Sterile Product Building, Facility and Clean Area Classification Guideline By USFDA has clearly mentioned what conditions to be followed to maintain the Sterile Conditions in Sterile Product Manufacturing site.
21 CFR 211.42(b) states, in part, that “The flow of components, drug product containers, closures, labeling, in-process materials, and drug products through the building or buildings shall be designed to prevent contamination.”
21 CFR 211.42(c) states, in part, that “Operations shall be performed within specifically defined areas of adequate size. There shall be separate or defined areas or such other control systems for the firm’s operations as are necessary to prevent contamination or mixups during the course of the following procedures: * * * (10) Aseptic processing, which includes as appropriate: (i) Floors, walls, and ceilings of smooth, hard surfaces that are easily cleanable; (ii) Temperature and humidity controls; (iii) An air supply filtered through high-efficiency particulate air filters under positive pressure, regardless of whether flow is laminar or nonlaminar; (iv) A system for monitoring environmental conditions; (v) A system for cleaning and disinfecting the room and equipment to produce aseptic conditions; (vi) A system for maintaining any equipment used to control the aseptic conditions.”
21 CFR 211.46(b) states that “Equipment for adequate control over air pressure, micro-organisms, dust, humidity, and temperature shall be provided when appropriate for the manufacture, processing, packing, or holding of a drug product.” 21 CFR 211.46(c) states, in part, that “Air filtration systems, including prefilters and particulate matter air filters, shall be used when appropriate on air supplies to production areas * * *.”
21 CFR 211.63 states that “Equipment used in the manufacture, processing, packing, or holding of a drug product shall be of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and maintenance.” 21 CFR 211.65(a) states that “Equipment shall be constructed so that surfaces that contact components, inprocess materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.” 21 CFR 211.67(a) states that “Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.” 21 CFR 211.113(b) states that “Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.”
As provided for in the regulations, separate or defined areas of operation in an aseptic processing facility should be appropriately controlled to attain different degrees of air quality depending on the nature of the operation. Design of a given area involves satisfying microbiological and particle criteria as defined by the equipment, components, and products exposed, as well as the operational activities conducted in the area.
Clean area control parameters should be supported by microbiological and particle data obtained during qualification studies. Initial cleanroom qualification includes, in part, an assessment of air quality under as-built, static conditions. It is important for area qualification and classification to place most emphasis on data generated under dynamic conditions (i.e., with personnel present, equipment in place, and operations ongoing). An adequate aseptic processing facility monitoring program also will assess conformance with specified clean area classifications under dynamic conditions on a routine basis.
The following table summarizes clean area air classifications and recommended action levels of microbiological quality (Ref. 1).
TABLE 1- Air Classificationsa
Clean Area Classification (0.5 um particles/ft3)
> 0.5 µm particles/m3
Microbiological Active Air Action Levelsc (cfu/m3 )
a- All classifications based on data measured in the vicinity of exposed materials/articles during periods of activity. b- ISO 14644-1 designations provide uniform particle concentration values for cleanrooms in multiple industries. An ISO 5 particle concentration is equal to Class 100 and approximately equals EU Grade A. c- Values represent recommended levels of environmental quality. You may find it appropriate to establish alternate microbiological action levels due to the nature of the operation or method of analysis.
The additional use of settling plates is optional.
Samples from Class 100 (ISO 5) environments should normally yield no microbiological contaminants.
Two clean areas are of particular importance to sterile drug product quality: the critical area and the supporting clean areas associated with it.
A. Critical Area – Class 100 (ISO 5)
A critical area is one in which the sterilized drug product, containers, and closures are exposed to environmental conditions that must be designed to maintain product sterility (§ 211.42(c)(10)). Activities conducted in such areas include manipulations (e.g., aseptic connections, sterile ingredient additions) of sterile materials prior to and during filling and closing operations.
This area is critical because an exposed product is vulnerable to contamination and will not be subsequently sterilized in its immediate container. To maintain product sterility, it is essential that the environment in which aseptic operations (e.g., equipment setup, filling) are conducted be controlled and maintained at an appropriate quality. One aspect of environmental quality is the particle content of the air. Particles are significant because they can enter a product as an extraneous contaminant, and can also contaminate it biologically by acting as a vehicle for microorganisms (Ref. 2). Appropriately designed air handling systems minimize particle content of a critical area.
Air in the immediate proximity of exposed sterilized containers/closures and filling/closing operations would be of appropriate particle quality when it has a per-cubic-meter particle count of no more than 3520 in a size range of 0.5 µm and larger when counted at representative locations normally not more than 1 foot away from the work site, within the airflow, and during filling/closing operations. This level of air cleanliness is also known as Class 100 (ISO 5).
We recommend that measurements to confirm air cleanliness in critical areas be taken at sites where there is most potential risk to the exposed sterilized product, containers, and closures. The particle counting probe should be placed in an orientation demonstrated to obtain a meaningful sample. Regular monitoring should be performed during each production shift. We recommend conducting nonviable particle monitoring with a remote counting system. These systems are capable of collecting more comprehensive data and are generally less invasive than portable particle counters. See Section X.E. for additional guidance on particle monitoring.
Some operations can generate high levels of product (e.g., powder) particles that, by their nature, do not pose a risk of product contamination. It may not, in these cases, be feasible to measure air quality within the one-foot distance and still differentiate background levels of particles from air contaminants. In these instances, air can be sampled in a manner that, to the extent possible, characterizes the true level of extrinsic particle contamination to which the product is exposed. Initial qualification of the area under dynamic conditions without the actual filling function provides some baseline information on the non-product particle generation of the operation.
HEPA-filtered air should be supplied in critical areas at a velocity sufficient to sweep particles away from the filling/closing area and maintain unidirectional airflow during operations. The velocity parameters established for each processing line should be justified and appropriate to maintain unidirectional airflow and air quality under dynamic conditions within the critical area (Ref. 3).
Proper design and control prevents turbulence and stagnant air in the critical area. Once relevant parameters are established, it is crucial that airflow patterns be evaluated for turbulence or eddy currents that can act as a channel or reservoir for air contaminants (e.g., from an adjoining lower classified area). In situair pattern analysis should be conducted at the critical area to demonstrate unidirectional airflow and sweeping action over and away from the product under dynamic conditions. The studies should be well documented with written conclusions, and include evaluation of the impact of aseptic manipulations (e.g., interventions) and equipment design. Videotape or other recording mechanisms have been found to be useful aides in assessing airflow initially as well as facilitating evaluation of subsequent equipment configuration changes. It is important to note that even successfully qualified systems can be compromised by poor operational, maintenance, or personnel practices.
Air monitoring samples of critical areas should normally yield no microbiological contaminants. We recommend affording appropriate investigative attention to contamination occurrences in this environment.
 A velocity of 0.45 meters/second (90 feet per minute) has generally been established, with a range of plus or minus 20 percent around the setpoint. Higher velocities may be appropriate in operations generating high levels of particulates.
Sterile Drug Products Produced by Aseptic Processing —Current Good Manufacturing Practice (USFDA)
Sterility Testing is defined as a testing which confirms that products are free from the presence of viable microorganisms. Sterility testing is very important for medical devices, pharmaceuticals, preparations, tissue materials and other materials that claim to be sterile or free from viable microorganisms.
A. Method Suitability Test
For all product types, follow current USP methodology in <71>, with the following additional instructions.
In all cases, even if the product does not include a preservative, the product itself may have growth inhibiting properties. All products should undergo a prescribed Method Suitability test.
Units selected for suitability testing should be subjected to the same disinfection procedure utilized in the sample analysis.
When developing the testing protocol for method suitability the volume of product as well as the concentration of the product should be evaluated such that the highest volume of product and the highest concentration of product should be used for the method suitability testing.
If multiple samples of the same product from the same manufacturer (same dosage and form) are collected, one sample may be used for method suitability for all the samples collected.
When to run Method Suitability:
Run the method suitability test prior to conducting the sterility test in accordance with USP requirements under the following conditions:
If insufficient information about the product exists to judge its probable growth inhibiting activity.
In all cases, when there is sufficient analytical time available,
i.e., survey type samples.
Run the method suitability test concurrently with product sterility tests when time is critical, and problems associated with 1. above have been resolved. However, it should be noted that if the Method Suitability Test is run concurrently with the product sterility test and the Method Suitability Test should fail, the results of the product test are invalid and the Method Suitability Test as well as the product test will need to be repeated with proper method modification to neutralize the inhibiting property.
If an insufficient amount of product is collected and the analysis is critical, the suitability test can be conducted at the end of the 14-day incubation period. Be sure to use best judgment and maximum neutralization approach when initially conducting the product sterility test. If the suitability results indicate inhibition then the results, if negative, are invalid. However, if the product test results indicate microbial presence and the suitability test shows inhibition, the results are still valid.
Method Suitability Test Procedures
Method Suitability and positive culture control tests which require the use of viable microorganisms, should be performed outside the clean room or isolator, in a biosafety cabinet or equivalent.
