Aseptic Processing [43,73–75]

Aseptic fill processes are validated by simulating production conditions and using a bacterial culture medium as the product. This process simulation test is commonly referred to as a “media fill.”

Production facilities must be checked to ensure that all installed equipment both satisfies the engineering and quality design criteria (installation qualifica- tion) and functions properly (operational qualification). In the performance of a media fill, it is important that everything be conducted just as a normal produc- tion run. All equipment normally used should be used. All equipment should be cleaned, sanitized, sterilized, handled, and assembled in a normal manner. All personnel normally involved in an aseptic process must participate in the media fill. Such personnel must have sufficient training in such areas as basic microbi- ology, personal hygiene, gowning techniques, manipulative techniques, safety, and cleaning procedures.

Table 10 provides a list of considerations for ensuring that every aseptic process is appropriately simulated during a media fill validation exercise.

Media fills are conducted to initially qualify a new filling line, a new product, and/or a change in product container configuration. Subsequent to ini-

Table 10 Considerations for Ensuring Media Fill Runs Adequately Simulate Actual Production Runs

Duration of longest run Multiple runs on separate days Worst-case environmental conditions

Number and type of interventions, stoppages, adjustments, transfers; both planned and unplanned (e.g., replacing filling needles, pumps, filters, stopper bowl stopping line, removing all containers, manual stoppering)

Aseptic assembly of equipment

Maximum number of personnel normally present Number of aseptic additions

Shift breaks, changes, multiple gownings

Number and type of aseptic equipment disconnections and connections Aseptic sampling

Line speed and configurations Manual weight checks Operator fatigue (work time)

Container/closure types run on the line Temperature and relative humidity extremes Conditions permitted before line clearance

tial qualification, media fills are required on a semiannual basis to provide mini- mal assurance that good aseptic conditions and practices have been maintained. Initial media fill qualification typically involves a minimum of three con- secutive, separate, and successful media fills. The definition of successful has evolved from allowing one contaminated unit out of 1000 containers to having zero contaminated units.

Periodic requalification of aseptic processes with media fills every 6 months applies to every filling line, every product container configuration, and every aseptic process operator. Sometimes a requalification media fill will need to be conducted in intervals of less than 6 months if environmental monitoring data start failing acceptance limits, if there is personnel change, if a major manufac- turing deviation occurred, or if equipment changes or modifications take place. A valid change control procedure needs to be in place to ascertain when changes in the manufacturing environment require a new media fill qualification.

Ideally, the maximum batch size should be simulated in the media fill. Practically, the number of units filled with media must be sufficient to reflect the effects of worst-case filling rates, including operator fatigue and the maxi- mum number of interventions and stoppages. While 3000 vials are needed to detect with 95% confidence a contamination rate of one in 1000, using 3000 vials as a minimum number of units to be filled is no longer considered to be sufficient. The current regulatory position is to fill a minimum of 4750 units three consecutive times with zero positives. One positive in each of three con- secutive runs is viewed as a serious process control problem. The International Organization for Standardization (ISO) has developed acceptance criteria (the maximum acceptable contaminated units in a media fill run) as a function of the number of media fill units.

Following a periodic requalification media fill, production may resume while media fill units are incubating. No product can be released until media fill data are analyzed and acceptance criteria are met, however.

Each lot of media must pass a growth promotion test (10 to 100 CFUs per container) following the media fill run and again after the end of the incuba- tion period. Typically incubation of media involves a period of 7 days at 30– 35°C to detect bacterial growth, followed by 7 days at 20–25°C for molds. Incubation conditions must be justified based on favored growth conditions for common environmental isolates. Prior to incubation, each unit must be in- spected, with any leaking or damaged units removed. Each unit also must be rolled or inverted prior to incubation for media to contact all interior surfaces of the container.

Media fill failures can and do occur. Standard operating procedures must be in place to provide action steps in case of a media fill failure. Typically, if one of three runs fail, the entire sequence of three separate, consecutive media fill runs is repeated unless a clear assignable cause can be given to the failed

media run. If a requalification media fill fails (one run), there should be criteria in the procedure to determine whether a single repeat run or a repeat of the initial qualification (three runs) should be done. All documentation involved in a production process (e.g., environmental monitoring data and trends, personnel monitoring data and trends, sterilization charts, HEPA filter certification, filter integrity test data, handling and storage of all equipment) must be reviewed after a media fill failure.

