Validation of Sterile Processes and Concept of Barrier Isolators

 

Sushruta Mulay* and Anubha Khale

H.K. College of Pharmacy, Jogeshawari (W), Mumbai.

*Corresponding Author E-mail: sushruta.mulay@hkcollege.ac.in, scientific.cell@hkcollege.ac.in.

ABSTRACT:

Current article reveals some of the important validation methods and the concept of Barrier Isolators which potentially control parenterals manufactured mainly by aseptic processing. Sterility in the absolute sense cannot be achieved even though greater levels of assurance can be achieved with current technology. All sterilization methods require validation and during validation they should demonstrate that products meet the required sterility assurance level, remain in function and are safe for use. Understanding the scope of this testing, the applicable standards and their application to the product produced with the designated sterilization methods are the essential requirements of validation. Thus Sound Validation Programme is an essential requirement of a company which affords a level of confidence in quality of products as there are fewer rejects, retests and reworks because of validation.

 

KEYWORDS: validation, sterility assurance, class 100 area, barrier isolators, unidirectional airflow devices.

 

 

INTRODUCTION:

Validation is established documented evidence providing a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes¹. Validation of sterilization procedures is done pertaining to the design of equipment, process used and confirmation with reproducible data on completion of process. Though concepts such as Quality By Design, (QBD), Process Analytical Technology (PAT) Quality Risk Management(QRM) and various statistical processes control methodologies may be advantageous but their application in sterility assurance is still in limitation². Effectiveness of each sterilization process has limitations. Microorganisms exhibit resistance to sterilization processes. Resistant spores require additional treatment designed to provide margin of safety against sterilization failure. All sterilization processes filtration, thermal, chemical and radiation must be validated. Validation has become an integral part of regulatory requirements as there are rigorous environmental expectations associated with sterile dosage forms.

 

Sterility assurance relates to process capability and is a routine procedure in sterile products manufacturing units. It is also an essential part of CGMP (Current Good Manufacturing Practices) involving production, quality control, quality assurance and engineering department. Following validation during routine production some testing is required but often manufacturers are not sure of what testing is required, the frequency necessary and how much flexibility they have in the testing. Environment control systems, personnel, equipments, sreilisation methods and water systems are the main areas where validation is required.

 

 

In validation process various microbiological and product tests are required. These tests must be compliant with recognized standards. Basically these tests involve bioburden, bacterial endotoxins, residual analysis, functional tests and sterility tests. Currently there are a variety of rapid microbiological techniques that are commercially available which significantly reduce testing time, minimize in process inventory  and better control over manufacturing processes. Seperative technologies such as Barrier Isolators, Restricted access barriers (RABS) and automation systems decrease personnel reliance. As aseptic process is carried out in classified and controlled environment, aseptic techniques are used to control level of microbial contamination to a degree that microorganisms can be excluded from the product during processing.

 

1.       Validation of environmental control systems :

 

Fig-1    Layout of aseptic area

 

In aseptic process all the operations are carried out in aseptic environment. The environmental control system plays an important role when products are aseptically manufactured (fig-1) where sterility assurance is must. Such concentration of airborne nonviable contamination (particulate matter) is to be considered as the main environmental parameter. Aseptic area is divided into two sections:

 

Critical area : It  is a core area (class 100 area) in which  there are not more than 100 particles per cubic feet of air of 0.2 micron and larger in size are present. In this area aseptic manipulations such as aseptic connections, addition of sterile ingredients and filtration of parenterals take place.

 

Supporting area:  This is a less critical area compared to core area in which non-sterile components, formulated products, in-process materials, equipments and accessories are cleaned and prepared. These environments are soundly designed where they minimize the level of particle contaminants in the final products and control the disburden of articles and components that are subsequently sterilized. It is again divided into Class 10000 area and Class 100000 area. Class 10000  is adjacent to core area  also called as clean room area in which not more than 10000 particles of 0.2 micron and larger in size per cubic feet of air are present. It involves operations such as products manufacturing, washing of ampoules, vials or plugs. In class 100000 area there are not more than 100000 particles of 0.2 micron and larger in size per cubic feet of air are present.

 

Air Handling Units (AHU): It is an essential part of aseptic area responsible for moving, filtering and conditioning the air to be distributed throughout controlled environment.