Pass product fluid through filter membrane. Rinse the membrane with three 100 ml portions (or more if applicable) of specified rinse fluid. Do not exceed a washing cycle of five times 100mL per filter. This step hopefully will neutralize and remove any antimicrobial residue on the filter membrane.
Add indicated test organisms in specified numbers (less than 100 CFU) into the last 100 ml rinse fluid used. iii. Filter the rinse fluid and aseptically cut the filter membrane into two equal parts, transfer one half into each of two suitable media. If conducting the sterility test using a closed canister system, rinse each canister with the inoculated rinse fluid.
If the available number of test vessels is insufficient for a complete challenge test for each individual microorganism, then the test organisms may be composited as necessary. However, confirmation of growth for the composited microorganisms will need to be performed.
Confirm composited microorganisms by Gram stain, microscopic examination, and identification after the completion of incubation.
See step c. below for additional considerations.
For direct inoculation, add the test microorganisms to separate test vessels of product and culture media if sufficient product is available. See step c. below for additional considerations.
The following test procedures apply to Direct Inoculation and Membrane Filtration:
Inoculate the same microorganism using the same medium without the product as a positive control.
For bacteria and fungi, incubate test vessels according to USP requirements. Ensure that seed-lot cultures used are not more than five passages removed from the original master seed-lot. For in-house prepared test strain suspensions of vegetative bacteria and yeast should be used within 2 hours,
or within 24 hours if refrigerated between 2ºC and 8ºC. Spore suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) can be prepared and maintained between 2ºC and 8ºC for up to seven days. Additionally, all bacterial and spore suspensions should be prepared to yield ≤100 CFU.
If growth comparable to that of the positive control vessel without product is obtained, then you may proceed with the sterility test. If comparable visible growth is not obtained, the antimicrobial activity of the product has not been eliminated under the conditions of the test. Modify the test conditions and repeat the Method Suitability test.
If product is found to exhibit growth inhibiting activity when determined concurrently with product testing, the sterility test must be repeated using a neutralizing agent (or increase media volume) to modify the conditions in order to eliminate the antimicrobial activity.
Cultures used for the method suitability test can be purchased commercially, ready to use, or can be prepared and maintained locally. Either procedure requires quantitative verification of actual CFU’s inoculated at time of use.
Open the outer sample packaging on a laboratory bench disinfected with a sporicidal antimicrobial agent. Refer to appropriate literature for choosing suitable antimicrobial agents for use in your facility.
Count the number of units received. Compare this number with the number of units collected.
Inside the clean room preparation area located outside the ISO 5 area (if available) remove all outer packaging from subsample units that will be tested without compromising the sterile integrity of the product. Remove sample units and place them on a tray or cart disinfected with an effective antimicrobial agent. Note: One or more units can be sacrificed to aid in the determination for how to aseptically remove test material if the number of the units received is sufficient.
Examine all units visually for container closure integrity, for the presence of any foreign matter in the product and other container closure defects. Note findings on analyst’s worksheet.
If foreign matter is observed within the primary container, discuss with supervisor the employment of ORS procedure Document ORA-LAB.015 entitled “Screening Protocol for Direct Staining on Products with Appearance of Visible Contamination” (see QMiS for Procedure).
If sample units are not identified by the collector, the analyst should identify unit with sample #, initials, date, and sub sample # as appropriate for sample traceability. Otherwise, date and initial each unit.
Unit Container Disinfection
a. Cleanse the exterior of all product primary containers using antimicrobial/sporicidal agents.
Depending on the clean room design, immediately move the sample to the clean room on a disinfected designated stainlesssteel cart or place it inside the clean room pass thru for final preparation. If conducting the sterility test in an isolator, place the sample on a designated stainless-steel cart. Allow exposure of the sample to the disinfectant for appropriate time before further handling. All units should be disinfected appropriately. The suggested disinfection procedures can be performed on commonly encountered units as follows:
Ampoules can be wiped with lint free sterile towel/wipes saturated with disinfectant. Ampoules may be soaked in disinfectant/sporicidal following manufacturer’s guidance or laboratory SOP.
Vials should not be soaked due to the possibility of migration of disinfectant under the closure and into the product.
Laminated Tyvek package composed of polyethylene/plastic laminate can be disinfected with sterile towel/wipes soaked in disinfectant. Tyvek portion lightly scrubbed with sterile particle free dry wipe and air dry in a HEPA filtered laminar flow hood before testing.
Paper Packages can be disinfected with UV light if possible. Wipe where applicable with sterile particle free dry wipes and air dry as above.
Number of units and/or amount of product tested:
Follow the current edition of the USP to determine the correct number of units to be tested and the amount of product to be analyzed from each unit. It is preferable to test the entire contents of each unit if possible. Follow laboratory policy if it requires testing more units than the USP requires.
If the number of units collected is less than the USP requirements, discuss with the laboratory supervisor before proceeding. Samples collected in a for-cause situation may be analyzed with a number of units less than the USP requirements.
Preparation for the Analysis
Media and Rinsing Fluid Preparation:
Follow current USP when preparing media used for sample analysis.
Commercially purchased media may also be used for the analysis. Both prepared and purchased media must meet the requirements of the USP growth promotion test of aerobes, anaerobes and fungi. Media used are:
Fluid Thioglycollate medium (FTM) This medium should be prepared in a suitable container to provide a surface to depth ratio so that not more than the upper half of the medium has undergone a color change indicative of oxygen uptake at the end of the incubation period. If more than the upper third of the medium has acquired a pink color, the medium may be restored once by heating until the pink color disappears. Care should be taken to prevent the ingress of non-sterile air during cooling.
Soybean Casein Digest medium (SCD medium) This media must be incubated under aerobic conditions
Alternative Thioglycollate medium This type of media must be incubated under anaerobic conditions.
Media for Penicillin and Cephalosporin containing drugs. Add sufficient quantity of sterile Beta-lactamase to the media to inactivate the effect of these antibiotics.
Diluting and rinsing fluids. These fluid rinses may be filtered before sterilization to avoid clogging of the filter membrane during testing.
For laboratory prepared media, do not use medium for longer storage period than has been validated.
For commercially purchased media, follow the manufacturer’s recommended storage requirements and expiration date.
Perform the following tests on the prepared media before use:
Sterility: The media batch may be used if the sterilization cycle is validated and monitored with the use of a biological indicator, and the batch passes other quality control testing. Also, if possible, prior to otherwise concurrently, incubate a portion of the media at the specified temperature for 14 days.
Growth promotion test; follow the current USP using recommended strains of organisms (Table 1, USP <71>). Do not use cultures that are more than five passages removed from the original master seed lot. Commercially prepared and standardized stable suspension cultures of the recommended organisms can also be used. Test strains suspensions of vegetative bacteria or yeast should be used within 2 hours, or within 24 hours if refrigerated between 2ºC and 8ºC. Spore suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) refrigerated between 2ºC and 8ºC may be kept for a validated period of time. If using commercially prepared organisms, follow the manufacturer’s instructions. Additionally, all bacterial and spore suspensions should be prepared to yield ≤100CFU. All bacterial counts must be verified at time of use.
Analytical equipment and tools used in sterility analysis and suitability should be cleaned and sterilized using a validated sterilization procedure. Commercially purchased equipment and tools should be labeled sterile and accompanied by a certificate of analysis for sterility.
Clean Room Activities
Personnel are critical to the maintenance of asepsis in the controlled environment. Thorough training in aseptic techniques is required. Personnel must maintain high standards each time they deal with sterile product.
Personnel gowning qualification should be performed by any analyst that enters the aseptic clean room. Personnel gowning qualification must consist of:
Training of gowning techniques by a qualified trainer.
Observation of trainee by trainer while gowning. iii. General growth media touch plates utilized to analyze if the trainee gowned correctly without contaminating the sterile outer gown, sterile gloves and sterile head cover.
b. Some consideration should be taken before entering the clean room (see below). Follow applicable specific procedures for the facility.
Proper gowning immediately prior to entry the clean room is required of all personnel without exception.
Non-linting clean room scrubs that cover as much skin as possible is the ideal inner-suit to wear before gowning up for an aseptic clean room. Street clothes are not permitted.
Remove jewelry and makeup. iv. Scrub hands (and arms when possible) before gowning.
v. Non-shedding sterile uniform components should be used all the time. vi. Use aseptic gowning procedure to don sterile uniform components.
Care should be taken to choose gowning that does not expose any skin to the aseptic clean room environment.
An appropriate sporicidal/disinfectant is used to sanitize the gloves. ix. If possible, post the gowning procedures in the gowning room or area to help individuals follow the correct order of gowning.
Should an analyst find it necessary to leave the room, he/she should discard all gowning components and put on new ones upon re-entry.
If an individual scheduled to enter the clean room for analysis feels sick or has compromised skin, he/she should talk to his/her supervisor to postpone entry into the clean room until fully healed.
