4202:Design and Operation of Clean Room

DESIGN AND OPERATION OF CLEAN ROOM

Introduction

Generally clean room is designed to reduce the contamination of processes and materials and is accomplished by removing or reducing the sources of contamination.

Definitions

Clean Room

A clean room or area is a room with different environmental control of particulate and microbial contamination, constituted and used in such a way as to reduce the introduction, generation and retention of contaminants within the area.

In another word, a clean room is a room in which the concentration of air hold particle is controlled to a specified limit.

Aseptic area

Aseptic area is a room or special area within the clean room designed, constructed, serviced and used with the intension of preventing microbial contamination of the product during production.

Sources of contamination

The possible sources of contamination are as follows

1. External Sources

2. Internal Sources: It encompasses the followings

1. Atmosphere: This includes

1. Dusts
2. Droplets
3. Free Organisms

2. Operator: This includes

1. Breaths
2. The hands
3. Clothing
4. The hair

3. The working surface
4. Equipment’s
5. Raw materials

1. External Sources

For any given space, external sources exists externally and influences the gross atmospheric contamination.

External contamination is brought primarily through

Contamination Sources: •People ~75% •Ventilation ~15% •Room Structure ~5% •Equipment ~5%

1. Air conditioning system
2. Make up air

In addition, external contaminants can in filtrate through

• Building door
• Windows
• Cracks and wall
• Pipes
• The cables and ducts

Primarily the external contamination is controlled by

1. High efficiency filtration
2. Space pressurization
3. Sealing of the space of penetration

2. Internal Sources

A. Atmosphere

The atmosphere has no flora of its own. Because it can’t support the growth of microorganisms, but it is usually heavily contaminated with particles. Microorganisms are usually associated many of these.

The environment may become contaminated by the following materials.

1. Dust

A higher percentage of dust particles in the outside air come from the soil and therefore may carry soil borne bacteria. For e.g. Saprophytes and Cocci.  In addition, pathogenic anaerobic sporing rods are quite common.

Indoor air may also contain organisms of human origin such as Staphylococcus and
B Hemolyticus

2. Droplets

Large numbers of droplets are excreted from the respiratory tract by coughing and sneezing and may contain organisms from the nose, mouth, throat and lungs. In this way common cold, Influenza, viral diseases, TB etc are transmitted easily.

3. Free organisms

Bacteria and viruses free from dusts-droplets are uncommon, but naked yeast and mold spores are often abundant.

B. Operator

This includes the followings

1. Breaths

In normal breathing few organisms are passed into the atmosphere, because they are retained in the fluids lining in the respiratory tract. But coughing and sneezing can cause contamination at a considerable distance by expelling droplets of these fluids.
For e.g.  Staphylococcus aureas.

2. The hands

These are major means of transmitting infections. It has been estimated that, there are not less than 10,000 organisms per square centimeter (100000/cm2) of skin.

3. Clothing

Part of the atmospheric dust that becomes engulfed in the fiber of the fabrics is dislodged by the body movement and can considerably raise the level of contamination around a person who is working carelessly.

4. The hair

Hair is constantly exposed to the atmospheric dust and may liberate dust particles and flaky patches during such motion as the shaking of the head.

C. The working surface

This is a potential source of contamination because organisms will sediment on to it from the air.

D. Equipment

Sources of particles and microbial contamination may be inherent in the design of the processing equipment. Dust may also be generated by the equipment during use. Working surface and the external surface of the equipment are potent sources of contamination.

E. Raw materials

Raw materials may account for a high proportion of microbial and inert particticulate contamination included into the pharmaceutical products.

Raw materials come from four sources

Natural sources Bacteria Yeast Mold

Plant origin Bacteria Animal source Pathogenic bacteria

4. Water (A prime source of contamination)

Classification of Clean room

Clean rooms are classified based on the particle counts per cubic fit of air and are defined in terms of the maximum number of particle is in the range of 0.5 μ or larger than 5 μ or larger size.

