Thursday, November 27, 2014

Why is an effective environmental monitoring program so important?

In the end, whether you are manufacturing a medical device or a drug API, the safety of end users is at stake. Having a thorough environmental monitoring program in place will allow you to know the status of your environment, giving you the power to control it and make changes to it.

The overall keys to implementing an environmental monitoring program

1. Determine what the monitoring strategy will be, including:
  • Limits and acceptance levels (often based on the product being manufactured and knowledge of your facility)
  • Decide what specifically should be monitored
  • Determine the frequency of monitoring
  • Identify where problem areas might be; note where limits are most likely to be challenged
2. Decide on the type of sampling necessary:
  •  Air – good for general environment, but be aware that air contamination is affected by levels of activity
  •  Surfaces – products may come into contact with surfaces so surface collection strips may be necessary
3. Implement a thorough documentation procedure – data must match procedures
4. Must validate the monitoring procedure and ensure that collection media and techniques are sufficient
5. If any change in the manufacturing process or area occurs then you must decide whether monitoring changes need to be implemented. Also, if results have been steady for a long period of time, the frequency of testing may possibly be reduced
6. Use the results of the data to determine whether changes can be implemented to minimize the possibility of contamination
These are just some of the basic keys to implementing a successful program. For a more thorough background, we recommend "Environmental Monitoring: Making the Invisible Environment Visible" in Contract Pharma magazine.

An Overview of Microbial QC Test Procedures

Why Is Microbial Testing Necessary?

“How do we determine that our substance or preparation complies with an established specification for microbial quality?” This is what many manufacturers or producers of pharmaceuticals or medical grade products ask themselves. When manufacturing a product for human use or consumption, there are many considerations involved, not least of which is proving that the product or sample is safe for use, and determining how long the product can stay safe without changing its original composition or nature. Much of this centers on ensuring that microbial contamination is within established limits and standards.

History of the Harmonized MLT

To be able to determine whether a substance, a product, or a preparation complies with an established specification for microbial quality, testing needs to be performed. Many standards have been created not only to guard the manner of production but ultimately protect consumers and avoid release of a product that is unsafe to use. Historically, the United States Pharmacopeia chapter <61> Microbial Limits Test, provided directives for the estimation of the numbers of viable aerobic microorganisms present in a manufactured nonsterile product. It contained instructions to perform testing for: Plate Count (TPC), which includes Aerobic Plate Count (APC) and Yeast/Mold Count (YMC), and Screening/ Detection for Specified Microorganisms. USP Chapter 61 was also equivalent to Chapter 35 “Microbial Limit Test (MLT)” of the Japanese Pharmacopoeia.
In order to provide for standards that are applicable not just in the United States, but around the world, and because certain chapters from the USP are nearly identical to sections of the European Pharmacopeia, a course of harmonization was decided upon. International harmonization of the chapters was initiated and made official by the USP on May 1, 2009. The previous version of the MLT, USP <61>, was separated into 2 chapters, USP chapter <61> Microbial Enumeration Tests (MET) which includes Total Aerobic Microbial count (TAMC) and Total combined Yeasts and Molds Count (TYMC); and USP chapter <62> Tests for Specified Microorganisms. These chapters are equivalent to European Pharmacopoeia (EP) chapters 2.6.12 Total Viable Aerobic Count (TAC) and 2.6.13 Tests for Specified Microorganism. Incidentally, these chapters are exactly identical to the British Pharmacopoeia (BP) chapter B1. Tests for Specified Microorganisms and B2. Total Viable Aerobic Count.  Because of these equivalencies, the new USP Chapters are commonly referred to as the “Harmonized MLT” or the “Harmonized Microbial Limits Test.”

Microbial Testing Procedures for Manufactured Products

An Overview of Microbial QC Test Procedures
Microbial Limits Testing must be carried out under conditions designed to avoid accidental extrinsic microbial contamination of the product to be examined during the test. However, any product/preparation with antimicrobial component(s) needs to be neutralized to remove its antimicrobial activity before testing. The validity of the test results depends largely upon demonstrating that the test articles do not inhibit the multiplication of microorganisms that may be present under any test condition.
Classification of product plays an important role in the selection of the test method (Plate Count Method, Most-Probable-Number (MPN), and Membrane Filtration), type of organisms that need to be screened, and acceptance criteria to be imposed according to the microbial quality prescribed by the standards. Before any testing to occur, Growth Promotion, Suitability of the Counting Method (Validation), and Suitability Tests for Specified Microorganisms (Validation) should be performed first. The ability of the test to detect microorganisms in the presence of the product to be tested must be established, and suitability must be confirmed if a change in testing performance or a change in the product is introduced that may affect the outcome of the test.

Antimicrobial Preservatives and Testing Considerations

Products may contain antimicrobial preservatives added to prevent proliferation or limit microbial contamination that may occur subsequent to the manufacturing process. Preservatives may also be added to inhibit the growth of microorganisms during normal conditions of storage and use, such as when microbes might be introduced inadvertently from repeated withdrawing of individual doses of a product from a containment vessel. Without preservatives, continued use could results in spoilage of the product or render it hazardous to users. Always keep in mind, however, that “Antimicrobial preservatives must not be used as a substitute for Good Manufacturing Practice.”
In order to support a claim that the added preservative is adequate to provide protection from adverse effects that may arise from microbial contamination or proliferation of microorganisms during storage and use, the USP Antimicrobial Effectiveness Test (AET), also known as Efficacy of Antimicrobial Preservation (EAP) - European (EP) and British Pharmacopoeia (BP) and Preservatives-Effectiveness Tests (PET)-Japan Pharmacopoeia (JP) should be tested to. This test is not intended to be used for routine control purposes, but instead is focused on challenging the preparation in its final container with prescribed inoculums of suitable microorganisms.
Compendial articles for testing have been divided into four categories based on route of administration. Each category has its own acceptable criteria for tested microorganisms. Individual product samples are inoculated with high concentrations of specific organisms and incubated at room temperature for 28 days. The population of any surviving microorganisms will be determined at a specific time interval depending on which standard is to be followed (each standard has its own intervals). Determination of the surviving microbial population will be performed using the Plate Count Method and calculating the log reduction of each microbial strain. However, prior to performing the actual test, a Plate Count Method Validation must be completed for the specified item.


The first step to successfully test any product is to know the product itself, and be able to classify it according to its intended final use. The nature and frequency of testing vary according to the products, as some may require freedom from one or more species of selected indicator microorganisms.  The significance of microorganisms in non-sterile pharmaceutical products should be evaluated in terms of the use and the nature of the product, and the potential hazard to the user as well. Certain categories of products should be tested routinely for total microbial count and for a specified indicator of microbial contaminants. Regardless, it is still essential to apply strict good manufacturing practices to assure the lowest possible load of microorganisms. For reliable results, testing should be performed by personnel with specialized training in Microbiology and in the interpretation of microbiological results and data.

Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; freezing, primary drying (sublimation), and secondary drying (desorption).
The advantages of lyophilization include:
Ease of processing a liquid, which simplifies aseptic handling
Enhanced stability of a dry powder
Removal of water without excessive heating of the product
Enhanced product stability in a dry state
Rapid and easy dissolution of reconstituted product
Disadvantages of lyophilization include:
Increased handling and processing time
Need for sterile diluent upon reconstitution
Cost and complexity of equipment
The lyophilization process generally includes the following steps:
  • Dissolving the drug and excipients in a suitable solvent, generally water for injection (WFI).
  • Sterilizing the bulk solution by passing it through a 0.22 micron bacteria-retentive filter.
  • Filling into individual sterile containers and partially stoppering the containers under aseptic conditions.
  • Transporting the partially stoppered containers to the lyophilizer and loading into the chamber under aseptic conditions.
  • Freezing the solution by placing the partially stoppered containers on cooled shelves in a freeze-drying chamber or pre-freezing in another chamber.
  • Applying a vacuum to the chamber and heating the shelves in order to evaporate the water from the frozen state.
  • Complete stoppering of the vials usually by hydraulic or screw rod stoppering mechanisms installed in the lyophilizers.
There are many new parenteral products, including anti-infectives, biotechnology derived products, and in-vitro diagnostics which are manufactured as lyophilized products. Additionally, inspections have disclosed potency, sterility and stability problems associated with the manufacture and control of lyophilized products. In order to provide guidance and information to investigators, some industry procedures and deficiencies associated with lyophilized products are identified in this Inspection Guide.
It is recognized that there is complex technology associated with the manufacture and control of a lyophilized pharmaceutical dosage form. Some of the important aspects of these operations include: the formulation of solutions; filling of vials and validation of the filling operation; sterilization and engineering aspects of the lyophilizer; scale-up and validation of the lyophilization cycle; and testing of the end product. This discussion will address some of the problems associated with the manufacture and control of a lyophilized dosage form.
Products are manufactured in the lyophilized form due to their instability when in solution. Many of the antibiotics, such as some of the semi-synthetic penicillins, cephalosporins, and also some of the salts of erythromycin, doxycycline and chloramphenicol are made by the lyophilization process. Because they are antibiotics, low bioburden of these formulations would be expected at the time of batching. However, some of the other dosage forms that are lyophilized, such as hydrocortisone sodium succinate, methylprednisolone sodium succinate and many of the biotechnology derived products, have no antibacterial effect when in solution.
For these types of products, bioburden should be minimal and the bioburden should be determined prior to sterilization of these bulk solutions prior to filling. Obviously, the batching or compounding of these bulk solutions should be controlled in order to prevent any potential increase in microbiological levels that may occur up to the time that the bulk solutions are filtered (sterilized). The concern with any microbiological level is the possible increase in endotoxins that may develop. Good practice for the compounding of lyophilized products would also include batching in a controlled environment and in sealed tanks, particularly if the solution is to be held for any length of time prior to sterilization.
In some cases, manufacturers have performed bioburden testing on bulk solutions after prefiltration and prior to final filtration. While the testing of such solutions may be meaningful in determining the bioburden for sterilization, it does not provide any information regarding the potential formation or presence of endotoxins. While the testing of 0.1 ml samples by LAL methods of bulk solution for endotoxins is of value, testing of at least 100 ml size samples prior to prefiltration, particularly for the presence of gram negative organisms, would be of greater value in evaluating the process. For example, the presence of Pseudomonas sp. in the bioburden of a bulk solution has been identified as an objectionable condition.
The filling of vials that are to be lyophilized has some problems that are somewhat unique. The stopper is placed on top of the vial and is ultimately seated in the lyophilizer. As a result the contents of the vial are subject to contamination until they are actually sealed.
Validation of filling operations should include media fills and the sampling of critical surfaces and air during active filling (dynamic conditions).
Because of the active involvement of people in filling and aseptic manipulations, an environmental program should also include an evaluation of microbiological levels on people working in aseptic processing areas. One method of evaluation of the training of operators working in aseptic processing facilities includes the surface monitoring of gloves and/or gowns on a daily basis. Manufacturers are actively sampling the surfaces of personnel working in aseptic processing areas. A reference which provides for this type of monitoring is the USP XXII discussion of the Interpretation of Sterility Test Results. It states under the heading of "Interpretation of Quality Control Tests" that review consideration should be paid to environmental control data, including...microbial monitoring, records of operators, gowns, gloves, and garbing practices. In those situations in which manufacturers have failed to perform some type of personnel monitoring, or monitoring has shown unacceptable levels of contamination, regulatory situations have resulted.
Typically, vials to be lyophilized are partially stoppered by machine. However, some filling lines have been noted which utilize an operator to place each stopper on top of the vial by hand. At this time, it would seem that it would be difficult for a manufacturer to justify a hand-stoppering operation, even if sterile forceps are employed, in any type of operation other than filling a clinical batch or very small number of units. Significant regulatory situations have resulted when some manufacturers have hand-stoppered vials. Again, the concern is the immediate avenue of contamination offered by the operator. It is well recognized that people are the major source of contamination in an aseptic processing filling operation. The longer a person works in an aseptic operation, the more microorganisms will be shed and the greater the probability of contamination.
Once filled and partially stoppered, vials are transported and loaded into the lyophilizer. The transfer and handling, such as loading of the lyophilizer, should take place under primary barriers, such as the laminar flow hoods under which the vials were filled. Validation of this handling should also include the use media fills.
Regarding the filling of sterile media, there are some manufacturers who carry out a partial lyophilization cycle and freeze the media. While this could seem to greater mimic the process, the freezing of media could reduce microbial levels of some contaminants. Since the purpose of the media fill is to evaluate and justify the aseptic capabilities of the process, the people and the system, the possible reduction of microbiological levels after aseptic manipulation by freezing would not be warranted. The purpose of a media fill is not to determine the lethality of freezing and its effect on any microbial contaminants that might be present.
In an effort to identify the particular sections of filling and aseptic manipulation that might introduce contamination, several manufacturers have resorted to expanded media fills. That is, they have filled approximately 9000 vials during a media fill and segmented the fill into three stages. One stage has included filling of 3000 vials and stoppering on line; another stage included filling 3000 vials, transportation to the lyophilizer and then stoppering; a third stage included the filling of 3000 vials, loading in the lyophilizer, and exposure to a portion of the nitrogen flush and then stoppering. Since lyophilizer sterilization and sterilization of the nitrogen system used to backfill require separate validation, media fills should primarily validate the filling, transportation and loading aseptic operations.
The question of the number of units needed for media fills when the capacity of the process is less than 3000 units is frequently asked, particularly for clinical products. Again, the purpose of the media fill is to assure that product can be aseptically processed without contamination under operating conditions. It would seem, therefore, that the maximum number of units of media filled be equivalent to the maximum batch size if it is less than 3000 units.
After filling, dosage units are transported to the lyophilizer by metal trays. Usually, the bottom of the trays are removed after the dosage units are loaded into the lyophilizer. Thus, the dosage units lie directly on the lyophilizer shelf. There have been some situations in which manufacturers have loaded the dosage units on metal trays which were not removed. Unfortunately, at one manufacturer, the trays warped which caused a moisture problem in some dosage units in a batch.
In the transport of vials to the lyophilizer, since they are not sealed, there is concern for the potential for contamination. During inspections and in the review of new facilities, the failure to provide laminar flow coverage or a primary barrier for the transport and loading areas of a lyophilizer has been regarded as an objectionable condition. One manufacturer as a means of correction developed a laminar flow cart to transport the vials from the filling line to the lyophilizer. Other manufacturers building new facilities have located the filling line close to the lyophilizer and have provided a primary barrier extending from the filling line to the lyophilizer.