Repeat disinfection procedure using appropriate
disinfectant/sporicidal immediately prior to placing product primary containers in a working certified laminar flow hood. Allow all disinfected containers to completely air dry in the laminar flow hood prior to opening for analysis. Alternatively, if conducting the testing in an isolator, place the disinfected items into the isolator and proceed with the local procedures for the proper decontamination of the interior of the isolator.
Room Cleaning After Analysis
Remove inoculated tubes of media and all controls from the analytical area by putting them in the pass-thru or on a stainlesssteel cart used for transporting materials in and out of the clean room.
After analysis, all sample containers, equipment wrap, used equipment and tools are to be removed from the clean room before the analyst exits.
Sample containers used in the analysis should be returned to the original outer containers for storage as part of the reserve sample.
Disinfect working area before exiting the clean room.
Clean room disinfection and surface monitoring must be conducted for both aerobic and anaerobic microorganisms on a routine basis. The frequency is to be determined by the local laboratory policy.
Method of Analysis
Follow the current edition of the USP for the amount of sample to be tested.
Follow the current edition of USP for the amount of sample and media to be used. For example: Use 200 ml of each medium when analyzing solid form products. If the membrane filter method is unsuitable, certain liquids may be tested by direct inoculation method.
All devices with only the pathways labeled as sterile are to be tested by the pathway with sterile Fluid D and testing the Fluid D via membrane filtration.
Incubation of Sterility Test Media
Incubate Fluid Thioglycollate (THIO) at 32.5 ± 2.5oC. Do not shake or swirl test media during incubation or during examination to minimize aeration of this broth.
Incubate Soybean-Casein Digest Broth (SCD) at 22.5 ± 2.5oC. Gentle swirling, on occasion is acceptable to increase aeration of media.
Incubation period for THIO and SCD:
Not less than 14 days except for products sterilized using ionizing radiation. If tubes are not read on day 14 due to holiday or weekend then record the results, even if positive, on the first available day to observe the tubes.
Additional incubation time may be warranted if the analyst is made aware of sterilization processes other than heat or filtration (e.g. 30 days (at minimum) for products sterilized using ionizing radiation). This is to allow repair of DNA of microorganisms injured by ionizing radiation, if any, that may be present).
Analysis of Medical Devices (ex. Purified Cotton, Gauze, Sutures and Surgical Dressings)
The USP method for analysis of surgical dressing/cotton/gauze (in packages) calls for a minimum quantity of 100 mg, to be tested in each medium. It is recommended that an entire unit shall be tested in each medium for individually packaged single-use articles.
1. Gauze, Purified Cotton, Sutures and Surgical Dressings
Using media containers as large as quart jars analyze entire unit of product.
If unit is too large for the container, analyze as much of unit as can be placed in container and covered by the medium.
2. Compositing of Medical Devices
Devices may be tested in composites (2 – 4 units/composite) as long as they meet the specifications of Chapter 71 of the current USP with regards to minimum quantity of a test unit and minimum number of units to be tested. All composited units must be the same lot number.
Devices may be composited only if they successfully pass the Method Suitability test. If composited units do not pass Method Suitability test, then the product cannot be composited.
The objective of a control system is to ensure the sterility, within designated limits, of all items, media, rinsing fluids, and equipment used in a sterility test.
The control systems which will accompany all sterility analyses are outlined below.
1. System Control
A “system control” is used to demonstrate maintenance of sample integrity during all analytical manipulations. Any piece of equipment that comes in contact with the product under analysis, along with any manipulations by the analysts, must be controlled. Thus, all equipment, fluids, and culture media for the “system control” must be handled in a manner which duplicates, as closely as possible, the manipulations of the actual sample being analyzed. All materials used as system controls must be sterilized by the analyzing laboratory. However, the method of sterilization need not be the same as for the product, but they must render the material sterile.
The first choice for the system control is the actual product, if enough test units are available. When complex medical devices must be sacrificed in order to design a suitable sterility test, consider using them for a system control after cleaning, repacking and sterilizing.
When there are viable alternatives, a product unit should not be sacrificed for use as a system control if this will reduce the number of units available for sterility testing below USP requirements or ORS policy requirements, except as provided in the preceding paragraph. If using a product unit would reduce the subsamples examined below the number required by USP or ORS policy, the analyzing laboratory should prepare a control from other material than a unit of the sample product whenever possible.
a. Membrane Filtration: A filter funnel from the vacuum source connection on each manifold used in the test is used for the system control. Alternatively, if a closed canister system is used to conduct the sterility test a canister set from the same lot used during the analysis should be used for the system control. i. Filterable Materials (liquids, soluble solids, etc.)
Use a material similar to the product under test. The control material must be of the same volume, and similarly packaged as the test product. Filtersterilized and autoclaved Peptone water (USP Fluid A) may be useful for this purpose in many cases.
ii. Devices with sterile Fluid Pathway
Use tubing or other containers similarly fitted with needles, valves, connectors, etc., as the product under test. Use USP Fluid D to flush lumens.
b. Materials tested by direct inoculation (devices, insoluble solids, and other non-filterable materials)
Use materials similar in size, shape, and texture, and similarly packaged as product under test. Replicate as nearly as possible pertinent, unusual features that may reflect on the credibility of the sterility test.
In designing “system controls” for sterility testing, care must be taken to duplicate the sample product for most aspects, as nearly as possible. Be novel and innovative to meet this requirement and make the system control meaningful.
2. Equipment Controls
All equipment items used in the analysis listed below will be controlled individually. One item from each autoclave lot of equipment is tested in each medium used in the test. Therefore, for a sample tested in THIO and SCD, one item from each sterilizer load (oven or autoclave) is tested in each medium giving a total of two controls for each forceps, syringe, etc., used in the test.
Membranes (dry, directly from the package). If membranes are sterilized in place, this control may be omitted. Hemostats
Other special items that may be required by a specific test.
Media and Rinse Fluid Controls
An uninoculated media and rinse fluid control are analyzed to ensure sterility at time of use.
Alternatively, controls for these materials are accomplished as part of the “system control” for each manifold. This will also include membrane cutters, and other items that contact the product but cannot be individually controlled.
Open Media Controls
Tubes of each medium (THIO and SCD) used in the sterility analysis are exposed to the immediate environment of the test (e.g., laminar flow hood) for the duration of the test. Alternatively, a laboratory may use agar settling plates as detailed in section b.
Agar Settling Plates
Plastic Petri dishes containing an effective non-selective medium (based on test requirements) are exposed in the hood for a period not to exceed four hours during the analysis. After four hours, plates should be replaced to continue monitoring (as appropriate).
Plates should be incubated for 48 hours at 35o C, and an additional 5 days at 25oC in order to detect mold contamination.
Controls within an Isolator
When conducting the sterility test within an isolator, if it has been designed to allow for a connection to an air sampler and particle counter this sampling may be performed for the duration of the sample analysis in lieu of the environmental samples described above. If the isolator is unable to accommodate an air sampler and/or particle counter or the instruments are unavailable the environmental controls described in section a. and b. should be used. Isolator gloves should be examined before and after a testing session to ensure integrity of the gloves were maintained. This examination should be documented. Additionally, prior to each decontamination cycle a leak test of the isolator system must be performed with passing results.
Personnel monitoring must be performed after analysts conclude sterility testing and prior to exiting the aseptic clean room. The analyst shall use general media touch plates to monitor the sterile condition of their clean room attire and to ensure aseptic techniques were followed.
For example, a minimum of five touch plates should be used for the following personnel gowning sites:
RH glove finger tips.
LH glove finger tips. Chest
General media touch plates will be incubated for 5 days at 30-35ºC.
NOTE: The numerical values for personnel monitoring limits and specifications are established on the basis of a review of actual findings within the facility. All isolates are to be identified by local laboratory procedure to ensure that the analyst did not contaminate the sample. Analysts should be sanitizing their gloves throughout the sterility analysis and changing gloves when needed. However, changing gloves prior to performing personnel monitoring is unacceptable. Each laboratory is required to monitor and trend data to ensure compliance and detect any abnormalities. H. Sub-culturing Primary Media
Daily observations of primary test media (THIO and SCD) containing product should be performed without unnecessary disturbance. All handling of positive tubes, streaked plates, or subsequent inoculations of additional media will be done outside the clean room. These culture transfers are to be performed within a HEPA filtered biosafety cabinet or equivalent outside the ISO5 area which has been cleansed with an effective sporicidal/disinfectant anti-microbial agent. The analyst should be gowned with at least sterile gloves, sterile sleeves and a mask to minimize any possible cross contamination.
Record on Analyst’s worksheets the day that the primary isolation media, Fluid Thioglycollate Broth (THIO), or Soybean-Casein Digest Broth (SCD) is turbid and inform supervisor. Streak tubes on the day they first appear positive and again at 14 days to determine the presence of other possible slow-growing (i.e., fungi) microorganisms.