Generally two systems exists for the classification of clean room

1. The British Standard
2. The Unites States Federal Standard  

These two systems are compared in the following table

Particle size in  Micron(μ)	Maximum no. of particle greater than the stated size
	British Standard		Unites States Federal Standard
	Class	No. per cubic fit	Class	No. per cubic fit
0.5	1	86	100	100
5		0		0
0.5	2	8,495	10,000	10,000
5		57		65
10		0.08		N/A
0.5	˟	˟	100,000	100,000
5		˟		700

Table: Comparison between British Standard and Unites States Federal Standard.

All clean room and working stations designed by this standard must be capable of meeting at least one of the following classes;

Class 100

Class 100 clean room is defined as a room in which the particle count in the air is not more than a total of 100 particles per cubic foot of the size 0.5μ and larger.

Class 10,000

Class 10,000 clean room is defined as a room in which the particle count in the air is not more than a total of 10,000 particles per cubic foot of the size 0.5μ and larger.

or 65 particles per cubic foot of the size 5μand larger.

Class 100,000

Class 100,000 clean room is defined as a room in which the particle count in the air is not more than a total of 100,000 particles per cubic foot of the size 0.5μ and larger.

or 700 particles per cubic foot of the size 5μand larger.

Air flow in a clean room or typical system of supplying air in the clean room

There are mainly four methods of controlling the atmospheric contamination.

Fig: Conventional air flow (Turbulent) and Unidirectional/Laminar air flow.

1. Conventional air flow

This is also known as turbulent air flow and is distinguished by its method of air supplying system.

2. Unidirectional air flow

This is also known as laminar air flow. In this system the air is supplied linearly from a high efficiency filter and passes in a unidirectional manner through the room.

3. Mix flow

This is a conventional type of air flow but where the product is exposed to contamination, a unidirectional cabinet or work-station is used.

4. Isolator or micro-environment flow

This is also used within a clean room to give the highest level of protection against contamination. The isolator is shown to have a unidirectional supply of air.

Conventional air flow

In the Conventional air flow system filtered air is pumped into the room to produce a positive pressure, compared to the exterior and in a turbulent fashion. The foreign particles already present may flash out and maintain a clean condition.

Air enters into the room through the filter in the ceiling or high up in the wall and leaves from the room trough carefully cited outlets or outlet ducts fitted in low down on the opposite wall or in the floor at a distance from the inlet.

Conventional air flow is defined in terms of the number of air changes per hour. The air flow should not be less than 2 poise per hour.

Unidirectional air flow (Laminar air flow)

Here the room is continuously swept by a filtered air. The total air within the room flows ideally with uniform velocity along parallel flow line which may be either horizontal or vertical.

UDA = Unidirectional Air-Flow

The air enters into the room through a bank of filter which comprises one complete wall or ceiling and exists through a bank of outlet.

Laminar air flow is defined in terms of air velocity.

A typical system for supplying air involves the following things

1. Intake of fresh air
2. Pre filtration
3. Temperature adjustment
4. Humidification
5. Final filtration

1. Intake of fresh air

The air should flow through an intake situated well above the ground level.

2. Pre filtration

This consists of a coarse filtration to remove the large particles from the air and protect the main filter.

3. Temperature Adjustment

The air is passed over a coil through which steam or refrigerant is circulated to permit the thermostatic control of the air temperature.

4. Humidification

The air is passed through a fine atomized spray of demineralized water to increase the humidity to an acceptable level.

5. Final filtration

Final filtration is achieved through a high efficiency particulate air (HEPA) filter positioned at or as close as possible to the inlet of the room.