In order to correct this type of problem, another manufacturer installed a vertical laminar flow hood between the filling line and lyophilizer. Initially, high velocities with inadequate return caused a contamination problem in a media fill. It was speculated that new air currents resulted in rebound contamination off the floor. Fortunately, media fills and smoke studies provided enough meaningful information that the problem could be corrected prior to the manufacture of product. Typically, the lyophilization process includes the stoppering of vials in the chamber.
Another major concern with the filling operation is assurance of fill volumes. Obviously, a low fill would represent a subpotency in the vial. Unlike a powder or liquid fill, a low fill would not be readily apparent after lyophilization particularly for a biopharmaceutical drug product where the active ingredient may be only a milligram. Because of the clinical significance, sub-potency in a vial potentially can be a very serious situation.
For example, in the inspection of a lyophilization filling operation, it was noted that the firm was having a filling problem. The gate on the filling line was not coordinated with the filling syringes, and splashing and partial filling was occurring. It was also observed that some of the partially filled vials were loaded into the lyophilizer. This resulted in rejection of the batch.
On occasion, it has been seen that production operators monitoring fill volumes record these fill volumes only after adjustments are made. Therefore, good practice and a good quality assurance program would include the frequent monitoring of the volume of fill, such as every 15 minutes. Good practice would also include provisions for the isolation of particular sections of filling operations when low or high fills are encountered.
There are some atypical filling operations which have not been discussed. For example, there have also been some situations in which lyophilization is performed on trays of solution rather than in vials. Based on the current technology available, it would seem that for a sterile product, it would be difficult to justify this procedure.
The dual chamber vial also presents additional requirements for aseptic manipulations. Media fills should include the filling of media in both chambers. Also, the diluent in these vials should contain a preservative. (Without a preservative, the filling of diluent would be analogous to the filling of media. In such cases, a 0% level of contamination would be expected.)
After sterilization of the lyophilizer and aseptic loading, the initial step is freezing the solution. In some cycles, the shelves are at the temperature needed for freezing, while for other cycles, the product is loaded and then the shelves are taken to the freezing temperature necessary for product freeze. In those cycles in which the shelves are precooled prior to loading, there is concern for any ice formation on shelves prior to loading. Ice on shelves prior to loading can cause partial or complete stoppering of vials prior to lyophilization of the product. A recent field complaint of a product in solution and not lyophilized was attributed to preliminary stoppering of a few vials prior to exposure to the lyophilization cycle. Unfortunately, the firm's 100% vial inspection failed to identify the defective vial.
Typically, the product is frozen at a temperature well below the eutectic point.
The scale-up and change of lyophilization cycles, including the freezing procedures, have presented some problems. Studies have shown the rate and manner of freezing may affect the quality of the lyophilized product. For example, slow freezing leads to the formation of larger ice crystals. This results in relatively large voids, which aid in the escape of water vapor during sublimation. On the other hand, slow freezing can increase concentration shifts of components. Also, the rate and manner of freezing has been shown to have an affect on the physical form (polymorph) of the drug substance.
It is desirable after freezing and during primary drying to hold the drying temperature (in the product) at least 4-5o below the eutectic point. Obviously, the manufacturer should know the eutectic point and have the necessary instrumentation to assure the uniformity of product temperatures. The lyophilizer should also have the necessary instrumentation to control and record the key process parameters. These include: shelf temperature, product temperature, condenser temperature, chamber pressure and condenser pressure. The manufacturing directions should provide for time, temperature and pressure limits necessary for a lyophilization cycle for a product. The monitoring of product temperature is particularly important for those cycles for which there are atypical operating procedures, such as power failures or equipment breakdown.
Electromechanical control of a lyophilization cycle has utilized cam-type recorder-controllers. However, newer units provide for microcomputer control of the freeze drying process. A very basic requirement for a computer controlled process is a flow chart or logic. Typically, operator involvement in a computer controlled lyophilization cycle primarily occurs at the beginning. It consists of loading the chamber, inserting temperature probes in product vials, and entering cycle parameters such as shelf temperature for freezing, product freeze temperature, freezing soak time, primary drying shelf temperature and cabinet pressure, product temperature for establishment of fill vacuum, secondary drying shelf temperature, and secondary drying time.
In some cases, manufacturers have had to continuously make adjustments in cycles as they were being run. In these situations, the lyophilization process was found to be non-validated.
Validation of the software program of a lyophilizer follows the same criteria as that for other processes. Basic concerns include software development, modifications and security. The Guide to Inspection of Computerized Systems in Drug Processing contains a discussion on potential problem areas relating to computer systems. A Guide to the Inspection of Software Development Activities is a reference that provides a more detailed review of software requirements.
Leakage into a lyophilizer may originate from various sources. As in any vacuum chamber, leakage can occur from the atmosphere into the vessel itself. Other sources are media employed within the system to perform the lyophilizing task. These would be the thermal fluid circulated through the shelves for product heating and cooling, the refrigerant employed inside the vapor condenser cooling surface and oil vapors that may migrate back from the vacuum pumping system.
Any one, or a combination of all, can contribute to the leakage of gases and vapors into the system. It is necessary to monitor the leak rate periodically to maintain the integrity of the system. It is also necessary, should the leak rate exceed specified limits, to determine the actual leak site for purposes of repair.
Thus, it would be beneficial to perform a leak test at some time after sterilization, possibly at the beginning of the cycle or prior to stoppering. The time and frequency for performing the leak test will vary and will depend on the data developed during the cycle validation. The pressure rise found acceptable at validation should be used to determine the acceptable pressure rise during production. A limit and what action is to be taken if excessive leakage is found should be addressed in some type of operating document.
In order to minimize oil vapor migration, some lyophilizers are designed with a tortuous path between the vacuum pump and chamber. For example, one fabricator installed an oil trap in the line between the vacuum pump and chamber in a lyophilizer with an internal condenser. Leakage can also be identified by sampling surfaces in the chamber after lyophilization for contaminants. One could conclude that if contamination is found on a chamber surface after lyophilization, then dosage units in the chamber could also be contaminated. It is a good practice as part of the validation of cleaning of the lyophilization chamber to sample the surfaces both before and after cleaning.
Because of the lengthy cycle runs and strain on machinery, it is not unusual to see equipment malfunction or fail during a lyophilization cycle. There should be provisions in place for the corrective action to be taken when these atypical situations occur. In addition to documentation of the malfunction, there should be an evaluation of the possible effects on the product (e.g., partial or complete meltback. Refer to subsequent discussion). Merely testing samples after the lyophilization cycle is concluded may be insufficient to justify the release of the remaining units. For example, the leakage of chamber shelf fluid into the chamber or a break in sterility would be cause for rejection of the batch.
The review of Preventive Maintenance Logs, as well as Quality Assurance Alert Notices, Discrepancy Reports, and Investigation Reports will help to identify problem batches when there are equipment malfunctions or power failures. It is recommended that these records be reviewed early in the inspection.
Many manufacturers file (in applications) their normal lyophilization cycles and validate the lyophilization process based on these cycles. Unfortunately, such data would be of little value to substantiate shorter or abnormal cycles. In some cases, manufacturers are unaware of the eutectic point. It would be difficult for a manufacturer to evaluate partial or abnormal cycles without knowing the eutectic point and the cycle parameters needed to facilitate primary drying.
Scale-up for the lyophilized product requires a knowledge of the many variables that may have an effect on the product. Some of the variables would include freezing rate and temperature ramping rate. As with the scale-up of other drug products, there should be a development report that discusses the process and logic for the cycle. Probably more so than any other product, scale-up of the lyophilization cycle is very difficult.
There are some manufacturers that market multiple strengths, vial sizes and have different batch sizes. It is conceivable and probable that each will have its own cycle parameters. A manufacturer that has one cycle for multiple strengths of the same product probably has done a poor job of developing the cycle and probably has not adequately validated their process. Investigators should review the reports and data that support the filed lyophilization cycle.