The main components of Air Handling Units (AHU) are,

1.        Blowers or fans: These deliver airflows at specified pressures to every point of use throughout the system. It is with a positive pressure 1.5mm of water.

2.        Heating and cooling coils: These are used to condition the air to every point of use throughout the system.

3.        Air filters: These control airborne contaminants to pre specified levels.

4.        Dehumidifiers: Humidity is controlled at specified level.

 

HEPA filters (Unidirectional airflow devices):

These are High Efficiency Particulate Air filters installed at both supply and return points in aseptic room in order to provide a clean, unilamellar flow with a positive pressure throughout in order to continuously sweep the particles coming in between. They function primarily by sieving the particles of any kind which are retained on the surface. The clean and aseptic air is distributed that it flows at the greatest volume flow rate thereby producing the positive pressure in these areas. This prevents unclean air rushing from temporarily opened doors. The air is controlled with respect to temperature and humidity. The air is exhausted from the opposite side. The air velocity is 100ft /min as optimum. They are capable of removing the particles in size greater than 0.2µm.The efficiency of these filters is 99.97%. Prefilters or intermediate filters are used which reduce the particle burden on main filters in order to achieve the maximum efficiency of the filters. A pre filter is made of glass fibers and able to retain 97% of particles of size 5µm or greater. The risk of contamination during aseptic processing or sterility testing is low. It is widely used as it increases the rate of filtration³.

 

Integrity Test:

This test is done to check integrity of filters. Dioctyl Pthalate (DOP) in aerosol form is introduced through surface of HEPA filter. It passes the test if deflection in the needle is more than 99.97%.  If there are any cracks in the surface the test fails as the filter is not capable of filtering 0.2 micron particles. This test is done twice in a month.

 

Airflow velocity and uniformity test:

Environment is divided into 60 x 60cm grid. Air velocity is measured using thermal or vane type anemometer. Reading is taken at each square. The airflow velocity should not exceed 15 to 20% from the unit average velocity.

 

Airflow parallelism test:

Air should flow in a parallel stream to prevent the flow of outside air into critical environment. This test determines prelims of the airflow throughout the work zone. The visible smoke is generated upstream from the work zone  and a reference point is established by using a plumb bob . The smoke is also generated over manufacturing environment. The direction of airflow is videotaped and is determined by comparing with direction of normal air flow from HEPA filter.

 

Temperature and Humidity control tests:

Temperature and relative humidity levels are changed according to seasonal conditions. In aseptic processing areas the normally system should be capable of maintaining temperature range 18 to 20^oc and relative humidity 45 to 55 %. Environment is divided into 60 x 60 cm grid. A multiple point chart recorder thermometer is used to record the temperature. Relative humidity is measured by dry and wet bulb thermometer.

 

Environmental microbiological monitoring:

This includes air, surface and personnel in aseptic production areas as well as sterility testing room.  The personnel working in these areas transfer microorganisms from one place to another through water droplets, dust particles, fibers, gloves or gowns. Organisms are carried from one place to another by air current. The effectiveness of HEPA filtration, disinfection procedures and aseptic handling procedures is monitored. The controlled areas are supplied with HEPA filters (vertical or horizontal) having 99.97 % efficiency. These are class 100 areas. These include aseptic processing filling and bulk manufacturing units. The frequency of testing, direction of air flow in HEPA, number of people working in the room, amount of activity, time of testing, media used for incubation, time and temperature of incubation decide alert and action levels.

 

In air testing active samplers are used to draw an air and collect organisms from it . The efficiency depends upon nature of collection medium, size of sampling port, the sampling rate and volume of air taken and sampling time. Air is drawn by vacuum through a thin slit and is collected on a 15cm diameter agar plate. The measured volume of air is sampled. The plate rotates for a specific amount of time .These are quantitative, easy and a probe can be used for remote and smaller areas. In passive samplers agar exposure plates are used. Organisms floating on the air get settled on these plates. Settling plates (gravity exposure plates) are qualitative methods. For surface testing plates of 5cm diameter agar are dome surfaced.  No. of microorganisms per square inch is detected. In finger swab test Cotton swabs are wetted with isotonic solution and incubated. Irregular and small spaces can be covered. Alert levels for process surfaces at 1 to 2cfu per test and action levels are 3 to 5cfu per test.