For an aseptic filling process the level of sterility assurance is a cumula- tive function of all the unit operations involved in the entire manufacturing system. The final level of sterility cannot be greater than the unit operation providing the lowest probability of sterility. Adherence to a program that will enable the validation of all steps in the aseptic process from the solution prepara- tion step to the final container closing/sealing step will provide the highest as- surance possible that all steps of the process are collectively functioning and controlled to yield a product that is microbiologically safe.

I. Other Sterile Process Systems Requiring Qualification

and Validation

Historically, and even today, emphasis on the validation of sterile products is placed mainly on the sterilization processes. No manufacturing operation can be considered under complete control without qualification of every system that can potentially affect product quality, however. The following discussion will touch upon other systems and processes involved in sterile product manufactur- ing expected to be validated. Much of this section relies on the following litera- ture sources [Refs. 43,73–80]. Also refer to other chapters in this book that discuss certain topics in much greater detail.

J. Facility Design and Construction

The Good Manufacturing Practice (GMP) regulations, FDA, and European Eco- nomic Community (EEC) guidelines on aseptic processing, and other documents provide comprehensive details on facility requirements for sterile drug produc- tion. The facility must

1. Use HEPA filters for filtering the air supply to reduce or eliminate particulate contaminants

2. Maintain higher air pressures (positive pressure) within the critical areas to minimize infiltration of airborne contaminants from outside air 3. Provide smooth, easily cleanable surfaces on equipment, floors, walls,

and ceilings to minimize the opportunity for collection of particulates and growth of micro-organisms

4. Provide temperature and humidity controls appropriate to the product being manufactured

K. Utility Qualification (seeChapter 12)

Facility design is critical. Likewise, individual utilities require qualification. The most important of these are heating, ventilation, and air conditioning (HVAC), water (including clean steam), and compressed gases.

Typical programs begin with installation qualification (IQ). The IQ is de- scribed in a written protocol that contains the following key elements:

1. Equipment or system specifications 2. Spare parts list

3. As-built drawings 4. Wiring diagrams 5. Piping and installation

6. Installation certification statement

Following completion of the IQ, the equipment or system is subjected to operational qualification (OQ). This is a more rigorous exercise in which the object is to ascertain that the equipment or system being tested performs in accordance with design specifications throughout the full operational range(s). The OQ protocol contains

1. A full system description

2. Calibration certification documents 3. Testing plans

4. Acceptance criteria

5. Full record of testing results 6. Certification statement

1. Heating, Ventilation, and Air Conditioning (HVAC)

Features of the HVAC system that affect product quality (sterility) and therefore require qualification include

1. HEPA filter integrity 2. Airborne particle control 3. Airflow direction

4. Room air pressure differentials 5. Temperature and humidity control

A popular method for certifying the integrity of the filter installation uses a polydisperse aerosol, created by blowing air through liquid (e.g., poly-alpha- olefin) introduced into the upstream ductwork, followed by scanning the entire downstream side of the filter face and periphery with a probe nozzle of an aerosol photometer. This testing will identify “leaks” caused by damage due to

mishandling or faulty construction. Small leaks can be repaired with a suitable silicone-based compound without removing the filter.

The importance of maintaining air pressure differentials in the enclosures of the aseptic suite within the ranges specified in the design plans cannot be overemphasized. Reversal of airflow, which can occur if the relative room pres- sures are upset, can allow contaminated air from a noncontrolled region into the clean room, thus defeating the purpose of the HEPA-filtered air supply.

Most enclosures in the aseptic processing suite are not airtight because of the need for conveyor lines and pass-through openings, so there is a very real opportunity for contamination from the noncontrolled adjacent manufacturing areas and particularly from overhead uncontrolled technical areas.