Differences between Conventional and Unidirectional air flow system

	Features	Non-unidirectional(Conventional)	Unidirectional(Laminar)
1	Room Condition	The filtered air pumped into the room to produce a positive pressure.	The room is continuously swept by a layer of filtered air.
2	The air is pumped	In a turbulent fashion.	In a uniform velocity.
3	The air enters into the room	In a multidirectional pattern.	Along parallel flow lines, this may be vertical or horizontal.
4	Air flow type is defined	In terms of the number of air changes per hour.	In terms of air velocity.
5	Air is supplied	Through the bank of highly efficient filter.	By air diffusion or filter in the ceiling.
6	Air flow pattern	Is less than HEPA.	Is HEPA.
7	Turbulence	Creates significant turbulence.	Uniformly minimizes turbulence.

Air filtration

Filtration is an important aspect of clean room. Most of the filters are define by their particle removal efficiency and air flow rate.

Mainly two types of filters are used in the clean room

1. HEPA Filter
2. ULPA Filter

HEPA filter

High-Efficiency Particulate Air (HEPA), also sometimes called high-efficiency particulate arresting or high-efficiency particulate arrestance, is a type of air filter.

Filters meeting the HEPA standard have many applications, including use in medical facilities, automobiles, aircraft and homes. The filter must satisfy certain standards of efficiency such as those set by the United States Department of Energy (DOE).

To qualify as HEPA by US government standards, an air filter must remove (from the air that passes through) 99.97% of particles that have a size of 0.3 µm.

ULPA Filter

ULPA is an acronym for "Ultra Low Penetration Air (filter)". An ULPA filter can remove from the air at least 99.999% of dust, pollen, mold, bacteria and any airborne particles with a size of 100 nanometres (0.1 µm) or larger.

Mechanisms of Filtration

There are four mechanism of filtration

1. Straining
2. Impingement
3. Attractive forces
4. Diffusion

Straining

If the pores of the filter medium through which the fluid is flowing are smaller than the material, which is to be removed then the material will be retained. In this case filtration occurs in the surface of the filter.

Impingement

As a flowing fluid approaches and passes an object, for example a filter fiber, the fluid flow pattern is disturbed as shown in the following figure.

Suspended particles with in-sufficient momentum may not follow the fluid path, but impinge on the filter fibre and are retained due to the attractive forces between the particle and the fibre.

The effectiveness of the impingement process depends on the following factors:

1. Dust particle size: larger the particle, greater the inertia.
2. Density of the dust: heavier the particle the greater the mass and more inertia.
3. Depth of the filter: the thicker the media, the greater the number of times a particle will be exposed to the probability of capture.
4. Velocity of the airflow

Attractive forces

Electrostatic and other surface forces may exert sufficient hold on the particles to attract and retain them on the filter medium. For e.g. air can be freed from the dust particles in an electrostatic precipitation by passing the air between two highly charged surfaces which attract the dust particles.

Diffusion

Particles are filtered through the filter media due to the concentration gradient.

Filter Effectiveness

The ability of a filter to remove particles from the air is reflected by its efficiency rating. The American Society of Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE) has developed a standard for measuring filter effectiveness. The standard describes test procedures to classify filters in terms of arrestance and efficiency. Two terms are commonly used.

Arrestance is the amount of dust removed by the filter, usually represented as a percentage. Since large particles make up most of the weight in an air sample, a filter could remove a fairly high percentage of those particles while having no effect on the numerous small particles in the sample. Thus, filters with an arrestance of 90 percent have little application in clean rooms.

Efficiency measures the ability of the filter to remove the fine particles. ASHRAE efficiencies of between 10 percent and 40 percent should remove 20 percent to 40 percent of the 1µ particles in the air, but hardly any of the 0.3 to 0.5-µ particles. ASHRAE efficiencies of 80 percent to 95 percent can remove 50 percent to 70 percent of the 0.3-micron particles.

Filter efficiency test

The following tests are performed to evaluate the efficiency of the filter.

1. DOP testing
2. Two flow testing
3. Leak test
4. Methylene blue test

DOP testing

DOP or Dioctyl Phthalate is a liquid chemical which produces mono or poly-dispersed test aerosol of sub-micron particles, generated to challenge (evaluate integrity) of HEPA filters. In this test DOP is thermally vaporized in the upstream of the filter to produce an aerosol of particles which is detected downstream of the filter by using a suitable photometer.