 

2. Validation of sterilizing grade filters:

The purpose of sterile filtration validation is to prove that a particular filtration process generates a sterile filtrate. This is achieved by choosing a sterilizing grade filter that is compatible with the process, nontoxic, integrity testable, sterilizable, that does not adsorb formula components or add extractable to the process and can remove the bioburden associated with the product.⁴ The filters are one of the most critical components and provide sterility assurance of the final product. Therefore the filter requires stringent controls to assure consistent and reproducible results.  The purpose of filter validation is that filter will reproducibly remove microbial disburden and particulate matter. The industry accepted rating for a sterilizing grade filter is 0.2 or 0.22 µm. depending on the manufacturer, which is validated as capable of removing.10⁷ cfu/cm² B. diminuta organism under certain extreme processing conditions. Flow rate, throughput, retention, filter inertness, any change in drug product processing conditions, concentrations may change the performance of filter. The validation study involves three basic parameters. Physical parameters involve particle retention, shedding of fibers and sterilization integrity. Inertness, consistency of filter and are the chemical criteria’s and Microbial retention, endoxins level and toxicity should be considered as a biological tests. While testing these parameters viscosity, surface tension, pH, density, ionic strength, osmolarity should be considered and controlled.

 

Integrity testing:

This test is done to check integrity of filters. It  is done routinely performed post use. Failure of the test indicates faulty sterilization. Bubble point and diffusion methods are used. Bubble point test is dependent upon vehicle surface tension and wetting contact angle with a membrane. 50 PSI pressure is considered optimum pressure for bubble point test.  Integrity test ratio is the ratio of value obtained with drug product to that of specified liquid. Ratio 1 is considered to be optimum. It is taken at particular pressure and temperature and time. Diffusion test depends upon solubility and diffusibility of the gas in liquid vehicle. Wetting fluids, test gases are used.  Time validation is important as in long processing times bacteria die on the filter and endotoxin levels are increased and start penetrating through filter matrix.  Operating temperature has a significant effect on filters ability to withstand differential pressure.   Sterilization temp is considered for sterilization of filters.

 

Inlet pressure is responsible for structural damage. Differential pressure across the membrane depends upon temperature, direction of applied pressure, sterilization pressure, hydraulic pressure (stress) . Surface area of filter (size of filter) is responsible for throughput and flow rate.

 

Particulates testing:

It is done by optical microscopy, scanning electron microscopy and light obstruction methods. For Large volume parenterals Nmt 25 particles  per ml of ≥ 10 µm, Nmt 3 particles  per ml of ≥ 25 µm, For small volume parenterals Nmt 6000 particles  per ml of ≥ 10 µm, Nmt 600 particles  per ml of ≥ 25 µm.  Fibers of no longer than 1 µm are allowed. Fibers greater than 2.5 µm should be eliminated.  Asbestos filters (Seitz filters) are used for prefiltration followed by membrane filters.

 

Microbial retention  test :

It is critical test in validation of filters. B. diminuta organism (Test organism) has a mean diameter of 0.3µm used for 0.2µm filter. Passage of bacterial colony on an assay filter shows the non retentitivity of the filter.  A test is not applicable for vaccines.

 

Other tests:

As filters should be inert trace elements of filter extractable are detected by analytical methods. pH and conductivity tests are done immediately before and after filtration. Oxidisable substances, gravimetric extractable, weight change test is done and nonvolatile filter extractable are measured. After measuring dry weight filters are soaked in drug product and water as a control is used. Soaking time mimics manufacturing conditions.  After soaking filter is dried to constant weight and checked against original weight. In total oxidisable carbon determination conductivity of bicarbonate ion (HCO3^-) is checked by Toc analyzers. Resultant CO₂ is measured. As adsorption causes loss of drug product, leading to reduced activity and stability, drug product is analysed immediately before and after filtration. Stability study is performed on samples after filtration. As filter should not add any endotoxin to the product. Bioburden can be reduced with good aseptic technique to minimize source of endotoxins for reusable filters. Extractable from any component of filter assembly should be quantified.

