Thermal Mapping Validation Studies An R&D sterilizer is smaller than a production facility sterilizer, but can simulate the sterilization cycles conducted in the larger production vessels. Container thermal mapping studies (when applicable) are typically performed in a laboratory sterilizer: 1. To locate the coldest zone or area inside a container. 2. To determine the cold zone in the container and its relationship to the location monitored during validation studies. 3. To generatedata thatmay beusedduring the setting of production sterilization control parameters. When conducting thermal mapping studies, there are various factors to be considered, and these are dependent upon the: 1. Type of container (flexible or rigid) 2. Container orientation, size and fill volume 3. Cycle type and temperature 4. Viscosity 5. Autoclave trays/design/surface contact 6. Autoclave spray patterns/water flow. Typical container mapping data obtained for lipid emulsions contained within a 1000 mL glass container are shown as an example in Tables 1 and 2. The following summarizes the process for obtaining heat map data from the glass intravenous container filled with approximately 1000 mL of lipid emulsion.
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Table 1
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Heat Input ( F 0 Units) 1000 mL glass I.V. containers-heat mapping study (lipid emulsion) Run CLHK00.049
TC number 1,12 2,13 (PC) 3,14 4,15 5,16 6,17 7,18 8,19 9,20 10,21 11,22 H–C PC–C
Run CLHK01.050
btl 1
btl 2
btl 1
btl 2
Average (SD)
7.91 7.79 C7.46 7.64 12.66 12.73 12.78 13.32 14.21 H15.87 15.47 8.41 0.33
C7.28 7.49 7.40 7.80 12.90 12.46 12.69 13.33 14.33 H17.24 16.56 9.96 0.21
8.13 8.02 C7.71 7.87 12.95 12.77 12.95 13.42 14.03 H15.18 14.77 7.47 0.31
C7.36 7.64 7.47 7.96 12.91 12.68 12.91 13.78 14.56 H16.09 16.07 8.73 0.28
7.67 (0.415) 7.74 (0.226) C7.51 (0.137) 7.82 (0.135) 12.86 (0.132) 12.66 (0.138) 12.83 (0.120) 13.46 (0.223) 14.28 (0.222) H16.10 (0.856) 15.72 (0.773) 8.64 (1.028) 0.28 (0.053)
Note :
H denotes hottest TC location; C denotes coldest TC location; PC denotes approximate location of the production profile TC; Data from TC#9 used with a postcalibration variance of C0.25 C at 100 C; All heat input values are calibration corrected. 8
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TC probes (Copper Constantan, type T, 0.005 in. diameter) were used to monitor 11 locations within the 1000 mL container. The TC probes were positioned at various distances (in inches) as depicted (Fig. 4). Each container was filled with approximately 1000 mL of the lipid emulsion, evacuated to 20 in. of mercury and sealed with an aluminum overseal. A flat perforated rack on a reciprocating shaker cart was used in the autoclave. The cycle’s target temperature was 123 C, recirculating water spray cycle with 70 rpm of axial agitation and 30 psig (pounds per square inch) of air overpressure. When the sterilization cycle was controlled to give a heat input of approximately 7.5 F 0 minutes in the coldest emulsion area, the average coldest emulsion area was found to be measured by TC number (TC#) 3,14. The average hottest emulsion area was measured by TC# 10, 21. The difference between the hottest and coldest emulsion areas ranged from 7.5 to 10.0 F0 minutes with an average of 8.6 F0 minutes. Therefore, when the coldest 8
Table 2
emulsion area registered 7.5 F0 minutes, the hottest emulsion area would average 16.1 F 0 minutes. The emulsion area approximating the validation TC location was measured by TC # 2,13 and averaged 7.7 F 0 minutes when the coldest emulsion was approximately 7.5 F 0 minutes (Fig. 5).
