Validation Of Aseptic Processes Using Media Fill

[Ena Marklund]

Asepticprocess simulation (APS) is essential for validation of an aseptic manufacturing process and is required by regulators to demonstrate the aseptic capability of such processes. A successful program of APS and aseptic manufacturing requires significant operator training, skills, and supervision; thorough maintenance; effective cleaning and disinfection; significant oversight of every aspect of the operation by quality assurance; and microbiological monitoring by quality control.

An overall validation of aseptic processing (as distinct from manufacturing process validation [PV]) is used to assess the contamination risk of an aseptic production process by simulating the manufacturing process using microbiological growth media instead of the drug solution. This is necessary in part because the sterility test used to release batches of sterile products has inherent limitations in detecting contaminated units in batches with low levels of microbial contamination, due to the limited number of samples that can be removed for destructive testing; this relationship has been evaluated statistically.1

APS consists of three consecutive media simulations with designated personnel in the specific cleanroom environment, followed by repeat media simulations at six monthly intervals. Any media fill failures require thorough investigation and root cause analysis, and further media simulations may be required to complete the validation.

Because of the high safety risk profile for parenteral drug products, the protocols, results, and reports for APS form an integral part of regulatory submissions for such products, meaning they are included in investigational new drug (IND) applications, new drug applications (NDAs), and marketing authorizations (MAs). Ancillary documents such as training records, environmental monitoring reports, deviations, and investigations are key topics of scrutiny during facility inspections, as well as the qualification of facility, the equipment and utilities, and the process validation.

The expectation in APS is twofold. First, it must achieve three consecutive media batches that meet target acceptance criteria. Second, the solution filtration process must be validated against a microbial challenge with 107 colony-forming units per square centimeter of filter medium (using Brevundimonas diminuta, a small-celled Gram-negative bacterium suspended in the drug solution).

Interventions to be included for simulation in the media fill protocol include routine and nonroutine manipulations by operators. The regulatory expectation is that interventions included in APS should be compliant with current GMPs, and APS must not be used to justify poor aseptic practice or equipment design.

Routine interventions include charging stopper and seal hoppers, removing jammed stoppers or toppled vials, taking environmental monitoring samples (settle plates, active air samples, and contact plates), and checking in-process control samples (e.g., manual weight checks). Routine interventions should be performed as described in the production standard operating procedure (SOP) or the batch record or environmental monitoring SOP. Procedures to be followed in the event of machine jams and spills may include partial line clearances, including removal of exposed units.

Nonroutine interventions may include changing the filling nozzles or handling unexpected events, such as breakdown maintenance, line stoppages, machine adjustments, and material transfers. Interventions can also be grouped by access point, and their risk assessed so that worst-case (highest risk) interventions are included in the study.

The chamber dwell time during APS does not impact risk because the higher chamber pressure required to avoid boiling of media does not require the use of a pressure control (gas injection) system. In the absence of airflow transport mechanism and turbulence, the chamber dwell time becomes immaterial during APS. Based on risk analysis, the aeration or vacuum-break step in the lyophilization cycle may have higher risk of contamination because it involves air turbulence18 and the possibility of entrained particles entering the containers. Because the application of full vacuum is not possible during APS, multiple partial vacuum steps should be considered to simulate the worst-case aeration. The media volume in the vials before lyophilization must ensure the wetted surface of the container mimics the production case.

Media simulation of the lyophilization step could involve loading the required number of media-filled vials as per the routine commercial production procedures, while assuring the time that the door is open to the cleanroom environment is at least as long as the maximum time incurred when loading a commercial batch of product.

Once the modified media lyophilization cycle has been completed, the chamber vacuum should be broken using sterile-filtered compressed air so that all units are stoppered under pressure to avoid inhibiting microbial recovery and growth. (Sterile-filtered nitrogen gas should not be used to break the vacuum unless a specific anaerobic media simulation is undertaken.)

After filling, stoppering, and sealing, 100% visual inspection is performed for defects such as the presence of visible foreign matter, high or low fill volumes, and damaged vials, stoppers, or seals. Such defective units would be normally removed (rejected) from product batches, but in the case of APS batches, such defective integral units must be retained and all such containers must be incubated. If filled containers are broken or otherwise damaged so that they are nonintegral and potentially contaminated, they must be recorded and reconciled with the batch record quantities. All appropriate media fill container units must be incubated.

The incubation conditions selected are optimal for recovery and to allow for detection of both slow-growing and normal contaminating organisms, i.e., adequate to detect microorganisms that might otherwise be difficult to culture. The incubation conditions used generally are 20C to 25C for seven days (lower temperature first) followed by 30C to 35C for a further seven days.

Containers are typically incubated on their sides, and while subjected to each incubation temperature, turned at least once to ensure that the entire interior surfaces of the vials and the stoppers are contacted by the growth medium.

During APS, all routine and normal processes (such as cleaning, disinfection, and maintenance) should be continued to maintain the cleanroom environment in qualified status. This includes particulate and microbiological environmental monitoring, which can demonstrate that the specified clean-room environment conditions are maintained. These monitoring results may provide key information for the investigation of a failed media run.

Particulate monitoring during aseptic product filling and APS consists of continuous monitoring for particulates in the The microbiological methods used should be described in an SOP, including a map of the locations at which the samples are to be taken or plates exposed. Each batch of environmental sampling plates must be tested for sterility and growth promotion capability against the recommended compendial strains of microorganisms before release for use.

The methods used for environmental monitoring are stated in China GMP3 and EudraLex, current Annex 1:6 active air sampling (1 m3 sample volume) onto 90 mm agar plates; settling plates 90 mm in diameter, with exposure up to 4 hours (if the APS or production filling lasts longer, new settling plates must be exposed for each subsequent 4-hour period); surface contact plates 55 mm in diameter (in which the plates are contacted against machine surfaces or cleanroom walls, floors, or operator gowns); gloved-finger samples performed by cleanroom operators during the filling period and upon leaving the cleanroom, taken by contacting four fingers and thumb onto the surface of a 90 mm tryptone soya agar (TSA) settle plate.

Media is usually TSA for viable aerobes or sabaroud dextrose agar (SDA) for fungi (molds) and yeasts. Surface contact plates may be TSA, usually incorporating a neutralizing agent to counter detergent residues from the sampled surfaces. Agar residues are removed from the sampling locations by wiping with 70% alcohol.

The expected (regulatory) action limits for the microbiological monitoring results of the Grade A cleanroom areas (Grade A LAF in Grade B back-ground; RABS; isolator), including during APS, in colony-forming units are tabulated in China GMP3 and EudraLex, current Annex 1.6 Adjacent Grade B, C, or D cleanrooms through which operator gowning and material transfer for the APS occur should also be monitored; the stated regulatory (action) limits for these cleanroom grades are also included in the China GMP3 and EudraLex, current Annex 1.6 The frequency of monitoring Grade C and D cleanrooms is to be determined based on quality risk assessment because such monitoring at the time of an APS may help investigate any discrepancy or failure.

In APS batches, the numbers of colony-forming units recorded on the environmental monitoring plates in Grade A (LAF, RABS, or isolator) and Grade B areas should be recorded. An isolate should be taken from each visually distinct microbial colony and identified by species using available biochemical and/or nucleic acid identification methods so it can be compared with organisms in contaminated units that arise during the APS. This information will be critical in investigating and determining corrective actions in the event of an APS media fill that exceeds acceptance criteria. Environmental samples (those with colonies) from Grade C and D cleanrooms should be enumerated and preferably also identified, as the information regarding the numbers, species, and locations of contaminating microorganisms may prove crucial in the investigation and resolution of a failed media fill.

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