 

3. Validation of Ethylene Oxide sterilization process:

In ethylene oxide gas sterilization concentration of ethylene oxide gas, relative humidity, temp and time of exposure is important for effective sterilization. During ethylene oxide gas sterilization gas interacts with materials processed by reaction, absorption or adsorption .Ethylene oxide gas is also trapped in the air spaces within the product. Unreached residual gas is rapidly removed through evacuation and air exchanges. Ethylene gas concentration used is 400mg to 600mg per liter. The following tests are important in ethylene oxide validation,

• Bioburden

• Bioburden Validation for Recovery Efficiency

• Test of Sterility

• Bacteriostasis-Fungistasis – Validation of a product test of     sterility

• Ethylene Oxide Residuals

• Bacterial Endotoxin Test

• Inhibition Enhancement – Validation of the BET

 

Most often, the validation of an ethylene oxide cycle follows the half-cycle or “over-kill” method using Biological indicators (BI’s) and product. This method demonstrates that the resistance of the microbiological challenge test system is equal to or greater than the product bioburden. The appropriateness of the Biological Indicator (106 Bacillus atrophaeus) should be evaluated. This can be done by characterization of the natural bioburden and using this information to determine D-values; determining that the bioburdenlevel is ≤100 CFU (colony forming units), indicating a lesser challenge than the BI; or, if characterization is not performed and the bioburden level is >100 CFU, by performance of a fractional exposure cycle using BI’s and product, followed by testing for comparison of any positive response yielded from the BI’s vs. the product. A half-cycle, the minimum exposure to ethylene oxide which yields no surviving microorganisms with all process parameters – except time – remaining the same, demonstrates a 6 log reduction. Two additional half-cycle experiments are performed as confirmation. A full cycle, providing a 12 log reduction (10-6), is performed for product release purposes.

 

The analytical methods are either gas chromatographic, IR spectroscopy or microwave detectors. These instruments are installed directly to sterilizers. Periodic gas samples are withdrawn from sterilizer or gas circulation lines and passed through the detectors. Some IR or microwave detectors are mounted on exterior chamber wall using an access port or in gas circulation system. Electronic signals are sent to control valves in the gas supply lines allowing makeup charges to maintain target gas concentration. Adequate moisture content is required for effective sterilization. It makes the reactive sites in microbial cells available for alkylation action of ethylene oxide. When the cells or the spores dry their proteineous and nuclear material and their active sites are physically withdrawn making reaction with ethylene oxide difficult. When these materials are hydrated they swell and expand. This exposes active sites and makes them available to alkylation by ethylene oxide. In indirect method the differential pressure is measured where pressure change takes place due to vapor pressure of water. In direct method electric hygrometer is used. These can be used inside the vessels. IR or GC systems require samples to be withdrawn from the sterilizer.

 

4. Validation of disinfectants:

Disinfectants destroy infectious microorganisms from the surfaces. Effective disinfection methods destroy vegetative forms of bacteria and fungi. Many times disinfection is not successful as disinfectants are not equally effective against all species of microorganisms. Bacterial endospores of gram positive microorganisms are highly resistant to many disinfectants. Microorganisms colonizing on a surface create a layer of organic materials serving as a matrix acts as a protective barrier which is very difficult to penetrate by disinfectants. PH, temperature and presence of particular ions have effect on resistance of microorganisms.  Formation of slimy layer around the cell (capsule) resists the cell permeability⁵. Encapsulated stains are more resistant to antiseptics and disinfectants than noncapsulated stains⁶. Various disinfectants used are ethyl alcohol, isopropyl alcohol, aldehydes such as formaldehyde, quaternary ammonium compounds, phenols and peroxides. Disinfectants are frequently rotated in order to avoid resistant strains of microorganisms. Verification of continued disinfectant effectiveness is done by inoculating surfaces of the same material as used in production room with a simulated microbial contamination. A contact plate method is used to show inactivation of contamination by a disinfectant. Qualitative evaluation is done or it can be studied by results of environmental monitoring. If the results are consistent and within the limits, it is assumed that disinfectant effectiveness is appropriate (table-1).

 

5.    Validation of steam sterilization system:

Steam sterilization or Autoclaving is the most effective and most efficient means of sterilization. It is frequently used method in sterilization of parenrerals. All autoclaves operate on a time/temperature relationship. The usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes⁷. If temperature in an autoclave does not reach 121°C, all spores will not be destroyed in the cycle time of 20 minutes. Validation is therefore required to ensure that the autoclave is operating correctly and that complete sterilization is being achieved (fig-2).