Solution/Product Moist Heat Resistance D - and z -Value Analysis A BIER vessel meets specific performance requirements for the assessment of BIs per American National Standards developed and published by AAMI (5). One important requirement for a BIER steam vessel is the capability of monitoring a square wave heating profile. Refer to Figure 6 for a schematic of the steam BIER vessel used to generate the D - and z -value data. D-value is the time in minutes required for a one log or 90% reduction in microbial population (Refer to Chapter 12, Fig. 1). The z-value is the number of degrees of
Solution Heat Rates (Minutes) 1000 mL glass I.V. containers-heat mapping study (lipid emulsion) Run CLHK00.49
Coldest location Thermocouple number Time to 100 C Time R100 C Time R120 C Time R120K100 C Maximum temperature ( C) Heat input ( F 0) Production profile TC location Thermocouple number Time to 100 C Time R100 C Time R120 C Time R120K100 C Maximum temperature ( C) Heat input ( F 0) 8
8 8
8
8
8
8 8
8
8
Note :
Run CLHK01.050
btl 1
btl 2
btl 1
btl 2
Average (SD)
3 19.0 21.0 4.0 4.0 120.82 7.46
12 19.0 21.0 3.0 5.0 120.77 7.28
3 19.0 21.0 4.0 4.0 120.92 7.71
12 19.0 21.0 3.0 5.0 120.77 7.36
– 19.00 (0.000) 21.00 (0.577) 3.50 (0.577) 4.50 (0.577) 120.82 (0.071) 7.45 (0.187)
2 19.0 22.0 4.0 5.0 120.91 7.79
13 19.0 21.0 3.0 5.0 120.82 7.49
2 19.0 22.0 4.0 5.0 120.91 8.02
13 19.0 21.0 3.0 5.0 120.92 7.64
– 19.00 (0.000) 21.50 (0.577) 3.50 (0.577) 5.00 (0.000) 120.89 (0.047) 7.74 (0.226)
H denotes hottest TC location; C denotes coldest TC location; PC denotes approximate location of the production profile TC; Data from TC#9 used with a postcalibration variance of C0.25 C at 100 C; All heat input values are calibration corrected. 8
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PSLR Values Lipid emulsion moist heat resistance values (D121 C and z-values) were generated in the steam BIER vessel using the BI C. sporogenes as shown in Table 3. The columns in the Table list the representative code or list number of the product, the emulsion or product name, its average D121 C value and z-value and finally the PSLR value. Those parenteral formulations with the lowest PSLR value(s) are those that should be used for the microbial validation at subprocess conditions, since these provide the most microbial resistance (6).
1000 mL Glass I.V. Container
8
- Heat Mapping Study Thermocouple Locations
8
1–1/12
1–1/2
1–1/2
1–1/2
Fill Level
5,16
10,21
11,22
6,17
7,18
8,19
2,13
3,14
4,15
3/4 9,20 1–1/12
1,12
Figure 4
Heat mapping study using a 1000 mL glass container.
temperature required for a 10-fold change in the D-value. (Refer to Chapter 11 for additional details on F-, D- and z-values.)