The sterilization of the lyophilizer is one of the more frequently encountered problems noted during inspections. Some of the older lyophilizers cannot tolerate steam under pressure, and sterilization is marginal at best. These lyophilizers can only have their inside surfaces wiped with a chemical agent that may be a sterilant but usually has been found to be a sanitizing agent. Unfortunately, piping such as that for the administration of inert gas (usually nitrogen) and sterile air for backfill or vacuum break is often inaccessible to such surface "sterilization" or treatment. It would seem very difficult for a manufacturer to be able to demonstrate satisfactory validation of sterilization of a lyophilizer by chemical "treatment".
Another method of sterilization that has been practiced is the use of gaseous ethylene oxide. As with any ethylene oxide treatment, humidification is necessary. Providing a method for introducing the sterile moisture with uniformity has been found to be difficult.
A manufacturer has been observed employing Water For Injection as a final wash or rinse of the lyophilizer. While the chamber was wet, it was then ethylene oxide gas sterilized. As discussed above, this may be satisfactory for the chamber but inadequate for associated plumbing.
Another problem associated with ethylene oxide is the residue. One manufacturer had a common ethylene oxide/nitrogen supply line to a number of lyophilizers connected in parallel to the system. Thus, there could be some ethylene oxide in the nitrogen supply line during the backfilling step. Obviously, this type of system is objectionable.
A generally recognized acceptable method of sterilizing the lyophilizer is through the use of moist steam under pressure. Sterilization procedures should parallel that of an autoclave, and a typical system should include two independent temperature sensing systems. One would be used to control and record temperatures of the cycle as with sterilizers, and the other would be in the cold spot of the chamber. As with autoclaves, lyophilizers should have drains with atmospheric breaks to prevent back siphonage.
As discussed, there should also be provisions for sterilizing the inert gas or air and the supply lines. Some manufacturers have chosen to locate the sterilizing filters in a port of the chamber. The port is steam sterilized when the chamber is sterilized, and then the sterilizing filter, previously sterilized, is aseptically connected to the chamber. Some manufacturers have chosen to sterilize the filter and downstream piping to the chamber in place. Typical sterilization-in-place of filters may require steaming of both to obtain sufficient temperatures. In this type of system, there should be provisions for removing and/or draining condensate. The failure to sterilize nitrogen and air filters and the piping downstream leading into the chamber has been identified as a problem on a number of inspections.
Since these filters are used to sterilize inert gas and/or air, there should be some assurance of their integrity. Some inspections have disclosed a lack of integrity testing of the inert gas and/or air filter. The question is frequently asked how often should the vent filter be tested for integrity? As with many decisions made by manufacturers, there is a level of risk associated with the operation, process or system, which only the manufacturer can decide. If the sterilizing filter is found to pass the integrity test after several uses or batches, then one could claim its integrity for the previous batches. However, if it is only tested after several batches have been processed and if found to fail the integrity test, then one could question the sterility of all of the previous batches processed. In an effort to minimize this risk, some manufacturers have resorted to redundant filtration.
For most cycles, stoppering occurs within the lyophilizer. Typically, the lyophilizer has some type of rod or rods (ram) which enter the immediate chamber at the time of stoppering. Once the rod enters the chamber, there is the potential for contamination of the chamber. However, since the vials are stoppered, there is no avenue for contamination of the vials in the chamber which are now stoppered. Generally, lyophilizers should be sterilized after each cycle because of the potential for contamination of the shelf support rods. Additionally, the physical act of removing vials and cleaning the chamber can increase levels of contamination.
In some of the larger units, the shelves are collapsed after sterilization to facilitate loading. Obviously, the portions of the ram entering the chamber to collapse the shelves enters from a non-sterile area. Attempts to minimize contamination have included wiping the ram with a sanitizing agent prior to loading. Control aspects have included testing the ram for microbiological contamination, testing it for residues of hydraulic fluid, and testing the fluid for its bacteriostatic effectiveness. One lyophilizer fabricator has proposed developing a flexible "skirt" to cover the ram.
In addition to microbiological concerns with hydraulic fluid, there is also the concern with product contamination.
During steam sterilization of the chamber, there should be space between shelves that permit passage of free flowing steam. Some manufacturers have placed "spacers" between shelves to prevent their total collapse. Others have resorted to a two phase sterilization of the chamber. The initial phase provides for sterilization of the shelves when they are separated. The second phase provides for sterilization of the chamber and piston with the shelves collapsed.
Typically, biological indicators are used in lyophilizers to validate the steam sterilization cycle. One manufacturer of a Biopharmaceutical product was found to have a positive biological indicator after sterilization at 121oC for 45 minutes. During the chamber sterilization, trays used to transport vials from the filling line to the chamber were also sterilized. The trays were sterilized in an inverted position on shelves in the chamber. It is believed that the positive biological indicator is the result of poor steam penetration under these trays.
The sterilization of condensers is also a major issue that warrants discussion. Most of the newer units provide for the capability of sterilization of the condenser along with the chamber, even if the condenser is external to the chamber. This provides a greater assurance of sterility, particularly in those situations in which there is some equipment malfunction and the vacuum in the chamber is deeper than in the condenser.
Malfunctions that can occur, which would indicate that sterilization of the condenser is warranted, include vacuum pump breakdown, refrigeration system failures and the potential for contamination by the large valve between the condenser and chamber. This is particularly true for those units that have separate vacuum pumps for both the condenser and chamber. When there are problems with the systems in the lyophilizer, contamination could migrate from the condenser back to the chamber. It is recognized that the condenser is not able to be sterilized in many of the older units, and this represents a major problem, particularly in those cycles in which there is some equipment and/or operator failure.
As referenced above, leakage during a lyophilization cycle can occur, and the door seal or gasket presents an avenue of entry for contaminants. For example, in an inspection, it was noted that during steam sterilization of a lyophilizer, steam was leaking from the unit. If steam could leak from a unit during sterilization, air could possibly enter the chamber during lyophilization.
Some of the newer lyophilizers have double doors - one for loading and the other for unloading. The typical single door lyophilizer opens in the clean area only, and contamination between loads would be minimal. This clean area, previously discussed, represents a critical processing area for a product made by aseptic processing. In most units, only the piston raising/lowering shelves is the source of contamination. For a double door system unloading the lyophilizer in a non-sterile environment, other problems may occur. The non-sterile environment presents a direct avenue of contamination of the chamber when unloading, and door controls similar to double door sterilizers should be in place.
Obviously, the lyophilizer chamber is to be sterilized between batches because of the direct means of contamination. A problem which may be significant is that of leakage through the door seal. For the single door unit, leakage prior to stoppering around the door seal is not a major problem from a sterility concern, because single door units only open into sterile areas. However, leakage from a door gasket or seal from a non-sterile area would present a significant microbiological problem. In order to minimize the potential for contamination, it is recommended that the lyophilizers be unloaded in a clean room area to minimize contamination. For example, in an inspection of a new manufacturing facility, it was noted that the unloading area for double door units was a clean room, with the condenser located below the chamber on a lower level.
After steam sterilization, there is often some condensate remaining on the floor of the chamber. Some manufacturers remove this condensate through the drain line while the chamber is still pressurized after sterilization. Unfortunately, some manufacturers have allowed the chamber to come to and remain at atmospheric pressure with the drain line open. Thus, non-sterile air could contaminate the chamber through the drain line. Some manufacturers have attempted to dry the chamber by blowing sterile nitrogen gas through the chamber at a pressure above atmospheric pressure.
In an inspection of a biopharmaceutical drug product, a Pseudomonas problem probably attributed to condensate after sterilization was noted. On a routine surface sample taken from a chamber shelf after sterilization and processing, a high count of Pseudomonas sp. was obtained. After sterilization and cooling when the chamber door was opened, condensate routinely spilled onto the floor from the door. A surface sample taken from the floor below the door also revealed Pseudomonas sp. contamination. Since the company believed the condensate remained in the chamber after sterilization, they repiped the chamber drain and added a line to a water seal vacuum pump.
There are several aspects of finished product testing which are of concern to the lyophilized dosage form. These include dose uniformity testing, moisture and stability testing, and sterility testing.