Within a HEPA filtered biosafety cabinet or equivalent outside the clean room, streak turbid tubes onto Modified Soybean-Casein Digest Medium [SCD broth + 1.5% agar] (Modified SCDA) or other non-selective agar plate.
All streaked plates are incubated for a period at least as long as required for growth in original isolation media (THIO or SCD) not to exceed seven days.
Subculturing from Fluid Thioglycollate Broth (THIO)
a. Subculture Thioglycollate broth to general medium agar plates in duplicate. Streak two plates; incubate one aerobically, and one anaerobically, each at 32.5 ± 2.5 ºC. NOTE: It is suggested to transfer an aliquot of media from close to the bottom of the tube to maximize the recovery of strict anaerobes.
Note if any growth is observed on the anaerobic plate which differs from growth on the aerobic plate. Pick a single representative colony and perform an aero-tolerance test in order to determine if a strict anaerobe has been recovered. Proceed with identification of any strict anaerobes recovered when isolation is complete.
Note if any growth is observed on aerobic plate and compare to growth on anaerobic plates. Proceed with identification when isolation is complete.
Subculturing from Soybean Casein Digest broth (SCD)
a. Sub culture SCD broth to general growth medium and incubate aerobically. Streak one plate; incubate aerobically at 22.5 ± 2.5 ºC.
Note if any growth is observed on general growth medium plate. Proceed with identification when isolation is complete.
Each organism should be identified to genus and species, if possible, using rapid identification kits or DNA sequencing.
I. Product-Induced Turbidity in Primary Test Media
When product-induced turbidity prevents the confirmation of visual observation of growth, the following instructions apply
Record “T” for any subsample which is turbid due to product-medium mixture.
On the daily observation page, indicate the meaning of “T” as: “T = product induced turbidity”.
At the end of the initial 14 days of incubation, transfer portions of the medium (not less than 1 ml) to a fresh container of the same medium and then incubate the original and transferred containers for not less than 4 days. Note: Follow the current edition of the USP for any changes concerning subculturing and incubation of turbid samples.
Examine original product inoculated media and the subcultured media for growth daily when possible for not less than 4 days of incubation and record the results on a new daily observation continuation sheet.
Antimicrobial Effectiveness testing is described in USP <51>. Previously this chapter was known as “Preservative Effectiveness Testing”. Detailed procedure for the performance of the test can be found in USP <51>.
For the cultivation of the test organisms, select agar medium that is favorable to the rigorous growth of the respective stock culture. The recommended media are Soybean Casein Digest Agar/Broth and Sabouraud’s Dextrose Agar/Broth. Add a suitable inactivator (neutralizer) for the specific antimicrobial properties in the product to the broth and/or agar media used for the test procedure whenever needed.
Growth Promotion of the Media
Media used for testing needs to be tested for growth promotion by inoculating the medium with appropriate microorganisms. It is preferable that test microorganisms be chosen for growth promotion testing (Section D).
Solid media tested for growth promotion is to be set up using the method that will be used to analyze the product (pour plate or spread plate) to determine a microbial plate count (CFU) which must be ≥ 50% of the microorganism inoculum’s calculated value.
Suitability of the Counting Method in the Presence of Product
For all product types, follow current USP methodology in chapter <51>, with the following additional instructions.
Prior to the Antimicrobial Effectiveness testing, determine if any antimicrobial properties exist by performing a Suitability testing utilizing microorganisms used for product testing (section D). Should the Suitability Test fail the results of Suitability test are invalid and will need to be repeated with proper method modification to neutralize the inhibiting property.
If multiple samples of the same product from the same manufacturer (same amount and form) are collected, one sample may be used for method suitability for all the samples collected.
All cultures must be no more than 5 passages removed from the original stock culture.
Candida albicans (ATCC No. 10231)
Aspergillus brasiliensis (ATCC No. 16404)
Escherichia coli (ATCC No. 8739)
Pseudomonas aeruginosa (ATCC No. 9027)
Staphylococcus aureus (ATCC No. 6538)
E. Preparation of Inoculum
Preparatory to the test, inoculate the surface of the appropriate agar medium from a recently grown stock culture of each of the above test microorganisms.
Use Soybean-Casein Digest agar for Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 and incubate at 32.5 ± 2.5° C for 3 – 5 days. Use Sabouraud Dextrose agar for Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 and incubate at 22.5 ± 2.5° C for 3 – 5 days for Candida albicans and 3 – 7 days for Aspergillus brasiliensis.
Harvest the cultures by washing the growth with sterile saline to obtain a microbial count of about 1×108 CFU/mL (see Microbial Enumeration Tests <61> and Tests for Specified Microorganisms <62>). For the A. brasiliensis ATCC 16404 culture, use sterile saline containing 0.05% polysorbate 80.
Alternatively, cultures may be grown in a liquid medium, i.e. Soybean Casein Digest Broth or Sabouraud’s Dextrose Broth, (except for the A. brasiliensis ATCC 16404 culture) and harvested by centrifugation, washing and suspending in sterile saline to obtain a count of about 1 X 108 colony forming units (CFU) per mL.
The estimate of inoculum concentration may be obtained by turbidimetric procedures for the challenge microorganisms and later confirmed by plate count.
Refrigerate the suspension if not used within 2 hours at 2-8° C.
Determine the number of CFU/mL in each suspension using the appropriate media and recovery incubation times to confirm the CFU/mL estimate.
Use bacterial and yeast suspensions within 24 hr. of harvest. The mold preparation may be stored under refrigeration (2-8° C) for up to 7 days. Note: Alternative commercially available standardized cultures may be used in lieu of in-house prepared cultures.
The procedure requires that the test be conducted with a suitable volume of product. It is advisable to begin with at least 20 mL of product. Use the original product containers whenever possible or five sterile, capped bacteriological containers of suitable size into which a suitable volume of product has been transferred. If the diluted product exhibits antimicrobial properties, specific neutralizers may need to be incorporated into the diluents or the recovery media. For purposes of testing, products have been divided into four categories:
Category 1 – Injections, other parenteral including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles.
Category 2 – Topically used products made with aqueous bases or vehicles, non-sterile nasal products, and emulsions, including those applied to mucous membranes.
Category 3 – Oral products other than antacids, made with aqueous bases or vehicles.
Category 4 – Antacids made with aqueous bases or vehicles.
Inoculate each container with one of the prepared and standardized inoculums and mix. The volume of the suspension inoculums used is 0.5% to 1.0% of the volume of the product. The concentration of the test organisms added to the product for Categories 1, 2 and 3 is such that concentration of the test preparation immediately after inoculation is between 1×105 and 1×106 colony forming organisms (CFU) per mL of product. If no suitable neutralizing agent or method is found and method suitability requires significant dilution, a higher level of inoculum (e.g., 107-108) may be used so that a 3-log unit reduction can be measured. For category 4 products (antacids) the final concentration of the test organisms is between 1×103 and 1×104 CFU/mL of product.
Immediately determine the concentration of viable organisms in each inoculum suspension and calculate the initial concentration of CFU/mL by the plate count method (see Microbial Enumeration Tests <61>).
Incubate the inoculated containers between 22.5 ±2.5°C in a controlled environment (incubator) and sample the container at specified intervals. The container sampling intervals include: Category 1 products are sampled at 7, 14, and 28 days and Category 2 – 4 products are sampled at 14 and 28 days. Refer to table 3 within USP <51>. Record any changes in appearance of the product at these intervals. Determine the number of viable microorganisms per mL present at each of these sampling intervals by the plate count method utilizing media with suitable inactivator (neutralizer). Calculate the change in log10 values of the concentration per mL based on the calculated concentration in CFU/mL present at the start of the test for each microorganism at the applicable test intervals and express the changes in terms of log reductions.
NOTE: The USP does not require a specific volume of product to be added to each of the five sterile tubes. It is recommended that 20 mL/tube be used to standardize testing for all ORS laboratories.
NOTE: All plate counts should be performed in duplicate (2 plates per dilution), and in a dilution series to detect growth inhibited by the preservative system at the lower dilutions. Carrying the test to the 10-3 dilution would be sufficient in most cases to overcome preservative inhibition. G. Interpretation
The criteria for microbial effectiveness are met if the specified criteria are met, see table below. No increase is defined as not more than 0.5 log10 unit higher than the previous value measured.
Note: This document is reference material for investigators and other FDA personnel. The document does not bind FDA, and does no confer any rights, privileges, benefits, or immunities for or on any person(s).
This guide discusses, primarily from a microbiological aspect, the review and evaluation of high purity water systems that are used for the manufacture of drug products and drug substances. It also includes a review of the design of the various types of systems and some of the problems that have been associated with these systems. As with other guides, it is not all-inclusive, but provides background and guidance for the review and evaluation of high purity water systems. The Guide To Inspections of Microbiological Pharmaceutical Quality Control Laboratories (May, 1993) provides additional guidance.