Master Solution/Product Concept Thefamily categoryof lipid emulsionsand their respective D121 C and z values as well as classification in terms of microbial resistance is shown in Table 3. A categorization of parenteral formulations with associated D121 C and z-values and their potential impact on microbial resistance using the BI, Clostridium sporogenes were previously reported (6). In addition, the methodologies used for Dand z-value analysis were likewise cited. The data in Table 3 indicate that the # 1 emulsion is at the top of the list, since it affords the most microbial moist heat resistance. It is therefore the emulsion that should be microbiologically challenged (inoculated with spores) as part of the emulsion validation scheme. D- and z-value data have been reported for other BIs such as Geobacillus stearothermophilus (6–8) and Bacillus subtilis 5230 (9). There are many factors that can affect moist heat resistance including a BI’s age, sporulation media used, as well as the particular spore strain employed (10). 8
8
Accumulated F bio for Lipid Emulsions Accumulated F bio and z-values (Table 4) were used to construct the PSLR ranking for lipid emulsions as previously discussed for Table 3. The F bio is the heat input for the biological solution based on the emulsion’s moist heat D- and z-values. By inputting the sterilizer temperatures from the coldest TC of an engineering run for a particular container/sterilization cycle, the emulsion can be ranked according to PSLR values. The combined D121 C and z-value allows comparison of moist heat rankings between emulsions. The data in Table 4 demonstrate that the #1 Emulsion has the lowest PSLR (7.105), thereby affording the highest moist heat resistance upon inoculation. Generation of this table allows prediction of which emulsion to microbiologically challenge as part of validation in the production sterilizer. 8
Microbial Closure Inactivation Validation in a Developmental Sterilizer In lieu of using the large type steam sterilizers in the production environment, microbial inactivation at the closure/bottle interface of an emulsion container can be assessed in a developmental sterilizer. The closure micro bial inactivation (kinetic) studies can determine how the size of the container, type of closure compound used as well as closure preparatory processes (e.g., leaching, washing, siliconizing, autoclaving) influence microbial inactivation. Microbial closure kinetic studies are conducted at various time intervals in a given sterilization cycle. DP count or F/N methodologies are used to
1000 mL Glass I.V. Container - Heat Mapping Study Average Heat Input (F 0) at Various Locations
Fill Level 16.1 12.9
7.7
15.7
12.7
12.8
13.5
7.7
7.5
7.8
14.3
Figure 5 Heat mapping study with average heat input ( F 0 ) at various locations in a 1000 mL glass container.
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195
VALIDATION OF TERMINAL STERILIZATION
Schematic of the Steam B.I.E.R Vessel
Instrument Well Vent
Top Chamber Vent
Thermocouples to Digital Display and Datalogger
Sample Chamber
Pressure Recording Controller
Steam Control Valve Steam Reservoir
Drain/Lower Chamber Vent
Table 3
Main Steam Supply
IV Lipid Emulsions Ranking
List #
1 2 3 4 5 6 7 8
Figure 6 Schematic of a steam biological indicator evaluator resistometer vessel used for generating moist heat resistance D - and z values for inoculated parenteral solutions or for biological indicators.
Solution
20% Emulsion 10% Emulsion w/increased linolenate 10% Emulsion w/100% soybean oil 20% Emulsion w/100% soybean oil 20% Emulsion w/increased linolenate 10% Emulsion w/50% safflower & 50% soybean oil 20% Emulsion w/50% safflower & 50% soybean oil 10% Emulsion
D121
z -value
Predicted spore log reduction
0.7 0.7 0.6 0.7 0.6 0.6 0.6 0.4
10.6 11.4 10.1 12.8 10.6 10.7 12.7 11.1
7.1 7.5 8.0 8.2 8.3 8.4 9.5 12.9
Table 4 Accumulated F bio by List Number and z Value Solution Temperature ( C) 8
105.4 110.1 114.1 116.2 118.1 119.1 119.4 119.2 118.5 117.8 116.2 114.1 110.6 105.9 101.7 Total F D value PSLR
Time (min)
F (PHY)