(a) Dose Uniformity
The USP includes two types of dose uniformity testing: content uniformity and weight variation. It states that weight variation may be applied to solids, with or without added substances, that have been prepared from true solutions and freeze-dried in final containers. However, when other excipients or other additives are present, weight variation may be applied, provided there is correlation with the sample weight and potency results. For example, in the determination of potency, it is sometimes common to reconstitute and assay the entire contents of a vial without knowing the weight of the sample. Performing the assay in this manner will provide information on the label claim of a product, but without knowing the sample weight will provide no information about dose uniformity. One should correlate the potency result obtained form the assay with the weight of the sample tested.
(b) Stability Testing
An obvious concern with the lyophilized product is the amount of moisture present in vials. The manufacturer's data for the establishment of moisture specifications for both product release and stability should be reviewed. As with other dosage forms, the expiration date and moisture limit should be established based on worst case data. That is, a manufacturer should have data that demonstrates adequate stability at the moisture specification.
As with immediate release potency testing, stability testing should be performed on vials with a known weight of sample. For example, testing a vial (sample) which had a higher fill weight (volume) than the average fill volume of the batch would provide a higher potency results and not represent the potency of the batch. Also, the expiration date and stability should be based on those batches with the higher moisture content. Such data should also be considered in the establishment of a moisture specification.
For products showing a loss of potency due to aging, there are generally two potency specifications. There is a higher limit for the dosage form at the time of release. This limit is generally higher than the official USP or filed specification which is official throughout the entire expiration date period of the dosage form. The USP points out that compendial standards apply at any time in the life of the article.
Stability testing should also include provisions for the assay of aged samples and subsequent reconstitution of these aged samples for the maximum amount of time specified in the labeling. On some occasions, manufacturers have established expiration dates without performing label claim reconstitution potency assays at the various test intervals and particularly the expiration date test interval. Additionally, this stability testing of reconstituted solutions should include the most concentrated and the least concentrated reconstituted solutions. The most concentrated reconstituted solution will usually exhibit degradation at a faster rate than less concentrated solutions.
(c) Sterility Testing
With respect to sterility testing of lyophilized products, there is concern with the solution used to reconstitute the lyophilized product. Although products may be labeled for reconstitution with Bacteriostatic Water For Injection, Sterile Water For Injection (WFI) should be used to reconstitute products. Because of the potential toxicities associated with Bacteriostatic Water For Injection, many hospitals only utilize WFI. Bacteriostatic Water For Injection may kill some of the vegetative cells if present as contaminants, and thus mask the true level of contamination in the dosage form.
As with other sterile products, sterility test results which show contamination on the initial test should be identified and reviewed.
The USP points out that it is good pharmaceutical practice to perform 100% inspection of parenteral products. This includes sterile lyophilized powders. Critical aspects would include the presence of correct volume of cake and the cake appearance. With regard to cake appearance, one of the major concerns is meltback.
Meltback is a form of cake collapse and is caused by the change from the solid to liquid state. That is, there is incomplete sublimation (change from the solid to vapor state) in the vial. Associated with this problem is a change in the physical form of the drug substance and/or a pocket of moisture. These may result in greater instability and increased product degradation.
Another problem may be poor solubility. Increased time for reconstitution at the user stage may result in partial loss of potency if the drug is not completely dissolved, since it is common to use in-line filters during administration to the patient.
Manufacturers should be aware of the stability of lyophilized products which exhibit partial or complete meltback. Literature shows that for some products, such as the cephalosporins, that the crystalline form is more stable than the amorphous form of lyophilized product. The amorphous form may exist in the "meltback" portion of the cake where there is incomplete sublimation.
The envelope of gases surrounding the earth, exerting under gravity a pressure at the earth's surface, which includes by volume 78% nitrogen, 21% oxygen, small quantities of hydrogen, carbon dioxide, noble gases, water vapor, pollutants and dust.
The pressure exerted at the earth's surface by the atmosphere. For reference purposes a standard atmosphere is defined as 760 torr or millimeters of mercury, or 760,000 microns.
A process that occurs at low chamber pressures where hydrocarbon vapors from the vacuum system can enter the product chamber.
This is the ultimate pressure the pump or system can attain.
BLOWER (see Mechanical Booster Pump)
This pump is positioned between the mechanical pump and the chamber. It operates by means of two lobes turning at a high rate of speed. It is used to reduce the chamber pressure to less than 20 microns.
Admitting air or a selected gas to an evacuated chamber, while isolated from a vacuum pump, to raise the pressure towards, or up to, atmospheric.
A pump for conveying the heat transfer fluid.
CONDENSER (Cold trap)
In terms of the lyophilization process, this is the vessel that collects the moisture on plates and holds it in the frozen state. Protects the vacuum pump from water vapor contaminating the vacuum pump oil.
In terms of refrigeration, this unit condenses (changes) the hot refrigerant gas into a liquid and stores it under pressure to be reused by the system.
The lowering of the temperature in any part of the temperature scale.
A device to pass thermocouple wires through and maintain a vacuum tight vessel.
ln the vacuum system, the introduction of water vapor into the oil in the vacuum pump, which then causes the pump to lose its ability to attain its ultimate pressure.
The removal of ice from a condenser by melting or mechanical means.
The ratio of the energy released during the freezing of a solution to that of an equal volume of water.
The number of degrees below the equilibrium freezing temperature where ice first starts to form.
A drying agent.
Free from liquid, and/or moisture.
The removal of moisture and other liquids by evaporation.
The temperature where ice will form in the absence of supercooling.
A point of a phase diagram where all phases are present and the temperature and composition of the liquid phase cannot be altered without one of the phases disappearing.
This tank is located in the circulation system and is used as a holding and expansion tank for the transfer liquid.
There are two systems that have their systems filtered or filter/dried. They are the circulation and refrigeration systems. In the newer dryers this filter or filter/dryer is the same, and can be replaced with a new core.
The free water in a product is that water that is absorbed on the surfaces of the product and must be removed to limit further biological and chemical reactions.
This is the absence of heat. A controlled change of the product temperature as a function of time, during the freezing process, so as to ensure a completely frozen form.
Used in the vacuum system on the vacuum pump to decontaminate small amounts of moisture in the vacuum pump oil.
GAS BLEED (Vacuum control)
To control the pressure in the chamber during the cycle to help the drying process. In freeze-drying the purpose is to improve heat-transfer to the product.
This exchanger is located in the circulation and refrigeration systems and transfers the heat from the circulation system to the refrigeration system.
A liquid of suitable vapor pressure and viscosity range for transferring heat to or from a component, for example, a shelf or condenser in a freeze-dryer. The choice of such a fluid may depend on safety considerations. Diathermic fluid.
This is a refrigeration system. To control the suction pressure of the BIG FOUR (20-30 Hp) compressors during the refrigeration operation.
This is a refrigeration system. To defrost the condenser plates after the lyophilization cycle is complete.
The solid, crystalline form of water.
Any gas of a group including helium, radon and nitrogen, formerly considered chemically inactive.
In a two stage compressor system, this is the cross over piping on top of the compressor that connects the low side to the high side. One could also think of it as low side, intermediate, and high side.
This valve controls the interstage pressure from exceeding 80 - 90 PSI. This valve opens to suction as the interstage pressure rises above 80 - 90 PSI.
A heat transfer fluid (high grade kerosene).
The liquid refrigerant leaving the condenser/receiver at cooling water temperature is sub-cooled to a temperature of +15oF (-10oC) to -15oF (-25oC).
A process in which the product is first frozen and then, while still in the frozen state, the major portion of the water and solvent system is reduced by sublimation and desorption so as to limit biological and chemical reactions at the designated storage temperature,
MAIN VACUUM VALVE (see Vapor Valve)
This valve is between the chamber and external condenser to isolate the two vessels after the process is finished. This is the valve that protects the finished product.
A matrix, in terms of the lyophilization process, is a system of ice crystals and solids that is distributed throughout the product.
A roots pump with a high displacement for its size but a low compression ratio. When backed by an oil-seal rotary pump the combination is an economical alternative to a two-stage oil-sealed rotary pump, with the advantage of obtaining a high vacuum.