 

Fig 2  Horizontal Large Scale Autoclave

 

The design of sterilization system and process of sterilization are key parameters for effective sterilization process⁸. In steam sterilization microorganisms are killed by using a saturated steam under pressure. Temperature, steam pressure, exposure time, load density are routine validation measures. The design of sterilization system and process of sterilization are key parameters for effective sterilization process. In cultural methods the Bio Indicators (BI) are used to validate steam sterilization method. In dry type BI, filter paper, glass or plastic is used to which spores are added, and dried. The packaging material should show good penetration of steam. It is placed at the place which is least affected by sterilization. Test organism used is B. Sterothermophilius and incubation medium used is casein soyabean digest medium. It is incubated for 7 days at temp. 50 – 60ºC.⁹ In chemical methods test tablets made up of lactose, starch and magnesium trisilicate are used which melt at 115^oc after 24 minutes. In Browne’s tubes method a sealed glass tubes contain a red fluid which changes to amber to green on heat are used. In Bowie Dick test huck back towels of size 1m x 0.6m are used folded. On it two pieces of autoclave tape which contain heat sensitive bars at intervals at about 15 mm are stuck from corner to corner in shape of  x. and a pile of 0.25 m high is made in a drum  and  then placed in steriliser¹⁰. In the centre of pile a sheet of paper with high permeability is used. The autoclave tapes become colored throughout the strips of if steam has fully penetrated the pack. If any air remains the bars in the centre show light colour.

 

The thermocouples are placed at all possible points where condensates get accumulated and chances of lowering of temperature are higher. The use of a  temperature control system also eases the multipoint temperature measurements allowing more precise timings of sterilization cycle.

 

Concept of  Barrier Isolators:

 

A Barrier Isolator

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A barrier isolator (Fig-3) system is high-tech aseptic system which eliminates contamination associated with the filling and sealing of vials, bottles, and syringes.¹¹ Barrier isolation involves a closed filling and packaging environment, which is considered to be superior to aseptic processing. The need for barrier isolation technology is that there are many biochemical drugs, which cannot be terminally sterilized. For products that can't be terminally sterilized, barrier isolation technology gives users the confidence that the package maintains its sterility; hence it is superior, to conventional aseptic processes. These units are prefabricated, self contained chambers that are capable of being sterilized in situ and are designed to prevent the direct exposure of sterile products to personnel and the surrounding environments.  As Isolators eliminate one of the primary sources of contamination-people they are widely implemented in sterile manufacturing areas. Aseptic Isolator, provides a safe and clean environment for compounding of non-hazardous, sterile drug preparations and IV admixtures. Isolators may be situated in an area subject to less severe environmental controls compared with open fronted clean air devices. isolators are a cost-effective solution especially for lower-volume pharmacies.

 

They reduce operating and renovation costs, take up less space, and are easier to maintain. The work zone and pass-thru interchange are under positive pressure to the room in order to maintain sterility in case of a breach in the barrier isolation system.  They provide positively pressured HEPA filtered ISO class 5 environment with a fully integrated Hydrogen peroxide vapour generator. ULPA(Ultra Low Particulate Air) filters  can also be used in isolators which are 99.9995% efficient in removing particles of 0.12 microns  and larger provide superior ISO Class 3 air cleanliness, 100 times better than HEPA Filters.  Transfer devices are to be designed to avoid direct contact of the chamber with the surrounding environment during the introduction or removal of components that are needed in aseptic process. Integrity of isolation chamber must be verified after construction and monitored continuously during operation.  These systems are arranged in such a way to accept the introduction of sterilizing gas or liquid needed for the Sterilization In Place (SIP) or Clean In Place (CIP) of the chamber and the components within. Isolators can afford the degree of reliability with aseptic processing. It cannot equal the terminal sterilization process. The use of sporicidal agent in the decontamination of an isolator providing multiple log reduction (less than 12 logs) is effective at the elimination of microorganisms than what is possible in the clean room. In isolators product contact parts should be sterilized while others are needed to be decontaminated.