Two flow testing

In this test an aerosol is produced from a solution of NaCl to generate a known concentration of NaCl particles of a controlled size, in the upstream of the filter. Retention efficiency is evaluated downstream of the filter by using Na- Flame photometry.

Fig: Photometer

Leak test

Leak test is performed by Bubble point test. In performing this experiment the membrane of the filter is wetted and usually the top of the filter is placed in contact with the liquid, the bottom with air, the filter holder is connected to a source of a regulated pressure. The air pressure is gradually increased and the formation of bubbles on the liquid side is noted.

Since the pores are full of liquid, there is no passage of gas at zero pressure. At pressures below the bubble point, gas passes across the filter only by diffusion, but when the pressure is high enough to dislodge liquid from the pores, bulk flow begins and bubbles will be seen. As the pressure is further increased, rapid bubbling begins to occur.

The initial bubble test pressure determines the size (and location) of the largest hole, the open bubble point pressure determines the mean pore size of the element. The latter can be affected by flow velocity as well as pressure.

Each membrane has a specific bubble point and at least a pressure of 20 psi (pound per square inch) is usually significantly detects the leaks.

Methylene blue test

Primarily this test is used for the high efficiency absolute filter. A methylene blue solution is atomized and the spray is discharged into an air stream which is passing through a duct containing the filter. The air arriving at the filter contains fine suspended particles of methylene blue.

The free and host (test) filter air is samples to bring out the colour. If no colour is present on the host filter air, then the efficiency of the filter is high. In case of absolute filter only 0.5-1% methylene blue may pass through the filter. This test is very costly because the filter paper may become entrapped very rapidly.

Fig: Leak test Apparatus (Arrangement of filter and filter holder)

Key issues to be considered in designing a clean room.

Cleanrooms and aseptic complexes should be designed for optimum flow of material, equipment, personnel, and waste streams so that raw materials and waste streams cannot cross over finished goods.

The following points should be considered:

1. Clarity about the GMP and Standard used.
2. Unidirectional air flow specifications and qualifications
3. Clean room classification
4. Air change rates and recovery time in non-unidirectional system.
5. Clean room configuration for the services and maintenance of the process equipment’s.
6. Validation of the building controlled system.
7. Real time particle monitoring system.
8. Retest and re-qualification frequency.
9. Bio decontamination by fumigation.
10. HEPA Filter led testing.
11. Floors, walls, and ceilings should be constructed of materials that are smooth, hard, and easy to clean.
12. Temperature and humidity controls in cleanrooms and supporting areas should be sufficient to maintain operator comfort when gowned and maintain adequate humidity for the process.
13. Cleanrooms should be designed to facilitate personnel, equipment, and material flows to prevent microbial contamination of sterilized or sanitized equipment and facilities, sterilized components, and sterile filtered drug product.

Necessities or purpose of clean room (The operation of clean aseptic room)

The aim of the aseptic technique is to prevent the access of microorganism during the preparation and testing of the pharmaceutical products.

There are several classes of pharmaceutical products for which the terminal heat treatment is not possible. For these preparations aseptic technique is used. For example

1. Thermolabile (Destroyed or changed easily by heat; unstable) soluble substance stable in solution

Solution of these substances can be filtered through a bacteria proof filter, but aseptic technique is required to prevent the contamination of the filtrate during the collection and while it is being filled & packed as well as sealed in the final container.
For e.g. Thyamine-HCl injection.

2. Thermolabile soluble substance unstable in solution

Filtration through a bacteria proof filter is impractible because these medications are not sufficiently stable in solutions for longer period. They must be preserved aseptically in a sterile solvent. For e.g. Chronic Gonadotropin injection.