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0.0269 0.0793 0.1991 0.3228 0.5000 0.6295 0.6745 0.6442 0.5483 0.4667 0.3228 0.1991 0.0889 0.0301 0.0115 4.7436
z
10.0
Z
z
1 10.6
Z
0.0330 0.0915 0.2181 0.3442 0.5200 0.6462 0.6897 0.6604 0.5672 0.4872 0.3442 0.2181 0.1020 0.0367 0.0148 4.9734 0.70 7.105
z
2 11.4
Z
0.0419 0.1082 0.2427 0.3709 0.5445 0.6663 0.7079 0.6799 0.5903 0.5124 0.3709 0.2427 0.1197 0.0463 0.0198 5.2646 0.70 7.521
z
3 10.1
Z
0.0278 0.0813 0.2023 0.3265 0.5035 0.6324 0.6772 0.6470 0.5515 0.4702 0.3265 0.2023 0.0911 0.0312 0.0120 4.7826 0.60 7.971
z
4 12.8
Z
0.0592 0.1380 0.2834 0.4134 0.5819 0.6966 0.7352 0.7092 0.6253 0.5513 0.4134 0.2834 0.1510 0.0648 0.0305 5.7366 0.70 8.195
z
5 10.6
Z
0.0330 0.0915 0.2181 0.3442 0.5200 0.6462 0.6897 0.6604 0.5672 0.4872 0.3442 0.2181 0.1020 0.0367 0.0148 4.9734 0.60 8.289
z
6 10.7
Z
0.0340 0.0935 0.2212 0.3476 0.5232 0.6489 0.6921 0.6630 0.5703 0.4905 0.3476 0.2212 0.1042 0.0379 0.0153 5.0107 0.60 8.351
z
7 12.7
Z
0.0579 0.1359 0.2806 0.4106 0.5794 0.6946 0.7334 0.7073 0.6230 0.5487 0.4106 0.2806 0.1487 0.0634 0.0296 5.7044 0.60 9.507
z
8 11.1
Z
0.0384 0.1019 0.2336 0.3611 0.5356 0.6591 0.7014 0.6729 0.5819 0.5033 0.3611 0.2336 0.1130 0.0426 0.0178 5.1573 0.40 12.893
196
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STERILIZATION, SANITIZATION AND STERILITY ASSURANCE
evaluate the surviving organisms. Test data has been generated demonstrating the value of using both a moist heat organism (C. sporogenes) and a dry heat organism (B. subtilis now known as Bacillus atrophaeus) as BIs for the sterilization validation of closure systems (11,12). A typical graphic representation of the inactivation kinetics is illustrated in Figure 7. The above studies may also be performed in a production sterilizer as engineering or feasibility studies.
closure systems of a parenteral container. This validation is performed to demonstrate that the closure system of a container is capable of maintaining the emulsion and fluid path in a sterile condition throughout the shelf life of the product. In a typical MOS study, the product container is sterilized at a temperature which is higher than the upper temperature limit of the chosen sterilization cycle and for a time that is greater than the maximum time limit for the cycle or producing an F 0 subzero level greater than the maximum F0 level for the cycle. The rationale for the selection of the maximum temperature and heat input level for the prechallenge sterilization is
ContainerClosure Integrity Validation Container–closure integrity or MOS validations are run on all moist heat terminally sterilized products with
1.0E+06 Study 1 Study 2 Linear Regression 1.0E+05
1.0E+04
1.0E+03
C. sporogenes 1.0E+02 s r o v i v r u S
1.0E+01 Fraction/Negative Zone
B. subtilis
1.0E+00 Enhanced Bioburden
Fraction of Negative Units 0.01 0.05 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90
1.0E−01
0.95
0.99
1.0E−02
1.0E−03 0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Time Above 100 C Average Count of 0 for Direct Plate Testing is Given a Value of 1.00E−01 Average Count of 0 for Fraction/Negative Testing is Given a Value of 1.00E −02
Figure 7 Microbialkineticinactivation of bioburden as compared to biological indicators, Bacillus atrophaeus (formerly named Bacillus subtilis ) and Clostridium sporogenes .
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PRODUCTION FACILITY STERILIZATION DEVELOPMENT
that rubber and plastic closures are subjected to thermal stresses during sterilization and those stresses are maximized at the highest temperature and the longest time allowed. In some cases, the closures, e.g., administration or additive port are claimed to be sterile by a radiation process. In such cases, the closures are sterilized in bulk exceeding the maximum end of the radiation process e.g., 40 kGy, then fabricated to the flexible container and exposed to steam sterilization cycle conditions exceeding the maximum temperature end of the cycle. Thus, the closures are stressed by a joint process of radiation as well as steam prior to performing the closure integrity test.