I. SYSTEM DESIGN
One of the basic considerations in the design of a system is the type of product that is to be manufactured. For parenteral products where there is a concern for pyrogens, it is expected that Water for Injection will be used. This applies to the formulation of products, as well as to the final washing of components and equipment used in their manufacture. Distillation and Reverse Osmosis (RO) filtration are the only acceptable methods listed in the USP for producing Water for Injection. However, in the bulk Pharmaceutical and Biotechnology industries and some foreign companies, Ultra Filtration (UF) is employed to minimize endotoxins in those drug substances that are administered parenterally.
For some ophthalmic products, such as the ophthalmic irrigating solution, and some inhalation products, such as Sterile Water for Inhalation, where there are pyrogen specifications, it is expected that Water for Injection be used in their formulation. However, for most inhalation and ophthalmic products, purified water is used in their formulation. This also applies to topicals, cosmetics and oral products.
Another design consideration is the temperature of the system. It is recognized that hot (65 – 80oC) systems are self sanitizing. While the cost of other systems may be less expensive for a company, the cost of maintenance, testing and potential problems may be greater than the cost of energy saved. Whether a system is circulating or one-way is also an important design consideration. Obviously, water in constant motion is less liable to have high levels of contaminant. A one-way water system is basically a “dead-leg”.
Finally, and possibly the most important consideration, is the risk assessment or level of quality that is desired. It should be recognized that different products require different quality waters. Parenterals require very pure water with no endotoxins. Topical and oral products require less pure water and do not have a requirement for endotoxins. Even with topical and oral products there are factors that dictate different qualities for water. For example, preservatives in antacids are marginally effective, so more stringent microbial limits have to be set. The quality control department should assess each product manufactured with the water from their system and determine the microbial action limits based on the most microbial sensitive product. In lieu of stringent water action limits in the system the manufacturer can add a microbial reduction step in the manufacturing process for the sensitive drug product(s).
II. SYSTEM VALIDATION
A basic reference used for the validation of high purity water systems is the Parenteral Drug Association Technical Report No. 4 titled, “Design Concepts for the Validation of a Water for Injection System.”
The introduction provides guidance and states that, “Validation often involves the use of an appropriate challenge. In this situation, it would be undesirable to introduce microorganisms into an on-line system; therefore, reliance is placed on periodic testing for microbiological quality and on the installation of monitoring equipment at specific checkpoints to ensure that the total system is operating properly and continuously fulfilling its intended function.”
In the review of a validation report, or in the validation of a high purity water system, there are several aspects that should be considered. Documentation should include a description of the system along with a print. The drawing needs to show all equipment in the system from the water feed to points of use. It should also show all sampling points and their designations. If a system has no print, it is usually considered an objectionable condition. The thinking is if there is no print, then how can the system be validated? How can a quality control manager or microbiologist know where to sample? In those facilities observed without updated prints, serious problems were identified in these systems. The print should be compared to the actual system annually to insure its accuracy, to detect unreported changes and confirm reported changes to the system.
After all the equipment and piping has been verified as installed correctly and working as specified, the initial phase of the water system validation can begin. During this phase the operational parameters and the cleaning/ sanitization procedures and frequencies will be developed. Sampling should be daily after each step in the purification process and at each point of use for two to four weeks. The sampling procedure for point of use sampling should reflect how the water is to be drawn e.g. if a hose is usually attached the sample should be taken at the end of the hose. If the SOP calls for the line to be flushed before use of the water from that point, then the sample is taken after the flush. At the end of the two to four week time period the firm should have developed its SOPs for operation of the water system.
The second phase of the system validation is to demonstrate that the system will consistently produce the desired water quality when operated in conformance with the SOPs. The sampling is performed as in the initial phase and for the same time period. At the end of this phase the data should demonstrate that the system will consistently produce the desired quality of water.
The third phase of validation is designed to demonstrate that when the water system is operated in accordance with the SOPs over a long period of time it will consistently produce water of the desired quality. Any variations in the quality of the feedwater that could affect the operation and ultimately the water quality will be picked up during this phase of the validation. Sampling is performed according to routine procedures and frequencies. For Water for Injection systems the samples should be taken daily from a minimum of one point of use, with all points of use tested weekly. The validation of the water system is completed when the firm has a full years worth of data.
While the above validation scheme is not the only way a system can be validated, it contains the necessary elements for validation of a water system. First, there must be data to support the SOPs. Second, there must be data demonstrating that the SOPs are valid and that the system is capable of consistently producing water that meets the desired specifications. Finally, there must be data to demonstrate that seasonal variations in the feedwater do not adversely affect the operation of the system or the water quality.
The last part of the validation is the compilation of the data, with any conclusions into the final report. The final validation report must be signed by the appropriate people responsible for operation and quality assurance of the water system.
A typical problem that occurs is the failure of operating procedures to preclude contamination of the system with non-sterile air remaining in a pipe after drainage. In a system illustrated as in Figure 1, (below) a typical problem occurs when a washer or hose connection is flushed and then drained at the end of the operation. After draining, this valve (the second off of the system) is closed. If on the next day or start-up of the operation the primary valve off of the circulating system is opened, then the non-sterile air remaining in the pipe after drainage would contaminate the system. The solution is to pro-vide for operational procedures that provide for opening the secondary valve before the primary valve to flush the pipe prior to use.
Another major consideration in the validation of high purity water systems is the acceptance criteria. Consistent results throughout the system over a period of time constitute the primary element.
III. MICROBIAL LIMITS
Water For Injection Systems
Regarding microbiological results, for Water For Injection, it is expected that they be essentially sterile. Since sampling frequently is performed in non-sterile areas and is not truly aseptic, occasional low level counts due to sampling errors may occur. Agency policy, is that less than 10 CFU/100ml is an acceptable action limit. None of the limits for water are pass/fail limits. All limits are action limits. When action limits are exceeded the firm must investigate the cause of the problem, take action to correct the problem and assess the impact of the microbial contamination on products manufactured with the water and document the results of their investigation.
With regard to sample size, 100 – 300 mL is preferred when sampling Water for Injection systems. Sample volumes less than 100 mL are unacceptable.
The real concern in WFI is endotoxins. Because WFI can pass the LAL endotoxin test and still fail the above microbial action limit, it is important to monitor WFI systems for both endotoxins and microorganisms.
Purified Water Systems
For purified water systems, microbiological specifications are not as clear. USP XXII specifications, that it complies with federal Environmental Protection Agency regulations for drinking water, are recognized as being minimal specifications. There have been attempts by some to establish meaningful microbiological specifications for purified water. The CFTA proposed a specification of not more than 500 organisms per ml. The USP XXII has an action guideline of not greater than 100 organisms per ml. Although microbiological specifications have been discussed, none (other than EPA standards) have been established. Agency policy is that any action limit over 100 CFU/mL for a purified water system is unacceptable.
The purpose of establishing any action limit or level is to assure that the water system is under control. Any action limit established will depend upon the overall purified water system and further processing of the finished product and its use. For example, purified water used to manufacture drug products by cold processing should be free of objectionable organisms. We have defined “objectionable organisms” as any organisms that can cause infections when the drug product is used as directed or any organism capable of growth in the drug product. As pointed out in the Guide to Inspections of Microbiological Pharmaceutical Quality Control Laboratories, the specific contaminant, rather than the number is generally more significant.
Organisms exist in a water system either as free floating in the water or attached to the walls of the pipes and tanks. When they are attached to the walls they are known as biofilm, which continuously slough off organisms. Thus, contamination is not uniformly distributed in a system and the sample may not be representative of the type and level of contamination. A count of 10 CFU/mL in one sample and 100 or even 1000 CFU/mL in a subsequent sample would not be unrealistic.
Thus, in establishing the level of contamination allowed in a high purity water system used in the manufacture of a non-sterile product requires an understanding of the use of the product, the formulation (preservative system) and manufacturing process. For example, antacids, which do not have an effective preservative system, require an action limit below the 100 CFU/mL maximum.
The USP gives some guidance in their monograph on Microbiological Attributes of Non-Sterile Products. It points out that, “The significance of microorganisms in non-sterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential harm to the user.” Thus, not just the indicator organisms listed in some of the specific monographs present problems. It is up to each manufacturer to evaluate their product, the way it is manufactured, and establish am acceptable action level of contamination, not to exceed the maximum, for the water system, based on the highest risk product manufactured with the water.
IV. WATER FOR INJECTION SYSTEMS
In the review and evaluation of Water For Injection systems, there are several concerns.
Pretreatment of feedwater is recommended by most manufacturers of distillation equipment and is definitely required for RO units. The incoming feedwater quality may fluctuate during the life of the system depending upon seasonal variations and other external factors beyond the control of the pharmaceutical facility. For example, in the spring (at least in the N.E.), increases in gram negative organisms have been known. Also, new construction or fires can cause a depletion of water stores in old mains which can cause an influx of heavily contaminated water of a different flora.