3. Thermolabile suspension stable in vehicle

Example: Propyliodon and Propyliodon Oily injection

4. Thermolabile powder that require dilution with other powder

Example: Antibiotic dusting powder

5. Thermolabile powder that require incorporation of a semisolid base

Example: The eye ointment BP

Filtration is impossible for the last three cases and therefore the medicaments must be aseptically mixed with the appropriate sterile vehicle.

Personnel Protective Clothing

Cleanrooms and aseptic complexes should ensure that, personnel cannot move from less clean to cleaner areas without gowning.

The following rules are to be observed by all persons qualified to work in the Cleanroom:

1. Only personal qualified through the Cleanroom Access Training program are allowed to enter the Cleanroom.

2. No makeup shall be worn inside the Cleanroom.
3. Food and drinks are prohibited in the Cleanroom.

4. No smoking is allowed before entering the Cleanroom.

5. Clothing Requirements. Everyone must wear full-length pants and closed shoes, such as safety shoes (no sandals, no open-toed shoes). In addition to that suitable cleanroom gowning, e.g. head-caps and shoe-covers have to be used.

6. Only use pens. Pencils are not allowed in the Cleanroom.

7. Hair nets, shoe covers, safety glasses, mustache/beard masks, and gloves must be worn at ALL times.

8. Nonessential items (tools, books, backpacks etc.) must be kept outside the Cleanroom or in the gowning room lockers.

9. Try not to sneeze, cough or breathe directly on a clean surface or into the product area.

10. Do not let your skin touch any surface in the Cleanroom. Do not touch your face with your gloved hand. Do not touch the outside of a glove (except for the wrist edge) with your ungloved hand.

11. Always clean up your work area before you leave.

Fig: Gowning for clean room

Fig: Material Exchange / Air-Showers

Key factors in clean room operations
(Facilities of premises for the manufacture of parenteral products)

The preparation of sterile pharmaceutical products requires special facilities, designed to eliminate microbial and microbial and particulate contamination at all stages of manufacture.

1. Premises
2. Storage area
3. Bottle washing area
4. Laundry or Changing area
5. Preparation
6. Filling area
7. Sterilization area
8. Inspection and Labelling
9. Bonded area and release

Premises
The premises in which the manufacturing of sterile products take place should provide sufficient space to allow efficient flow of work through all the necessary operation.

Storage area

Storage space is required for raw materials, Empty containers, Packaging and labeling materials.

Weighing area

The main use of this area is to measure raw materials used for the product.

Fig: Design of a Typical Clean room

Bottle washing area

All bottles should be washed prior to entry into the clean room. Clean washed areas within the clean room are sometimes used for washing the ampoules, vials and various components just prior to use.

Laundry or cleaning area

Access to clean room or areas is permitted only via the changing area. The operators must exchange their outdoor clothing for working un the clean room.

Preparation

There are Clean room areas for the collection of water, Preparation of solution and final bulk product

Filling area

This is the most critical area. The product is filled into the final container via a filler of suitable size and then sealed and send out of the clean room area.

Sterilization area

If the product is not aseptically produced, it has to be sterilized in this area.

Inspection and labeling

Once the product has been sterilized, the contents are inspected for defects and then labeled.

Bonded area and release

Lockable area where the product is kept until it passes all the requirements of Q.C. After qualifying the requirements of QC &QA the product is released for use.

Test of clean and aseptic area

The tests applied to clean or aseptic room can be classified into two classes

1. Commissioning test

1. Final filter installation test
2. Induction leak test
3. Filter efficiency test
4. Particulate contamination controlling test
5. Air pressure test
6. Temperature and Humidity
7. Air flow test
8. Noise level test
9. Lighting test
10. Microbiological test

2. Monitoring test

Commissioning test

British standard 5295 list the tests and procedures which should be used to confirm that the room meets the required designated specifications. These tests and procedures includes the followings

1. Final filter installation test

This test is carried out to demonstrate that, the filter is not damaged, filter holding frame do not leak at the connection. This test can be performed by the bubble point test.