The production list below depicts some of the sterilization engineering and microbiological activities associated with a parenteral product as it moves into the production environment. These studies occur in a production environment as appropriate. Production facility activity
Activity statement
Heat P&Ds
Product Validation for Endotoxin Endotoxins are lipopolysaccharides from the outer cell membrane of gram-negative bacteria. Endotoxins can be detected by the manual gel-clot method known as the LAL test. There are also various quantitative methods (turbidimetric and chromogenic) which use more rapid automated methodologies. All final product formulations have regulatory requirements to be tested for endotoxins and the method must be validated using three different lots of final product. LAL testing should be performed on final product formulations per FDA Guidelines and other regulatory compendia. Emulsion formulations, if colored or opaque cannot be tested by the turbidimetric method and therefore may use a comparable test e.g., LAL, chromogenic or kinetic. The LAL test is for products other than oral and topical products (e.g., parenteral solutions, some devices, etc). Endotoxin testing is usually required at three different times in the cycle of the product. First, endotoxin testing should be performed on the lot of drug being used in clinical studies to ensure that the product is safe for the patients with respect to endotoxin. Second, in the developmental stages, endotoxin testing is usually required at the beginning and end of the stability studies. Finally, once the product is ready to be marketed, each lot of the product requires endotoxin testing prior to release. To improve in-process control, a process should also be in place to decide if endotoxin testing should be performed on the APIs and/or excipients used in the product. In order to determine this, the ICH guidelines for quality should be used; i.e., Q7A “Good Manufacturing Practice Guidance for APIs.”
Table 5
197
VALIDATION OF TERMINAL STERILIZATION
Solution (master) microchallenge validation
Container–closure microchallenge validation as applicable
Hold time studies
Perform triplicate studies for minimum and maximum loading conditions using temperature probes within the product containers and outside the containers to measure the sterilizer heating medium temperature Perform microbial validation of a parenteral solution or master solution at subprocess conditions in the production sterilizer Perform microbial validation of the container–closure system at sub-process conditions in the production sterilizer Microbial, chemical and endotoxin studies are performed to establish the longest time that a product can be held following manufacture but prior to filling and sterilization
Engineering P&D Validation Perform triplicate studies with minimum and maximum loading configurations with temperature probes penetrating the product containers as well as temperature probes distributed outside the product containers in a production sterilizer at nominal operating process parameters. Microbial Solution Validation in a Production Sterilizer Table 5 shows the microbial solution validation conducted at subprocess conditions in a fully loaded
Lipid Emulsion Microbial Solution Validation Fraction negative method
Organism
Code
Average no. spores/bottle
No. positivea / no. positive controls
C. sporogenes C. sporogenes G. stearothermophilus G. stearothermophilus
5C6 15C6 5B2 15B2
4.8!105 6.4!105 7.6!101 7.7!101
2/2 2/2 2/2 2/2
c
No. positivea / no. negative controls
No. positive / no. test samples
0/4 0/4 0/4 0/4
0/20 0/20 0/20 0/20
a
Sporeb logarithmic reduction O7.0 O7.1 O3.2 O3.1
F 0 Range : 5.8–7.6; Temperature Range: 120–125 C; Agitation: 67–73cpm a Positive for the indicator microorganism. b Spore logarithmic reduction log a Klog b ; where a , initial population of spores; b , 2.303 log (N / q) ln (N / q) ; where N , total number of units tested; q , number of sterile units. c F , integrated lethality or equivalent minutes at 121.1 C for hottest and coldest thermocoupled containers. 8
Z
Z
8
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III:
Table 6
Lipid Emulsion Microbial Closure validation
STERILIZATION, SANITIZATION AND STERILITY ASSURANCE
Microorganism
Initial population/stopper
No. positivea / no. positive controls
No. positivea / no. negative controls
No. positivea / test samples
C. sporogenes B. subtillis
8.4!103 3.0!104
2/2 2/2