 

Essential features of barrier isolators are:

·         Main work zone remains sterile during insertion and removal of items because of Airlock pass-thru interchange.

·         Robust dual-wall construction.

·         All positive pressure work zones are surrounded by negative pressure plenums at the sides and back.

·         Ergonomically angled front and oval glove ports improve reach and comfort.

·         Safe-change cuff rings permit glove changes with zero risk of contaminating the work zone.

·         One piece work zone liner with no crevices is easy to clean.

·         Antimicrobial coating on all painted surfaces minimizes contamination.

 

Sharp disposal system and hydraulic height-adjustable stand  facilitate function of isolators.

 

Requirements for validation in Isolators:

Sterility assurance in isolators depends upon proper installation, operation and performance of isolators. As installation and operational structures are similar to those in aseptic room, the tests such as integrity testing of HEPA or ULPA, pressure maintenance are the same. As these systems are specially prepared with connections to accept the introduction of sterilizing agent leak testing is important. Bio indicators are located in sufficient numbers inside the controlled environment to demonstrate lethality and penetration of sterilizing agent. At all critical locations within the unit. The use of chemical agents and pressurization of chamber are used as STD tests for integrity.

Residuals:

Hydrogen peroxide gas in concentration greater than 4%  is widely used for the decontamination of isolators. Hydrogen peroxide is a sporicidal oxidizing agent. A safe level of sanitizing agent residual is needed to be validated. Residues on product contact material are measured directly.  

 

Material

Issues:

The sterilizing agents are highly reactive chemicals it is important to recognize adverse material effects. In order to attain a greater kill the dwell period where materials are in contact with the agent is extended. Long exposure times have a negative effect on materials with contact.  Corrosion of metal surfaces, brittleness of polymers takes place. These adverse material effects should be evaluated by suitable methods.

 

Aeration:

Establishing the aeration period at the conclusion of treatment step is important as this ensures that materials exposed to that agent are not adversely affected by the treatment. Before activities in isolator, required to be given. Aeration is important in sterility testing where residual agent can impede microbial recovery.  The value of 5ppm of H₂O₂ at the end of aeration is fully acceptable. Aeration can be improved by increasing the air changes in the isolator. The purpose of H₂O₂ process is to kill residual bioburden in the isolator enclosure. Therefore the direct demonstration of spore killing effectiveness is the best way to confirm the effectiveness of H₂O₂ cycle. Chemical indicators are available that can demonstrate that H₂O₂ vapour is well distributed within the enclosure.  It is the qualitative method.  The conditioning phase  in cycle development that selecting appropriate  conditioning injection rates  so that the exposure phase decontamination  concentration can be reached  relatively rapidly.

 

CONCLUSION:

Thus it can be concluded that the reliance on end product testing can be minimized and a successful process validation effort builds quality into the process. Hence validation becomes a tool in success of product and the technical talent to recognize and solve problems is fundamental to validation.

 

REFERENCES:

1         FDA guideline on General principals of process validation May 1987.

2         Dr. James Akers, James Agalloco, Macho Raja Sage In Scientific aspects of aseptic processing pharmatimes vol 42 issue 10 page 16.

3          S J Carter dispensing for pharmaceutical students 12 the edition page 516.

4         Mcburmie Leesa and Bardo Barry -Validation of sterile filtration In Pharmaceutical Technology Filtration 2004 page 13.

5         Russell AD, Gould GW. Resistance of entero bacteriaceae to preservatives and disinfectants. J Appl Bacteriol symp Suppl 1988; 17:1678-95.

6         Costerton JW, Irwin Rt,Cheng K-J. The bacterial glycocalix in nature and disease. Ann Rev microbial 1981; 35:299-324).

7         Kenneth Todar-Control of microbial growth by www.textbookofbacteriology.net

8         Guy Snelling -Autoclave validation what is really required?  from internet.

9         Tsugo Sasaki and Fumi Yamamoto from Japanese regulatory requirements, page 692.

10      S.J. Carter -Dispensing for pharmaceutical students Twelfth edition page 431.

11      Karen G Beagley – Improving parenteral packaging from internet.

 

 

 

Received on 17.02.2011          Modified on 15.03.2011

Accepted on 21.03.2011         © RJPT All right reserved