Sterility Testing in Pharmaceutical Product as per USFDA Guideline

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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.

Sterility Testing

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.

  1. When to run Method Suitability:
    1. Run the method suitability test prior to conducting the sterility test in accordance with USP requirements under the following conditions:
      1. If insufficient information about the product exists to judge its probable growth inhibiting activity.
      1. 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.

  1. Membrane filtration
    1. 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.
    1. 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.
    1. 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. 
    1. Confirm composited microorganisms by Gram stain, microscopic examination, and identification after the completion of incubation.
    1. See step c. below for additional considerations.
    1. Direct inoculation:

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.

  1. 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.   
  2. 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.  
  3. 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.   
  4. Sample Analysis
    1. Sample Containers
      1. 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.
      1. Count the number of units received.  Compare this number with the number of units collected.
      1. 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.
      1. 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.
      1. 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).
      1. Sample Identification

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:

  1. 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.  
    1. Vials should not be soaked due to the possibility of migration of disinfectant under the closure and into the product.
      1. 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.
      1. Paper Packages can be disinfected with UV light if possible.  Wipe where applicable with sterile particle free dry wipes and air dry as above.
    1. 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:

  1. 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.
    1. Soybean Casein Digest medium (SCD medium) This media must be incubated under aerobic conditions 
    1. Alternative Thioglycollate medium This type of media must be incubated under anaerobic conditions.
    1. Media for Penicillin and Cephalosporin containing drugs.  Add sufficient quantity of sterile Beta-lactamase to the media to inactivate the effect of these antibiotics.
    1. Diluting and rinsing fluids. These fluid rinses may be filtered before sterilization to avoid clogging of the filter membrane during testing.
    1. Media storage

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. 

  • Media qualification: 

Perform the following tests on the prepared media before use: 

  1. 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.
    1. 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.
    1. Equipment Preparation

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
    • Gowning

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. 

  1. Personnel gowning qualification should be performed by any analyst that enters the aseptic clean room.  Personnel gowning qualification must consist of:
    1. Training of gowning techniques by a qualified trainer.
    1. 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.

  1. Proper gowning immediately prior to entry the clean room is required of all personnel without exception.
    1. 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.
    1. 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. 
  • Sample Preparation

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
    • Membrane Filtration

Follow the current edition of the USP for the amount of sample to be tested. 

  • Direct Inoculation

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.

  • Devices

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

  1. Using media containers as large as quart jars analyze entire unit of product.
    1. 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

  1. 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.
    1. 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.
  2. Control Systems

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.

Forceps

Syringes

Scissors

Scalpels Swabs

Pipettes

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.

  • Environmental Controls
    • 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

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

Left Forearm

Right Forearm

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.

  1. 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.
  2. 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.
  3. 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.
  4. 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.  

  1. 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.  
    1. Note if any growth is observed on aerobic plate and compare to growth on anaerobic plates.  Proceed with identification when isolation is complete. 
  2. 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.

  1. Note if any growth is observed on general growth medium plate.  Proceed with identification when isolation is complete. 
    1. 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 

  1. Record “T” for any subsample which is turbid due to product-medium mixture. 
    1. On the daily observation page, indicate the meaning of “T” as:  “T = product induced turbidity”. 
    1. 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.
    1. 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.

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