A water system should be designed to operate within these anticipated extremes. Obviously, the only way to know the extremes is to periodically monitor feedwater. If the feedwater is from a municipal water system, reports from the municipality testing can be used in lieu of in-house testing.
Figures 3-5 represent a typical basic diagram of a WFI system. Most of the new systems now use multi-effect stills. In some of the facilities, there has been evidence of endotoxin contamination. In one system this occurred, due to malfunction of the feedwater valve and level control in the still which resulted in droplets of feedwater being carried over in the distillate.
In another system with endotoxin problems, it was noted that there was approximately 50 liters of WFI in the condenser at the start-up. Since this water could lie in the condenser for up to several days (i.e., over the weekend), it was believed that this was the reason for unacceptable levels of endotoxins.
More common, however, is the failure to adequately treat feedwater to reduce levels of endotoxins. Many of the still fabricators will only guarantee a 2.5 log to 3 log reduction in the endotoxin content. Therefore, it is not surprising that in systems where the feedwater occasionally spikes to 250 EU/ml, unacceptable levels of endotoxins may occasionally appear in the distillate (WFI). For example, recently three new stills, including two multi-effect, were found to be periodically yielding WFI with levels greater than .25 EU/ml. Pretreatment systems for the stills included only deionization systems with no UF, RO or distillation. Unless a firm has a satisfactory pretreatment system, it would be extremely difficult for them to demonstrate that the system is validated.
The above examples of problems with distillation units used to produce WFI, point to problems with maintenance of the equipment or improper operation of the system indicating that the system has not been properly validated or that the initial validation is no longer valid. If you see these types of problems you should look very closely at the system design, any changes that have been made to the system, the validation report and the routine test data to determine if the system is operating in a state of control.
Typically, conductivity meters are used on water systems to monitor chemical quality and have no meaning regarding microbiological quality.
Figures 3-5 also show petcocks or small sampling ports between each piece of equipment, such as after the still and before the holding tank. These are in the system to isolate major pieces of equipment. This is necessary for the qualification of the equipment and for the investigation of any problems which might occur.
VI. HEAT EXCHANGERS
One principal component of the still is the heat exchanger. Because of the similar ionic quality of distilled and deionized water, conductivity meters cannot be used to monitor microbiological quality. Positive pressure such as in vapor compression or double tubesheet design should be employed to prevent possible feedwater to distillate contamination in a leaky heat exchanger.
An FDA Inspectors Technical Guide with the subject of “Heat Exchangers to Avoid Contamination” discusses the design and potential problems associated with heat exchangers. The guide points out that there are two methods for preventing contamination by leakage. One is to provide gauges to constantly monitor pressure differentials to ensure that the higher pressure is always on the clean fluid side. The other is to utilize the double-tubesheet type of heat exchanger.
In some systems, heat exchangers are utilized to cool water at use points. For the most part, cooling water is not circulated through them when not in use. In a few situations, pinholes formed in the tubing after they were drained (on the cooling water side) and not in use. It was determined that a small amount of moisture remaining in the tubes when combined with air caused a corrosion of the stainless steel tubes on the cooling water side. Thus, it is recommended that when not in use, heat exchangers not be drained of the cooling water.
VII. HOLDING TANK
In hot systems, temperature is usually maintained by applying heat to a jacketed holding tank or by placing a heat exchanger in the line prior to an insulated holding tank.
The one component of the holding tank that generates the most discussion is the vent filter. It is expected that there be some program for integrity testing this filter to assure that it is intact. Typically, filters are now jacketed to prevent condensate or water from blocking the hydrophobic vent filter. If this occurs (the vent filter becomes blocked), possibly either the filter will rupture or the tank will collapse. There are methods for integrity testing of vent filters in place.
It is expected, therefore, that the vent filter be located in a position on the holding tank where it is readily accessible.
Just because a WFI system is relatively new and distillation is employed, it is not problem-free. In an inspection of a manufacturer of parenterals, a system fabricated in 1984 was observed. Refer to Figure 6. While the system may appear somewhat complex on the initial review, it was found to be relatively simple. Figure 7 is a schematic of the system. The observations at the conclusion of the inspection of this manufacturer included, “Operational procedures for the Water For Injection system failed to provide for periodic complete flushing or draining. The system was also open to the atmosphere and room environment. Compounding equipment consisted of non-sealed, open tanks with lids. The Water for Injection holding tank was also not sealed and was never sampled for endotoxins.” Because of these and other comments, the firm recalled several products and discontinued operations.
Pumps burn out and parts wear. Also, if pumps are static and not continuously in operation, their reservoir can be a static area where water will lie. For example, in an inspection, it was noted that a firm had to install a drain from the low point in a pump housing. Pseudomonas sp. contamination was periodically found in their water system which was attributed in part to a pump which only periodically is operational.
Piping in WFI systems usually consist of a high polished stainless steel. In a few cases, manufacturers have begun to utilize PVDF (polyvinylidene fluoride) piping. It is purported that this piping can tolerate heat with no extractables being leached. A major problem with PVDF tubing is that it requires considerable support. When this tubing is heated, it tends to sag and may stress the weld (fusion) connection and result in leakage. Additionally, initially at least, fluoride levels are high. This piping is of benefit in product delivery systems where low level metal contamination may accelerate the degradation of drug product, such as in the Biotech industry.
One common problem with piping is that of “dead-legs”. The proposed LVP Regulations defined dead-legs as not having an unused portion greater in length than six diameters of the unused pipe measured from the axis of the pipe in use. It should be pointed out that this was developed for hot 75 – 80o circulating systems. With colder systems (65 – 75oC), any drops or unused portion of any length of piping has the potential for the formation of a biofilm and should be eliminated if possible or have special sanitizing procedures. There should be n o threaded fittings in a pharmaceutical water system. All pipe joints must utilize sanitary fittings or be butt welded. Sanitary fittings will usually be used where the piping meets valves, tanks and other equipment that must be removed for maintenance or replacement. Therefore, the firm’s procedures for sanitization, as well as the actual piping, should be reviewed and evaluated during the inspection.
X. REVERSE OSMOSIS
Another acceptable method for manufacturing Water for Injection is Reverse Osmosis (RO). However, because these systems are cold, and because RO filters are not absolute, microbiological contamination is not unusual. Figure 8 shows a system that was in use several years ago. There are five RO units in this system which are in parallel. Since RO filters are not absolute, the filter manufacturers recommend that at least two be in series. The drawing also illustrates an Ultraviolet (UV) light in the system downstream from the RO units. The light was needed to control microbiological contamination.
Also in this system were ball valves. These valves are not considered sanitary valves since the center of the valve can have water in it when the valve is closed. This is a stagnant pool of water that can harbor microorganisms and provide a starting point for a biofilm.
As an additional comment on RO systems, with the recognition of microbiological problems, some manufacturers have installed heat exchangers immediately after the RO filters to heat the water to 75 – 80oC to minimize microbiological contamination.
With the development of biotechnology products, many small companies are utilizing RO and UF systems to produce high purity water. For example, Figure 9 illustrates a wall mounted system that is fed by a single pass RO unit.
As illustrated, most of these systems employ PVC or some type of plastic tubing. Because the systems are typically cold, the many joints in the system are subject to contamination. Another potential problem with PVC tubing is extractables. Looking at the WFI from a system to assure that it meets USP requirements without some assurance that there are no extractables would not be acceptable.
The systems also contain 0.2 micron point of use filters which can mask the level of microbiological contamination in the system. While it is recognized that endotoxins are the primary concern in such a system, a filter will reduce microbiological contamination, but not necessarily endotoxin contamination. If filters are used in a water system there should be a stated purpose for the filter, i.e., particulate removal or microbial reduction, and an SOP stating the frequency with which the filter is to be changed which is based on data generated during the validation of the system.
As previously discussed, because of the volume of water actually tested (.1ml for endotoxins vs. 100ml for WFI), the microbiological test offers a good index of the level of contamination in a system. Therefore, unless the water is sampled prior to the final 0.2 micron filter, microbiological testing will have little meaning.
At a reinspection of this facility, it was noted that they corrected the deficient water system with a circulating stainless steel piping system that was fed by four RO units in series. Because this manufacturer did not have a need for a large amount of water (the total system capacity was about 30 gallons), they attempted to let the system sit for approximately one day. Figure 9 shows that at zero time (at 9 AM on 3/10), there were no detectable levels of microorganisms and of endotoxins. After one day, this static non-circulating system was found to be contaminated. The four consecutive one hour samples also illustrate the variability among samples taken from a system. After the last sample at 12 PM was collected, the system was resanitized with 0.5% peroxide solution, flushed, recirculated and resampled. No levels of microbiological contamination were found on daily samples after the system was put back in operation. This is the reason the agency has recommended that non-recirculating water systems be drained daily and water not be allowed to sit in the system.