The mechanical pumping system that lowers the pressure in the chamber to below atmospheric pressure so that sublimation can occur.
That temperature where mobile water first becomes evident in a frozen system.
MICRON (see Torr)
A unit of pressure used in the lyophilization process. One micron = one Mtorr or 25,400 microns = 1" Hg., or 760,000 microns = one atmosphere.
A mixture of gases such as nitrogen, hydrogen, chlorine, and hydrocarbons. They may be drawn into the system through leaks when part of the system is under a vacuum. Their presence reduces the operating efficiency of the system by increasing the condensing pressure.
The formation of ice crystals on foreign surfaces or as a result of the growth of water clusters.
In vacuum terminology a filter attached to the discharge (exhaust) of an oil-sealed rotary pump to eliminate most of the "smoke" of suspended fine droplets of oil which would be discharged into the environment.
A standard type of mechanical vacuum pump used in freeze-drying with a high compression ratio but having a relatively low displacement (speed) for its size. A two-stage pump is effectively two such pumps in series and can obtain an ultimate vacuum.
Separates the oil from the compressor discharge gas and returns the oil through the oil float trap and piping to the compressor crankcase.
A real leak is a source of atmospheric gases resulting from a penetration through the chamber.
The dissolving of the dried product into a solvent or diluent.
Used for safety purposes to prevent damage in case excessive pressure is encountered.
A mechanical pumping system with sliding vanes as the mechanical seal. Can be single or two stages.
SHELF COMPRESSOR (Controlling Compressor)
Used for controlling the shelf temperature, either cooling or from overheating.
The transfer of heat from the shelf fluid to the refrigeration system through tubes in the exchanger causing compressor suction gas to warm.
In terms of the lyophilization process, they are a form of heat exchanger, within the chamber, that have a serpentine liquid flow through them, entering one side and flowing to the other side. They are located in the circulation system.
This is a normal type compressor used in refrigeration. In the lyophilization process it is used to control the shelf temperature, both for cooling and keeping the shelf temperature from overheating using a temperature controller.
A heat transfer fluid.
The use of steam and pressure to kill any bacteria that may be able to contaminate that environment or vessel.
The conversion of a material from a solid phase directly to a vapor phase, without passing through the liquid phase. This is referred to as the primary drying stage.
SUB-COOLED LIQUID (See Liquid Sub-cooler Heat Exchanger)
The liquid refrigerant is cooled through an exchanger so that it increases the refrigerating effect as well as reduces the volume of gas flashed from the liquid refrigerant in passage through the expansion valve.
To provide adequate refrigerant liquid slug protection (droplets of liquid refrigerant) from returning to the compressor, and causing damage to the compressor.
Trichloroethylene - A heat transfer fluid.
The degree of hotness or coldness of a body.
A metal-to-metal contact between two dissimilar metals that produces a small voltage across the free ends of the wire.
An automatic variable device controlling the flow of liquid refrigerant.
TORR (See Micron)
A unit of measure equivalent to the amount of pressure in 1000 microns.
This is a specially built compressor. Its function is to be able to attain low temperatures by being able to operate at low pressures. It is two compressors built into one. A low stage connected internally and a high stage connected externally with piping, called interstage.
This valve connects the interstage with suction to equalize both pressures during pump-down.
Strictly speaking, a space in which the total pressure is less than atmospheric.
To assist in the rate of sublimation, by controlling the pressure in the lyophilizer.
A mechanical way of reducing the pressure in a vessel below atmospheric pressure to where sublimation can occur. There are three types of pumps, rotary vane, rotary piston and mechanical booster.
A target shaped object placed in the condenser to direct vapor flow and to promote an even distribution of condensate.
The vacuum valves used are of a ball or disk type that can seal without leaking. The balI types are used for services to the chamber and condenser. They are also used for drains and isolation applications. The disk types are used in the vacuum line system and are connected to the vacuum pump, chamber and condenser.
VAPOR VALVE (See Main Vacuum Valve)
The vacuum valve between the chamber and external condenser. When this valve is closed the chamber is isolated from the external condenser. Also known as the main vapor valve.
A small glass bottle with a flat bottom, short neck and flat flange designed for stoppering.
In the vacuum system a virtual leak is the passage of gas into the chamber from a source that is located internally in the chamber.
Once a sterilization method has been validated for a particular product, and the product is being manufactured, routine sterility assurance tests must be performed. These included bioburden tests, quarterly dose audits, cleaning and disinfection, and environmental monitoring, among others. DETAILED MEDICAL DEVICE STERILITY TESTING INFORMATION: Sample Item Portion (SIP) Preparation Bioburden Method Validation Bioburden Enumeration Bacteriostasis/Fungistasis Test AAMI/ISO Dose Audit Sterility Testing Microbial Environmental Monitoring Package Intergrity Testing Bacterial Endotoxins (LAL) Test Sample Item Portion (SIP) Preparation A sample item portion is a specially prepared portion of a medical device that is used in AAMI/ISO dose setting procedures. Some large or complex devices cannot practically be tested in their entirety. Using nonsterile samples, a defined portion of the device is aseptically removed and packaged. This portion (SIP) is used for the bioburden and verification dose studies. For some complex devices, it may be necessary to disassemble or cut up the device so that it can fit into rinse fluid and media containers used for bioburden and verification dose testing. This disassembly or cutting must be done aseptically so that the natural bioburden of the product is not affected. It also must not reduce the challenge to the sterilizing process. The adequacy of the SIP must be demonstrated by means of a sterility test of 20 non-sterile SIP samples. The SIP is considered adequate if at least 17 of the samples test positive in the sterility test. Please submit a product sample to Pacific BioLabs for evaluation so that an appropriate SIP preparation procedure can be determined. Bioburden Method Validation ANSI/AAMI/ISO Guideline 11737-1, Sterilization of Medical Devices – Microbiological Methods, Part 1: Determination of a Population of Microorganisms on Products, requires that the bioburden test method be validated for each medical device. The purpose of this validation is to insure that the bioburden test method which will be used to determine the product bioburden level, is effective in a) recovering microorganisms that are present on the product and b) does not inhibit growth of the recovered microorganisms. Insufficient recovery or inhibition would result in underestimation of a product's true bioburden and could lead to an inadequate sterilization cycle or dose. The recovery data from validation testing will indicate if a recovery factor should be applied to results obtained by routine bioburden testing. The bioburden method validation must be performed prior to proceeding with actual bioburden testing of the product. If any changes affecting materials, assembly or configuration are made to the product, the bioburden method should be revalidated. Repetitive (Exhaustive) Recovery This method uses the naturally occurring bioburden of the product. A bioburden test is performed on the same device three or more times. The counts obtained from the replicate extractions are used to calculate a percent recovery. Pacific BioLabs recommends this method for devices which have a moderate to high bioburden level and do not contain absorbent materials. Product Inoculation (Simulated) Recovery This method simulates a product bioburden by inoculating a known amount of spores onto a sterile device. The device is then tested for bioburden with the same method proposed for use in routine analysis. The recovered level is compared to the known inoculation level and the percent recovery is calculated. Pacific BioLabs recommends this method for devices with low bioburden levels or complex configurations. It also may be used for devices which absorb the rinsing fluid used in bioburden tests. The method does have limitations, because the spore inoculation may not reproduce the adherence properties of the natural bioburden. Please note that sterile samples are usually required for simulated recovery tests. Screening for Release of Substances Adversely Affecting Bioburden Estimates Some medical devices are manufactured with materials that adversely affect the bioburden testing of that device. These materials, or substances that are extracted from these materials during bioburden testing, have bacteriostatic, bacteriocidal, fungistatic and/or fungicidal properties which damage, inhibit the growth of, or kill microorganisms removed from the product during the extraction phase of the bioburden test. ANSI/AAMI/ISO Guideline 11737-1 includes a method that screens for the presence of these adverse substances. This method requires the inoculation of a known population of test organisms into a test container with the product and the bioburden extraction fluid. Following a holding period equivalent to that which occurs during routine bioburden testing, the test organisms are enumerated and compared to the initial population. If the counts are not similar, modification of the bioburden procedure is necessary. This screening test should be performed when: The product contains materials which could have biocidal or biostatic effects. The repetitive or product inoculation results indicate low recovery. The product is liquid, gel or powder. Bioburden Enumeration Bioburden is the population of microorganisms on a raw material, product component or finished medical device just prior to sterilization. For