Special monitoring devices known as Magnahelic or Photohelic gauges measure the pressure differentials across a diaphragm and depict the value in terms of inches of water or some other convenient scale. These instruments are very accurate and sensitive to very small changes in pressure differential. Typically they are connected directly to an alarm system that will cause a visual signal (flashing light) or an audible signal (alarm buzzer) and/or trigger a recording de- vice to report a deviation outside a prescribed range of pressure differential. 2. Water

Water quality is usually defined in terms of chemical and bacteriological purity, particulate matter content, and endotoxin levels. Potable water is normally from the municipal water system, which may have been treated with chlorine to con- trol microbiological growth. Soft water and deionized water have undergone ion exchange or similar treatment to eliminate unwanted ionic species, such as Mg2+ and/or Ca2+. Purified water, water for injection, and other types of water meeting compendial specifications are produced by ion exchange, reverse osmosis, dis- tillation, or a combination of such treatments.

The validation protocol provides a detailed description of sampling loca- tions and requirements, testing methodology, and test limits or specifications. Sampling and testing can be performed daily during qualification and validation. When the system is in routine use, following the validation the testing frequency can be reduced to a weekly schedule for monitoring purposes.

An action guideline of not more than 10 CFUs/100 ml for bacteriological purity is suggested. As with the purified water system, the sampling and testing frequency for the water for injection (WFI) system is defined in the protocol and can be reduced after the system is qualified and validated.

3. Compressed Gases

Various kinds of compressed gases (e.g., nitrogen, oxygen, and carbon dioxide) may be found in the sterile drug manufacturing plant; however, as an example only compressed air will be discussed.

Compressed air is one of the utilities that may have direct or incidental product contact and therefore requires qualification. The types of contaminants found in compressed air, not surprisingly, are the same as those found in the ambient environment. These may include micro-organisms (e.g., bacteria, molds, and viruses), moisture, particulate matter, and possibly pyrogens. Undesirable levels of hydrocarbons from compressor lubricants may be found if the compres- sor is not of the oil-free type.

A well-designed compressed air system eliminates or substantially reduces the levels of these contaminants. Components of such a system include the following:

1. An oil-free compressor—typically a rotary screw, multiple-stage de- sign

2. An oil-coalescing filter to trap any liquid hydrocarbons or water 3. A dryer to remove condensed moisture and reduce levels of gaseous

hydrocarbons

4. A filtration unit to eliminate gross particulate matter, such as fibers and metal particles

5. A sterilizing filter rated at 0.2µm

6. A sanitary design receiver tank and distribution piping sloped for proper drainage

7. Instrumentation suitable for monitoring the temperature, pressure, and volume or flow rate in the system

Installation and operational qualification work includes verification of tempera- ture, pressure, and flow rates, instrument calibration, and thorough flushing of the entire system to remove oil, metal particles, and other contaminants. The type of testing and acceptance limits listed in the validation protocol may vary from firm to firm; however, compressed air with product contact should be tested for such quality attributes as hydrocarbons, water vapor, and microbial content (typically less than 0.1 CFU/cu. ft.)

L. Equipment Qualification/Validation (seeChapter 13)

1. Container Preparation

Parenteral drug containers are typically fabricated from glass (bottles, vials, sy- ringes, or ampules) or plastic (bottles, bags, vials, or syringes). Regardless of the nature of the container, contaminating substances such as paper fibers, glass fragments, viable microbes, and pyrogenic materials must be eliminated from the containers before they are used in the filling operation.

The suitability of the design and utility services is established during the IQ and OQ phases of qualification discussed earlier in this chapter. Important criteria for a typical washer include the following:

Water: quality, temperature, pressure, and flow rate Steam: quality and pressure

Compressed air: quality and pressure

The duration of the prewash, washing, final rinse, and flush cycles must be established during validation and maintained within suitably narrow ranges to ensure repeatability.

One practical approach to validating the cleaning process is to establish a known level of challenge contaminant, which is applied or “spiked” into num- bered or otherwise identified containers, which then undergo a typical cleaning cycle. Typical contaminants include visible and subvisible particluate matter and chemical, microbiological, and pyrogen challenges.

After the wash cycle, the spiked container is evaluated by suitable testing to determine the amount of residual contaminant. The “before” and “after” num- bers can be compared to establish an efficiency number based on the original level of contaminant.

Bioburden loading levels were determined by a membrane filtration proce- dure prior to washing and also after the spiking to confirm that the desired challenge level was achieved. Following the cleaning cycle, the same procedure was used to evaluate residual bioburden. To recover the residual contaminants, sterile peptone water USP is used to rinse the entire inner surface of each vial. Results are reported as CFU per vial.