XI. PURIFIED WATER SYSTEMS
Many of the comments regarding equipment for WFI systems are applicable to Purified Water Systems. One type system that has been used to control microbiological contamination utilizes ozone. Figure 10 illustrates a typical system. Although the system has purported to be relatively inexpensive, there are some problems associated with it. For optimum effectiveness, it is required that dissolved ozone residual remain in the system. This presents both employee safety problems and use problems when drugs are formulated.
Published data for Vicks Greensboro, NC facility showed that their system was recontaminated in two to three days after the ozone generator was turned off. In an inspection of another manufacturer, it was noted that a firm was experiencing a contamination problem with Pseudomonas sp. Because of potential problems with employee safety, ozone was removed from the water prior to placing it in their recirculating system. It has been reported that dissolved ozone at a level of 0.45 mg/liter will remain in a system for a maximum of five to six hours.
Another manufacturer, as part of their daily sanitization, removes all drops off of their ozonated water system and disinfects them in filter sterilized 70% isopropyl alcohol. This manufacturer has reported excellent microbiological results. However, sampling is only performed immediately after sanitization and not at the end of operations. Thus, the results are not that meaningful.
Figure 11 and Figure12 illustrate another purified water system which had some problems. Unlike most of the other systems discussed, this is a one-way and not recirculating system. A heat exchanger is used to heat the water on a weekly basis and sanitize the system. Actually, the entire system is a “dead-leg.”
Figure 11 also shows a 0.2 micron in line filter used to sanitize the purified water on a daily basis. In addition to the filter housing providing a good environment for microbiological contamination, a typical problem is water hammer that can cause “ballooning” of the filter. If a valve downstream from the filter is shut too fast, the water pressure will reverse and can cause “ballooning”. Pipe vibration is a typical visible sign of high back pressure while passage of upstream contaminants on the filter face is a real problem. This system also contains several vertical drops at use points. During sanitization, it is important to “crack” the terminal valves so that all of the elbows and bends in the piping are full of water and thus, get complete exposure to the sanitizing agent.
It should be pointed out that simply because this is a one-way system, it is not inadequate. With good Standard Operational Procedures, based on validation data, and routine hot flushings of this system, it could be acceptable. A very long system (over 200 yards) with over 50 outlets was found acceptable. This system employed a daily flushing of all outlets with 80oC water.
The last system to be discussed is a system that was found to be objectionable. Pseudomonas sp. found as a contaminant in the system (after FDA testing) was also found in a topical steroid product (after FDA testing). Product recall and issuance of a Warning Letter resulted. This system (Figure 13) is also one-way that employs a UV light to control microbiological contamination. The light is turned on only when water is needed. Thus, there are times when water is allowed to remain in the system. This system also contains a flexible hose which is very difficult to sanitize. UV lights must be properly maintained to work. The glass sleeves around the bulb(s) must be kept clean or their effectiveness will decrease. In multibulb units there must be a system to determine that each bulb is functioning. It must be remembered that at best UV light will only kill 90% of the organisms entering the unit.
XIII. PROCESS WATER
Currently, the USP, pg. 4, in the General Notices Section, allows drug substances to be manufactured from Potable Water. It comments that any dosage form must be manufactured from Purified Water, Water For Injection, or one of the forms of Sterile Water. There is some inconsistency in these two statements, since Purified Water has to be used for the granulation of tablets, yet Potable Water can be used for the final purification of the drug substance.
The FDA Guide to Inspection of Bulk Pharmaceutical Chemicals comments on the concern for the quality of the water used for the manufacture of drug substances, particularly those drug substances used in parenteral manufacture. Excessive levels of microbiological and/or endotoxin contamination have been found in drug substances, with the source of contamination being the water used in purification. At this time, Water For Injection does not have to be used in the finishing steps of synthesis/purification of drug substances for parenteral use. However, such water systems used in the final stages of processing of drug substances for parenteral use should be validated to assure minimal endotoxin/ microbiological contamination.
In the bulk drug substance industry, particularly for parenteral grade substances, it is common to see Ultrafiltration (UF) and Reverse Osmosis (RO) systems in use in water systems. While ultrafiltration may not be as efficient at reducing pyrogens, they will reduce the high molecular weight endotoxins that are a contaminant in water systems. As with RO, UF is not absolute, but it will reduce numbers. Additionally, as previously discussed with other cold systems, there is considerable maintenance required to maintain the system.
For the manufacture of drug substances that are not for parenteral use, there is still a microbiological concern, although not to the degree as for parenteral grade drug substances. In some areas of the world, Potable (chlorinated) water may not present a microbiological problem. However, there may be other issues. For example, chlorinated water will generally increase chloride levels. In some areas, process water may be obtained directly from neutral sources.
In one inspection, a manufacturer was obtaining process water from a river located in a farming region. At one point, they had a problem with high levels of pesticides which was a run-off from farms in the areas. The manufacturing process and analytical methodology was not designed to remove and identify trace pesticide contaminants. Therefore, it would seem that this process water when used in the purification of drug substances would be unacceptable.
XIV. INSPECTION STRATEGY
Manufacturers typically will have periodic printouts or tabulations of results for their purified water systems. These printouts or data summaries should be reviewed. Additionally, investigation reports, when values exceed limits, should be reviewed.
Since microbiological test results from a water system are not usually obtained until after the drug product is manufactured, results exceeding limits should be reviewed with regard to the drug product formulated from such water. Consideration with regard to the further processing or release of such a product will be dependent upon the specific contaminant, the process and the end use of the product. Such situations are usually evaluated on a case-by-case basis. It is a good practice for such situations to include an investigation report with the logic for release/rejection discussed in the firm’s report. End product microbiological testing, while providing some information should not be relied upon as the sole justification for the release of the drug product. The limitations of microbiological sampling and testing should be recognized.
Manufacturers should also have maintenance records or logs for equipment, such as the still. These logs should also be reviewed so that problems with the system and equipment can be evaluated.
In addition to reviewing test results, summary data, investigation reports and other data, the print of the system should be reviewed when conducting the actual physical inspection. As pointed out, an accurate description and print of the system is needed in order to demonstrate that the system is validated.
Checklist for review of microbiology data generated during the different tests of microbiology i.e. Antibiotic Assays, Particulate matter test, BET test, Preservative effectiveness testing ( Antimicrobial effectiveness testing), Microbial limit test (MLT), sterility, water analysis, and preservative effectiveness testing, Sterility testing. etc.
Checklist for Microbiological Analytical Data and Reports
Followings are the checkpoints during the review of microbiology data during various tests performed…
1.0 Product Information (Review of Microbiology Data) :
Name of material (Brand name, Generic name)
Pharmacopoeial status if any.
Manufacturer supplier name.
Batch No., A.R. No., Batch size.
Mfg. Date, Exp. Date.
2.0 General Check (Review of Microbiology Data) :
The Analytical method, effective date, revision number.
Calibration status of Instrument/Equipment.
Instrument/Equipment code No.
Instrument/Equipment usage log entry.
Name and grade of reagents used in the analysis.
Solution code no.
Balance ID used in the analysis.
Dilution, sonication time,filter, centrifuge of sample as per ATP .
Weight slip print out and any other print out with B.No. /A.R No. and signature of the analyst.
Date of analysis.
Countersignature where ever applicable.
2.0 General Check – MLT, Sterility, Water Analysis, and P.E. Testing :
Media used (check against respective ATP/Pharmacopoeia).
Media preparation date and its validity.
Sterilization reference no. / lot of media.
The pH of media.
Autoclave /DHS starting and end time.
Autoclave /DHS temperature.
Incubator number, calibration status of the incubator.
A microbial culture (microbiological culture) is a procedure of growing microbial organisms (reproduction) by allowing them to breed in programmed culture medium under controlled laboratory conditions.
Microbial cultures are initial and basic diagnostic methods used as a research tool in molecular biology.
Microbial Culture Management
Standard Operating Procedure (SOP)
To lay down the procedure for the management of microbial cultures.
This Standard Operating Procedure is applicable at Microbiology Department.
PROCEDURE FOR MICROBIAL CULTURING
Procurement of Cultures:
Prepare the list of ATCC / NCTC / NCYC / MTCC cultures required in the Microbiology section as per the details mentioned in Annexure-1.
Raise the purchase requisition for the cultures for procurement.
Procure the required cultures once in a year.
All cultures shall be procured from the authorized sources with certificate-based on permissible subculturing periods.
Ensure that the cultures shall not be more than 2 passages removed from the reference.
Upon receipt of the cultures, enter the details along with in house identification no. in culture inward record as per Annexure-2.
Store these cultures in the refrigerator between 2º-8ºC or as per manufacturer recommendation.
For Example, E. coli received on 01/10/20 then given in house number should be E.coli 011020.
Reconstitution of Freeze-Dried Cultures:
Sanitized the surface of ampoule or vial or slant or loops using 70 % IPA.
Transfer the ampoule or vial or slant or loops under LAF/Biosafety cabinet and open the culture aseptically.