0/4 0/4
0/20 0/20
Sporeb logarithmic reduction O5.2 O5.8
F 0 Rangec: 5.8–7.6; Temperature Range: 120–125 C; Agitation: 67–73 cpm Sterilization validation of 200 mL bottle inoculated closure surface coated with I.V. fat emulsion in cycle with agitation. a Positive for the indicator microorganism. b Sporelogarithmic reduction log a Klog b ; where a , initial population of spores; b , 2.303 log (N / q) In (N / q ); where N , total number ofunitstested; q , number ofsterile units. c F , integrated lethality of equivalent minutes at 121.1 C for hottest and coldest thermocoupled containers. 8
Z
Z
8
production sterilizer. The acceptance criteria of 6 SLR was setup for the BI C. sporogenes and a 3 SLR for the higher moist heat-resistant BI, G. stearothermophilus. Each emulsion (20 containers) is inoculated with the appropriate BI at a target level of 1.0!106 and 1.0!102 for C. sporogenes and G. stearothermophilus, respectively. The 20 inoculated containers are distributed throughout the production sterilizer for sterilization at subprocess conditions. The test containers are then returned to the lab for testing by the F/N test method.
conditions in the production sterilizer and subsequently tested in the lab by the F/N test method. The data demonstrate that a O3 SLR was achieved at subminimal process conditions. Replicate test samples (e.g., 3 to 5) should be considered foruse to verify microbial kill in cold zone locations.
ANCILLARY SUPPORT PROCESS TESTING Bioburden Analysis for Closures and Commodities Determine microbial load on closures and commodities as well as their moist heat resistance analysis. As part of the microbiological quality control program, products and commodities are routinely sampled during the production process in order to assess the microbial load. This assessment is performed via the bioburden test for terminally sterilized product. The bioburden test method is developed during the product development stage prior to transfer to the production plant. This test assesses the microbial load of a solution prior to terminal sterilization (In-Process Bioburden Test). Micro R&D is responsible for the validation of the bioburden method prior to transfer to the production plant. The validation will demonstrate that
Microbial Closure Validation in a Production Sterilizer Table 6 shows the microbial closure validation at subprocess conditions in a fully loaded production sterilizer. The BIsused were C. sporogenes and B. subtilis. Acceptance criteria of three SLR must be achieved for moist heat (C. sporogenes) and dry heat (B. subtilis indicators). The surface of the stopper that comes into direct contact with the sidewall of the bottle was inoculated with the appropriate BI, dried and then a few drops of emulsion were placed over the inoculum to simulate manufacturing conditions. The inoculated closure was assembled to the finished container, exposed to subprocess steam
Production Environment Bioburden Screening Program Heat Shock Positives Gram Stain/Plate Morphology
Gram Positive Rods Yeasts, Molds Gram Negative Rods Cocci
Retesting with 30-min Heat Shock not Routinely Required
Second Heat Shock
10 min (+) 30 min (–) Calculate Probability of Non-Sterility
10 min (+) 30 min (+) Moist Heat Resistance Analysis Required
Figure 8 Representative production environment bioburden screening program.
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recovery of microbial load at a relatively low level can be achieved. The Microbial Limits Test is essentially a bioburden test of raw materials used to make the final product. The test method and validation are conducted in much the same manner as the bioburden test. The limit for the microbial limits test is calculated as follows: final Product Action Level/maximum concentration of API in the final product. This limit is then “normalized: by dividing by the total amount of APIs in the final product.” In addition, the production bulk solution is monitored for total bioburden load including spore formers. The screening allows the plant quality lab to ascertain if there are any moist heat-resistant microflora present in the bulk solution prior to the terminal sterilization of the parenteral solution in its finished container (Fig. 8).
Antimicrobial Preservative Efficacy Perform on those formulations containing a preservative and those container configurations that have a multidose claim. This validation is performed per compendial requirements.