finished medical devices, the bioburden test data is used to establish parameters for an effective sterilization process. To insure the ongoing safety of the sterilization process, it is necessary to verify that the bioburden level remains consistent over time. There are two important aspects of product bioburden control – maintaining consistency from lot to lot and avoiding spikes within a single lot. A bioburden spike occurs when the bioburden for an individual product is 2 or more times greater than the group average. A significant increase in the device bioburden would reduce the sterility assurance level of the sterilization process. For new medical devices, ten randomly selected samples from three separate newly manufactured lots should be tested for bioburden. It is useful to track samples by date and time of assembly or packaging to determine when and where any inconsistencies may be occurring. After initial data is generated, bioburden tests should be conducted monthly to quarterly, depending on frequency and volume of production, as part of an ongoing environmental monitoring program. It is also important to check bioburden levels whenever any changes are made in packaging locations, manufacturing processes, raw material vendors, or personnel involved with production. If bioburden data increases significantly or shows extreme variability, the manufacturing process should be investigated so that corrective measures can be implemented. Bioburden studies are also used to monitor microorganism levels on materials that could affect the bioburden of the finished device, such as product components, manufacturing fluids and product packaging. The bioburden test data may provide useful information in the investigation of bioburden spikes and dose audit failures. Bioburden testing can also be used as a material qualifications tool. For additional information refer to Sterilization of medical devices—Microbiological methods, Part 3: Guidance on evaluation and interpretation of bioburden data (ANSI/AAMI/ISO 11737-3:2004). To perform a bioburden test, a sample is aseptically transferred to an appropriate volume of extraction fluid and then mechanically agitated to remove microorganisms. Membrane filtration is the preferred method for the culturing and microbial enumeration of the extraction fluid. Pacific BioLabs uses this method for products with filterable extraction fluid. When the extraction fluid cannot be filtered, the plate count method is used. Bioburden results are reported on individual samples showing total aerobic count with a breakdown of bacteria and fungi. Anaerobic bioburden and heatshocking methods for enumeration of spores are also available upon request. Bioburden Test Method Validation Recovery Study – Repetitive Treatment (3 to 5 extractions/sample) Recovery Study – Simulated (spore inoculation of products) Screening for the Release of Adverse Substance Affecting Bioburden Estimates Bioburden – Total Aerobic Bacteria and Fungi by Filtration Method Small Devices (low level bioburden) Medium Devices (moderate level bioburden) Large Devices, Small Kits, Papers, Fabrics (higher level bioburden) Bioburden – Total Anaerobic Bacteria Bioburden – Total Aerobic Spores Bioburden – Process Fluids (Aerobic Bacteria by Filtration Method) Bacteriostasis/Fungistasis Test The bacteriostasis/fungistasis test is designed to validate the procedure used to test a product for sterility by demonstrating that microorganisms present on the product will be detected in the course of the sterility test. The USP (and FDA) requires this test because some products contain substances that inhibit the growth of microorganisms. Although a product may harbor microorganisms and be nonsterile, the presence of growth inhibition substances can cause a falsely negative sterility test. The test is conducted by performing a simulated sterility test, then adding low levels of selected bacteria and fungi as challenge microorganisms to the culture media. The organisms will remain viable, grow and be detectable in the culture media if the product does not exert a bacteriostatic or fungistatic effect. If the product is found to be bacteriostatic or fungistatic, the sterility test procedure must be modified and another bacteriostasis/fungistasis test must be performed. This testing should be performed on all new products and when any significant changes are made in the manufacturing or materials of an existing product. Pacific BioLabs strongly recommends repeating the B/F test biannually to account for any possible changes to the product or manufacturing process. For medical devices, three to six sterile samples are required for the B/F test. Bacteriostasis/Fungistasis Test – Direct Transfer Method 3 organisms in SCDM (Radiation Dose Audits) 6 organisms – 3 in FTM and 3 in SCDM (USP; EtO sterilized products) AAMI/ISO Dose Audit A dose audit is a sterility test of samples which have been irradiated at a defined kGy level determined as part of an AAMI/ISO dose validation study. The AAMI/ISO standard requires that dose audits be performed quarterly (or with each lot if production is less frequent than quarterly) for all devices that been validated according to ANSI/AAMI/ISO 11137 Method 1 or AAMI TIR 33 VDmax. (The dose audit interval may be extended to semiannually or annually if it is demonstrated over time that the product bioburden is stable with respect to levels, microorganism types, and microorganism resistance.) All samples are tested using Soybean Casein Digest Medium and incubated for 14 days at 30º±2ºC. The information below may be used to determine a likely dose audit fee. Compare the sample measurements with the container dimensions. The cost of a dose audit is usually based on the amount of medium required to test the sample. Samples which are difficult to aseptically handle will incur an additional charge. Please contact Pacific BioLabs to request a quote. Samples which can be easily cut with scissors can be aseptically divided and possibly tested in smaller containers. To minimize the chance of a sample being compromised in sterility testing, it is highly desirable to minimize sample manipulation. We prefer to use the smallest container and volume which will allow the test sample to be submerged in media (although it is not always possible to completely submerge all samples). An appropriate media volume must be validated for each product by bacteriostasis-fungistasis testing. If the product cannot be cut to fit into any of the following containers, SIP preparation will be required. Media containers used for Dose Audits Media Container Dimensions Orifice I.D. Media Volume Culture Tube 25 mm D x 150 mm H 25 mm 40 mL Culture Tube 25 mm D x 250 mm H 25 mm 80 mL Culture Tube 38 mm D x 200 mm H 38 mm 100 – 120 mL 0.9 L Glass Jar 90 mm D x 165 mm H 75 mm 500 mL 1.8 L Glass Jar 135 mm D x 150 mm H 95 mm 1000 mL Nalgene Jug 150 mm Square x 250mm H 82 mm 1000 mL Nalgene Jug 150 mm Square x 250mm H 82 mm 2000 mL D = Diameter, H = Height In a Nalgene jug, 1000 mL of media is about 65 mm deep; 2000 mL is about 130 mm deep. Some large or long samples and SIP preparations will only fit in the Nalgene jugs. The higher fees reflect increased costs for media, significantly greater time for media production and media quality control, extended time for sterility testing and significantly greater incubator space requirements. Notes: To expedite processing of your dose audit samples, please call us prior to sending samples so that we can schedule your tests. Some samples are difficult to aseptically cut and transfer to media containers. If possible, please include 3 to 5 additional samples with your dose audit sample submission. Sterility Testing Sterility testing of products and/or biological indicators (i.e. spore strips) exposed to a sterilization process is an important part of all sterility assurance programs. Most manufacturers of EO sterilized medical devices monitor their validated EO sterilization loads with B. atrophaeus spore strips and release their products for distribution based on negative sterility test results of the spore strips. The spore strips may be placed inside or outside the product depending on the type of spore strip testing performed during the validation. The spore strips should be distributed in locations throughout the sterilization chamber. A positive control (i.e. unprocessed spore strip) should be included with all spore strip sterility tests. These routine sterility tests must be supplemented periodically with more extensive cycle validations. Routine lot release of terminally sterilized medical devices by means of end product sterility testing is not recommended for several reasons. The bioburden of most medical devices generally is a lesser challenge to the sterilization process than biological indicators, and overkill cannot be demonstrated. Statistically, the probability that a sterility test of 20 or 40 product samples will detect nonsterile samples among a much larger number of products is very limited. Also, it is generally recognized that the process of sterility testing has a significantly lower sterility assurance level than most validated terminal sterilization processes. However, end product sterility testing of medical devices is occasionally performed as part of investigations or to support other information in making a lot release decision. Spore Strips Only This test is appropriate for devices sterilized by steam or ETO in a validated cycle. Generally 10 to 20 spore strips are used to monitor a cycle. Spore strips are cultured in SCDM and then usually incubated for 7 days. Shorter incubation times can be validated. Product with Spore Strips This test is generally used for fractional and half cycles in ethylene oxide and steam sterilization validations. Spore strips are normally cultured in SCDM and incubated for at least 7 days. Products are usually tested in SCDM and FTM and incubated for 14 days. Product Only – Direct Transfer or fluid path Entire devices or portions of devices are rinsed with or submerged in Soybean Casein Digest Medium and Fluid Thioglycolate Medium. Usually 40 product samples are required, unless a product is large such that it can be divided to provide for each medium type, in which case 20 samples are required. Incubation time is 14 days. Inoculated Product For this test, product samples which have been inoculated with a microorganism that is resistant to the sterilization process are used as biological indicators. This test is used most frequently