Pyroburden was determined by validated limulus amebocyte lysate (LAL) techniques both before and after treatment in the washer to confirm pre-existing and challenge levels. It is expected that pretreatment pyroburdens will be low, and removal of a known challenge of pyrogen in the cleaning will be low. Removal of a known challenge of pyrogen in the cleaning procedure provides assurance that subsequent dry-heat depyrogenation will eliminate any pre-exist- ing contamination.

2. Closure Preparation

The most common type of primary closure used in conjunction with glass con- tainers for parenteral drugs is the elastomeric closure. As with the container itself, the closure must be sterile, pyrogen-free, and free from contaminants that could adulterate the drug substance, because the closure is likely to be in direct contact with the drug at some time during the storage, handling, or use of the dosage unit.

A number of undesirable substances could be present on the surface or sorbed into the matrix of the closures, but the predominant contaminants are particles of the closure matrix itself, other rubber compounds, metallic particles, micro-organisms, endotoxins, and template lubricants, which are usually organic in nature. In addition, various extractable substances used in the formulation of

the elastomeric closure can present problems. These extractable substances in- clude such activators as ZnO, MgO, and stearic acid; such curing agents as sulfur and phenolic compounds; such accelerators as amines and thiazoles; and such antidegradants as dithiocarbamates and various ketones and aldehydes.

Closure sterilization, following the cleaning cycle, is typically done by autoclaving with saturated steam. The temperatures achieved in such treatments are not sufficient to eliminate significant endotoxin contamination.

The validation of any cleaning procedure must therefore include testing for residual endotoxin, particulate matter, and any adventitious contaminant de- termined during the pretreatment examination. Achieving sterility during the cleaning cycle is not an absolute requirement; however, the bioburden remaining should not present a significant challenge to the subsequent sterilization process and should be considered in the development of those treatments.

Many manufacturers use equipment that combines the steps of washing, siliconization, and sterilization in a continuous operation. Such a treatment is desirable because it minimizes the time the closures are held in a wet condition. If sterilization does not follow the washing step immediately, the components must be thoroughly dried to eliminate the likelihood of microbial growth and/ or formation of pyrogens. Closures should be handled in such a manner as to minimize the potential for contamination from the cleaning operation through the filling and sealing steps.

3. Filling Equipment

Validation protocols for filling accuracy should specify the number and duration of filling runs for each size and fill configuration, the filling rates, and the limits for filling variability considered acceptable to the manufacturer. The purpose of the validation work is to determine a filling configuration (i.e., line speed, fill quantity, and container size combination) that will provide the optimum line speed while maintaining acceptable filling variability. Generally, the higher the filling rate, the poorer the filling accuracy.

4. Sealing/Capping Equipment

Adequacy of the container-closure system is determined through stability studies during the development work and is not the subject of the validation project for the equipment. It is the objective of this phase to demonstrate that the sealing/ capping equipment will consistently apply the overcap in such a manner that the integrity of the unit is ensured.

Container-closure integrity studies also can be conducted to validate the sealing efficiency of the capping equipment.

5. Lyophilization (seeChapter 9)

During the OQ the following specialized checks should be conducted: Maximum chamber vacuum under no load

Chamber leak rates under vacuum and pressure Shelf temperature control (i.e., temperature variation) Vacuum pumping rate

Chamber heating and cooling rates under no-load conditions to establish a reference point for future study

Condenser cooling rate

Refrigerant integrity test to verify that coolant does not leak into the chamber

Condenser drying rate to establish the maximum drying rate of which the unit is capable

Stoppering mechanism functionality to verify that the mechanism will properly insert the vial stoppers over the entire range of vials to be used In addition to the product specifications other attributes peculiar to lyophilized products should be verified. These may include: uniformity of cake, cake color, cake height, reconstitution time, moisture content (if not a product specifica- tion), and short-term (accelerated) and long-term stability.

Validation of the lyophilizer cleaning and sterilization processes should be accomplished. Particular care should be taken to verify that there is no back- migration of contaminants, whether from adjuvant fluids integral to the equip- ment of by cross-contamination from previous product. Typically, an overkill