Add 0.5 ml to 1.0 ml of sterile water to the vial/ampoule/ slant to reconstituting the lyophilized / slant cultures or reconstitute the cultures as per the recommendation of the provider.
This culture will serve as mother Culture.
Record the details in Culture Maintenance Record as per Annexure-3.
Revival and Maintenance of Cultures:
Streak the mother culture on agar plates for confirmation of purity as per SOP for Isolation and identification of microorganisms (Annexure-4) or
Direct by automated identification system and simultaneously inoculate the total content of vial/ ampoule in 100 ml of sterilized Soya bean casein digest medium.
After transfer the mother culture in to the medium,
Dispose the remaining content and vial as per current version of SOP for Disposal of used media and cultures. and record the details in Annexure-8.
Use agar plate for purity check and
Use the liquid medium for preparation of Seed lot cultures.
Incubate the media containing
Bacteria (Cultures of Bacteria) at 32.5 ± 2.5º C for 24-48 hrs,
Molds (Cultures of Molds) at 22.5± 2.5º C for 72-120 hrs and
Yeasts (Cultures of Yeasts) at 22.5 ± 2.5º C for 24- 48 hrs.
Media and incubation conditions shall be followed for different cultures as recommended in Annexure-1.
After completion of incubation check the purity as per SOP on Isolation and identification of microorganisms of
Culture by colonial characteristics,
Biochemical characterization or
through automated identification System (BD Phoenix).
Add 10% v/v sterile glycerol in culture suspension in 1:1 ratio, mix well and dispense 2-3 ml into the sterile cryo vial prepare 14 such vials which serves as Seed lot Culture (SLC).
Mark culture ID number as SLC-1, SLC-2, and SLC-3 and so on and store the cryo vials (Cryoprotective medium) at -30°C or below until use.
Label each cryo vial of SLC with the details like (as per the Annexure-6.)
Name of culture,
Date of subculturing,
Sub cultured by and
Use 12 cryovials of seed lot culture for subculturing up to 12 months (yearly) and Keep 2 cryovials as a stock which shall be used if any vial gets damaged or spillage.
Ensure that the cryovials shall not be used after one year.
Discard the remaining two cryovials after completion of yearly subculturing as per the current version of SOP on Disposal of used media and cultures.
Subculturing: (Microbial Culture)
Maintain the cultures as per the Schematic Flow for Subculturing as per Annexure-5.
For first-month subculture streak five slants of agar medium from the cryovial of SLC-1 and mark culture ID numbers as
3.0 SLC-1 WC-3,
4.0 SLC-1WC-4, and
Simultaneously streak on the plates of agar medium for purity check as per SOP for “Isolation and identification of microorganisms” of culture by
Biochemical characterization or
through an automated identification System (BD Phoenix).
Incubate the slants and plates containing cultures of
Bacteria at 32.5±2.5ºC for 24-48 hrs,
Molds at 22.5± 2.5ºC for 72-120 hrs. and
Yeast at 22.5± 2.5ºC for 24- 48 hrs.
When proper growth observed on the transferred slants discard SLC-1 following the current version of SOP on “Disposal of used media and cultures” and record the details in Annexure-8.
Label each slant of working culture with the details like
Name of culture,
Seed lot culture no.,
Date of sub culturing,
Sub cultured by and
Use before as per Annexure-7 and
Store the working cultures at 2 – 8 ºC.
Use one working culture for each week for routine lab work for up to one month.
Discard the working culture at the end of the week or before using a new working culture.
The fifth working culture shall also be discarded at the end of the month if it remains unused and details shall be recorded in Annexure-8.
Start the same procedure with SLC-2 and so on, well before completing the cycle of previous SLC to get the working cultures ready to use for next month.
Check the purity of seed lot culture and working culture as per SOP on Isolation and identification of microorganisms at the time of use by
Biochemical examination and
Record the observations in Annexure-4 or
through an automated identification system (BD Phoenix).
Record the details of subculturing in Annexure-3 at every step of sub-culturing.
Tentative Schedule for Maintenance of Microbial Cultures shall be prepared for subculturing at the time of the first revival of new cultures as per Annexure-9.
Ensure that the inoculates used shall not be more than 5 passages removed from the certified reference cultures.
Process Description: Live Cultures
During working with live cultures always use Gloves.
Segregate all live cultures from areas used for sample testing and optimally, handled in a different area of the laboratory within a Biosafety Cabinet.
Perform positive control dilutions and inoculation in a biological safety hood/cabinet.
Seal Agar plates containing fungal cultures with para-film to prevent spread of spores.
Surfaces in areas where a live culture plate, tube, bottle, pellet, etc., was opened shall be sanitized immediately after use by using an approved sanitizer for the appropriate contact time.
List of Microbial Cultures. (Annexure-1)
Microbial Culture Inward Record. (Annexure-2)
Culture Maintenance Record. (Annexure-3)
Purity Check of Microbial Culture. (Annexure-4)
Schematic flow for Sub-Culturing. (Annexure-5)
Seed Lot Culture Label. (Annexure-6)
Working Culture Label. (Annexure-7)
Culture Disposal Record. (Annexure-8)
Schedule for Maintenance of Microbial Cultures. (Annexure-9)
The Purpose of this SOP to lay down the procedure for preparation of Culture dilutions.
procedure is applicable for Culture Maintenance
in the Microbiology Laboratory at Pharmaceutical Manufacturing Industry.
For the purpose of preparing appropriate
microorganism from the weekly- transferred working culture slant, inoculate a
loopful of culture to 10 ml of Nutrient Agar for Bacteria & Fungi.
Incubate the bacterial suspension at
32.5 ± 2.5°C for 24 to 48 hours & fungal suspension at 22.5 ± 2.5°C for 24
to 72 hours for enrichment.
Serially dilute the enriched suspension in 0.9% sterile saline solution in a
ratio of 1: 10 dilution by transferring as follows.
Transfer the culture
suspension in a sterile test tube.
Collect the suspension in
a sterile test tube.
Vortex the culture
suspension to obtain a uniform suspension.
out serial dilution so as to obtain a culture suspension of 10-100 cfu/ml
by following the steps given below.
Transfer 1 ml of the suspension to 9
ml sterile normal saline solution – 101
ml of 101 Dilution to 9 ml sterile normal saline solution – 102
1 ml of 102 Dilution to 9 ml
sterile normal saline solution – 103 Dilution.
1 ml of 103 Dilution to 9 ml
sterile normal saline solution – 104 Dilution.
1 ml of 104 Dilution to 9 ml
sterile normal saline solution – 105 Dilution.
1 ml of 105 Dilution to 9 ml
sterile normal saline solution – 106 Dilution.
1 ml of 105 Di Dilution to 9 ml
sterile normal saline solution – 107 Dilution.
Check the CFU/ml from the above-prepared dilutions by transferring 1 ml of the inoculum on Nutrient agar plate for Bacteria & Fungi by pour plate method. Incubate the plates for 18 to 24 hours at respective Temperature as mentioned above. On observation of visible microbial growth on each plate, count & record the average number of CFU in Preparation of culture suspension register. Count the CFU/ml from each Dilution & select the dilution tube which gives 10-100 CFU/ml. Store the same Tube for daily use of Culture for Positive Control. Record all Dilutions Result In annexure. Use it for one week & discard (from date of preparation) as per SOP for disposal of media and record in the format. Take 1 ml of the above prepared culture suspension for Positive control for environmental monitoring, MLT and fertility test.
The Purpose of this SOP is To lay down the procedure for Microbial testing of drain water in the production area of the Pharmaceutical Manufacturing Site.
SOP is applicable to describe the procedure of Drain monitoring Microbial
Contamination for Production area and microbiology lab
at of Pharmaceutical Manufacturing Site.
All drains in the production area
shall be checked to monitor the effectiveness of cleaning and disinfecting agents
once a month.
Sample 100 ml of drain water from each drain and collect it in a sterile glass
container with the help of previously sterilized pipette.
Following tests shall be performed on
the drain water collected:
a) Total Microbial Count.
b) Absence of Pathogens.
Filter the sampled drain water through 0.45 membrane filter. For Phenolic disinfectants, dilution of drain water with sterile distilled water and filtration through 0.45 micron is sufficient. However, whenever other types of disinfectants. Wash the filter paper 2 times with sterile Normal Saline 50 ml 1st time and 50 ml Second. Inoculate the filter paper in 100 ml of Normal Saline. Shake thoroughly (so that the contents of filter paper comes in Normal Saline). Perform the Total Microbial Count and test for absence of Pathogens as per the standard.
Count and record the results in
Drain Water (Microbiological Testing) Record.
Necessary feedback shall be given to Production for corrective action if
For Bacteria 1000 CFU/ plate
For Fungi 100 CFU/Plate
Training is as per Training SOP
Annexure I: Microbial Testing of Drain
Annexure II: Microbial Testing of Drain