to verify steam or EtO penetration into an area of a medical device that is too small to be monitored with a spore strip. It is often used as part of the sterilization validation of a reusable medical device. Ten or more samples are usually required. Incubation time is 7 days. Microbial Environmental Monitoring Medical device manufacturers use microbial environmental monitoring programs to evaluate the effectiveness of cleaning and disinfection procedures and to assess the overall microbial cleanliness of their manufacturing environment. An effective program to control microorganism levels in the manufacturing environment is essential to minimize the bioburden on the medical device being manufactured and reduce potential for bioburden spikes. Spikes in the bioburden of finished medical devices can cause a reduction in the sterility assurance level for the product. Air and surface samples are taken during routine production operations to obtain a microbiological profile of the manufacturing environment. Observation of work practices are made during the survey. Test data and other information are evaluated to determine what actions can be taken to reduce or stabilize the bioburden of the medical device. Once an intensive survey has been conducted and strategic sampling locations are determined, samples can be taken by the manufacturer's personnel on a regular schedule. Pacific BioLabs can provide the necessary supplies for microbiological sampling. Exposed materials are returned to Pacific BioLabs for enumeration and reporting. If any major changes are made at the facility or in the manufacturing process, or if product bioburden levels increase significantly, a re-evaluation of environmental conditions should be conducted. For additional information about environmental monitoring, refer to USP general chapter <1116> Microbiological Evaluation of Clean Rooms and other Controlled Environments or PDA TR 13 (revised 2001) Fundamentals of a Microbiological Environmental Monitoring Program. Pacific BioLabs works with many of its clients to establish and maintain cost-effective programs to meet FDA requirements for monitoring the microbial cleanliness of their manufacturing environments and assessing the efficacy of production area disinfection procedures. Our microbiologists can train quality assurance and manufacturing personnel to collect microbial environmental samples. Following is an overview of aspects of a microbial environmental monitoring program. Please call the Microbiology/Sterility Assurance departments at any of our facilities to order supplies or for more information. Microbial Environmental Monitoring Plan for Production Areas Review manufacturing procedures movement of materials personnel practices cleaning and disinfection procedures Visit production and packaging areas. Observe manufacturing process. Evaluate possible sources of microorganisms and potential to impact product bioburden. water and ancillary fluid used in production HVAC systems manufacturing equipment manufacturing personnel Formulate sampling plan. prepare a production area schematic identify sites for air and surface samples determine if water or other process fluid sampling is required Collect environmental samples. identify each with a site code and the date and time collected Ship samples for next day delivery to Pacific BioLabs. Pacific BioLabs incubates samples at temperatures appropriate for the various environmental samples. Following incubation, bacteria and mold colonies are enumerated. microorganisms will be characterized or identified if desired Results are reported to client. Results are evaluated by a Pacific BioLabs microbiology manager and the client. Appropriate follow-up action is recommended. Materials used for Microbial Environmental Monitoring BiotestTM Air Sampler: A mechanical instrument which pulls in a preset volume of air, impacting microorganisms onto a strip filled with a nutrient agar. Contact Plate: A petri dish with an elevated convex surface of nutrient agar. It is used for taking samples of flat surfaces. The lid of the dish is removed. The agar surface is pressed lightly against the surface to be sampled. The lid is then immediately replaced on the dish. Fallout Plate: A petri dish containing a nutrient agar. It is used for semi-quantitative air sampling. The dish is placed in the desired location, the lid is removed and the agar is exposed for a defined amount of time, usually for 30 minutes to 2 hours. Organisms falling from the air settle on the surface of the agar. DE Neutralizing Agar: A neutralizing agar formulated with Tween 80 (a surfactant), lecithin (a general purpose neutralizer), sodium thiosulfate and sodium thioglycollate. This combination is used to neutralize chlorine, glutaraldehyde, iodophor, phenolic and quat based disinfectants.This agar is used to culture many types of bacteria and some fungi. Letheen Agar: A neutralizing agar formulated with Tween 80 and lecithin. This combination is used to neutralize alcohol, phenolic and quaternary ammonium chloride based disinfectants. Rose Bengal Agar: A neutral pH agar which contains stain that inhibits bacterial growth. It is used to select for yeast and molds. Sabouraud Dextrose Agar: A high sugar, low pH agar used to select for yeast and molds. Sodium Thiosulfate: A chemical added to agar to neutralize halogen based disinfectants, such as bleach. Sterile Buffer Solution with Swab: The buffer solution is supplied in a 10 mL screw cap test tube. The sterile swab is provided in a paper pouch. Swabs are used for taking samples of non-flat surfaces. The swab is moistened by dipping it in the sterile buffer solution. The surface to be sampled is swabbed. The tip of the swab is then cut or broken into the test tube of solution and the cap is replaced on the test tube. Tryptic Soy Agar: A general purpose nutrient agar used to culture many bacteria and some fungi. Microbial Environmental Services and Supplies Microbiological Environmental Survey Microbial Samples Enumeration and Report: Microbial Samples Enumeration and Report: Fallout Plate Count Contact Plate Count Biotest Air Sampler Strip Count Buffer Solution - Aerobic Count (Membrane Filtration) Microbial Environmental Monitoring Supplies Fallout Plate, 100 mm, Tryptic Soy Agar (TSA) Fallout Plate, 100 mm, Sabouraud Dextrose Agar (SDA) Contact Plate, DE Neutralizing Agar Contact Plate, Tryptic Soy Agar (TSA) Contact Plate, Sabouraud Dextrose Agar (SDA) Biotest Air Sampler Strip, Tryptic Soy Agar (TSA) Biotest Air Sampler Strip, Sabouraud Dextrose Agar (SDA) or Rose Bengal Agar Biotest Centrifugal Air Sampler – Daily Rental Sterile Buffer Solution with Dacron Swab Sterile Specimen Cups, Screw Cap, 120 m Package Intergrity Testing Package integrity tests are used to detect packaging problems that could adversely affect the sterility of a medical device. Sterile products are subjected to an environmental stress intended to simulate extreme conditions that a product might encounter in shipping or storage. The product packaging is then subjected to microbial challenge or dye penetration testing to determine if it has retained its properties as a microbial barrier. Thirteen samples are recommended for this test. Package Integrity by Microbial Challenge (13 products/test station are recommended) Package Integrity by Dye Penetration (13 products/test station are recommended) Bacterial Endotoxins (LAL) Test A pyrogen is the product of the action of heat on an organic substance and, in medical terms, is frequently described as a fever producing substance. The most potent pyrogens originate from gram negative bacteria, which are common water-borne organisms. Although not entirely accurate, the terms pyrogen and endotoxin are often used interchangeably. Detection of bacterial endotoxin contamination is essential to insure the safety of certain medical devices. The Bacterial Endotoxins Test using Limulus Amebocyte Lysate (LAL) is recommended for the detection of endotoxins in medical devices. Any product that is labeled as nonpyrogenic must be tested to verify that claim. Medical devices with bloodstream or cerebrospinal fluid contact must also be tested for the presence of bacterial endotoxins. Whether or not a device is considered pyrogenic is based on the amount of endotoxin the device contains in correlation to the accepted human tolerance of 5 endotoxin units (EU) per kilogram of body weight. Nonpyrogenic water is used to extract medical devices. Historically, the USP rabbit pyrogen test was used to test the extract. It specified a 40 mL extract per device with a 10 mL/kg injection volume. This is equivalent to 40 mL tested against 0.5 EU/mL LAL reagent sensitivity, and an allowable limit of 20 EU per device. The LAL test is usually performed on a composite of the extracts of 10 samples. It is possible that one device of the composite could contain > 20 EU, when others in the composite would contain < 20 EU, and the composite test would pass. The most endotoxin one device could contain, if all others in a 10 sample composite contained zero endotoxin, would be 200 EU. This is still below the human tolerance of 350 EU, based on an average human body weight of 70 kg (70 kg x 5 EU/kg = 350 EU). In a composite test, 20 EU is the average endotoxin limit for most medical devices. However, the limit for the devices which contact cerebrospinal fluid is 2.15 EU per device. The maximum allowable extraction volume is calculated to insure that the average endotoxin burden per device in the LAL test does not exceed these limits. FDA has published guidelines outlining validation procedures for endotoxin testing of finished products using the LAL test. This document is called Guideline on the Validation of the Limulus Amebocyte Lysate Test for Human and Animal Parenteral Drugs, Biological Products and Medical Devices, December 1987. A more current AAMI reference document is ANSI/AAMI ST72 Bacterial endotoxins – Test methodologies, routine monitoring, and alternatives to batch testing, 2002. For medical devices, the following sampling strategy is recommended for the LAL test: 2 samples for lot sizes less than 30, 3 samples for lot sizes 30-100, and 3% of the lot not to exceed 10 samples for lot sizes equal to or greater than 101. One test is performed on a composite of the test samples. Because water is a source of pyrogens, it is important to routinely monitor water systems using the bacterial endotoxins test. For process water samples, the amount of sample required is 10 mL.