2. Induction leak test

This test demonstrates that, particle cannot enter into the room through the leak in the construction joint. This is carried out by measuring the size of the leak or by microscopic examination.

3. Filter efficiency test

This is also carried out by the bubble point test.

4. Particulate contamination controlling test

This is used to demonstrate that, the number and size distribution of particulates in the room air do not exceed the level specified for the particular class of room.

Microscopic examination and direct reading light scattering photometer are used for such measurement.

5. Air pressure test

This test determines the pressure difference between the clean room and adjacent area. This is usually carried by using a sensitive monometer.

6. Temperature and Humidity

This test demonstrates that the specified limit for temperature and humidity can be achieved, maintained and usually carried out by a psycometer.

7. Air flow test

This test is carried out to demonstrate that the air velocity or uniformity or laminar air flow in the room, comply with the required standard.

8. Noise level test

Noise level measurement is taken to determine the sound frequency or noise sound for equipment, especially for oscillary equipment. The measurements are taken at one meter from air inlet and at work benches.

9. Lighting test

The quality of the general illumination within the area and also at the work benches is measured by using a potable photoelectric photometer.

10. Microbiological test

This test is carried out to determine the number of the microbial contamination resulting from the introduction of the personnel, equipment or other things into the area.

Monitoring test

Monitoring test is carried out to ensure the performance of the room as well as to ensure that the designed conditions are maintained.

The test should be carried out repeatedly and regularly. The British standard recommends minimum interval for repeated testing.

Fig: Equipment’s for Room monitoring system

The testing parameters are given below

1. Air pressure, temperature and humidity measurements should be recorded continuously.
2. Particulate contamination should be determined daily for aseptic room and weekly for clean room.
3. Tests for air flow velocity and uniformity should be carried out at the three months interval.
4. Tests for filter efficiency should be conducted yearly. But, tests should be repeated after repair and maintenance.
5. It is recommended that, the settle plate, Agar plate, contact plate, sterility test and swab should be carried out during each workshop.
6. The process simulation should be conducted at three three months interval.

Cleaning methods

Clearly defined and vigorously enforced cleaning and disinfection procedures are essential elements in the operation of clean and aseptic areas.

The objectives of these procedures are to remove microbial and particulate contamination that arises in the area during normal use.

Recommendations

• Daily cleaning should take place immediately after the workshop ends and involves cleaning of all work surface and floor. Moreover ‘through cleaning’ should be arranged on a weekly basis.

• The cleaning materials should be selected to be compatible with the surface being cleaning and should not cause corrosion. Suitable cleaning agents are the alkaline detergents and non-ionic & ionic detergents.

• Cleaning materials should always be stored in dry place to prevent microbial growth and all cleaning solutions should be maintained at 65◦C temperature during use.

The cleaning method involves

1. Fumigation of the room with formaldehyde (HCHO) vapor at high relative humidity for a 12 hours period and is undertaken on a weekly or monthly basis, depending upon the extent to which the room is used.

2. Disinfectants: Sodium Hypochloride or Organochloride compounds at the concentration of    50-100 ppm; Quartarnary ammonium compounds at 0.1-0.2%W/V; 70% Ethanol or Isopropyl alcohol and 1% W/V Formaldehyde solutions. All these are suitable disinfectants for use in the work surface in the clean room.
3. Bactericides: They are used to remove bacteria.

4. UV light is passed through the areas to destroy the microbes.

REFERENCES

1. US Food and Drug Administration, Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice, 2004.

2. European Commission: Enterprise and Industry Directorate-General, EudraLex–The Rules Governing Medicinal Products in the European Union – Volume 4 – EU Guidelines to Good Manufacturing Practice – Medicinal Products for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products (corrected version), 2008.

3. International Organization for Standardization (ISO), ISO14644-4, Cleanrooms and Associated Controlled Environments–Part 4:  Design, Construction and Startup, 2001.

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