Sterility Testing (if Required) Once parametric release is approved by regulatory authorities, then sterility testing is no longer required nor can it be used as an alternative in case parametric release parameters are not used.
Biological Testing Support of R&D and Marketed Product Stability Programs There are a number of analytical and microbiological tests performed over the shelf life of a product. A number of microbiological tests include BET, Container–Closure Integrity and APE if applicable.
CONCLUSION As one reviews the final configuration that a terminally sterilized parenteral product is packaged in, it is not surprising that a similar evaluation occurred when one was contemplating how to present the new product as being sterile and non-pyrogenic. The product development team focused on the various designs of sterilizers and the various manufacturing site locations for the support of currently marketed products. Once the team decided the appropriate facility for manufacture, then the various sterilization cycles discussed in this chapter were evaluated in order to select the appropriate one best suited for that parenteral product in its final container configuration. If a product is destined for the international market, then R&D personnel will follow the
VALIDATION OF TERMINAL STERILIZATION
199
EMEA decision tree to determine if the product can be sterilized at 121 C for 15 minutes minimum. If it cannot, then a justification is documented explaining the reason for selection of an alternate sterilization cycle. Personnel perform the applicable studies in a developmental sterilizer as detailed in this chapter, as well as feasibility studies in development or production sterilizers to monitor and test the physical attributes of the final designed container. Once the parenteral product’s designs as well as sterilization processes have been finalized, then the plant/site can perform their standard penetration, distribution and microbiological studies in the production sterilizer. 8
REFERENCES 1. ISPE, GAMP 4 Guide for Validation of Automated Systems. December 2001. 2. Committee for Proprietary Medicinal Products (CPMP) Decision Trees for the Selection of Sterilisation Methods (CPMP/QWP/054/98), Annex to Note for Guidance on Development Pharmaceutics, EMEA 2000, 1–3. 3. Young JH. Sterilization with steam under pressure. In: Morrissey RF, Phillips GB, eds. Sterilization Technology. New York: Van Nostrand Reinhold, 1993:120–51. 4. Owens JE. Sterilization of LVP’s and SVP’s. In: Morrissey RF, Phillips GB, eds. Sterilization Technology. New York: Van Nostrand Reinhold, 1993:254–85. 5. AAMI. BIER/Steam Vessels (ST45). Arlington, VA: American National Standard for the Advancement of Medical Instrumentation, 1992. 6. Berger TJ, Nelson PA. The effect offormulation of parenteral solutions on microbial growth-measurement of D- and z-values. PDA J Pharm Sci Technol 1995; 49(1):32–41. 7. Feldsine PT, Schechtman AJ, Korczynski MS. Survivor kinetics of bacterial spores in various steam heated parenteral solutions. Dev Ind Microbial 1977; 18:401–7. 8. Moldenhauer J. How does moist heat inactivate microorganisms?. In: Moldenhauer J, ed. Steam Sterilization, A Practitioner’s Guide. Bethesda, MD/Godalming, UK: PDA, 2003:1–15. 9. Caputo RA, Odlaug TE, Wilkinson RL, et al. Biological validation of a sterilization process for a parenteral product-fractional exposure method. J Parent Drug Assoc 1979; 33:214–21. 10. Pflug IJ, Holcomb RG. Principles of thermal destruction of microorganisms. In: Block SS, ed. Disinfection, Sterilization and Preservation. 3rd ed. Philadelphia, PA: Lea and Febiger, 1983:759–66. 11. Berger TJ, May TB, Nelson PA, et al. The effect of closure processing on the microbial inactivation of biological indicators at the closure–container interface. PDA J Pharm Sci Technol 1998; 52(2):70–4. 12. Berger TJ, Chavez C, Tew RD, et al. Biological comparative analysis in various product formulations and closure sites. PDA J Pharm Sci Technol 2000; 54(2):101–9.
