Cleanroom Classification (Ref: ISO 14644)

A cleanroom consists or either a single room or a number of interconnected rooms, where the concentration of airborne and work surface particles are known and limited to pre-defined levels in addition to the control of related environmental factors such as “viable and non-viable” particles, temperature, air pressure, airflow, …

Cleanrooms are widely utilized in the development and manufacture of pharmaceutical and biotechnology products, medical devices and sensitive electronics.

Is a “clean room” a regulatory requirement, what are the regulations?

Where a product being processed is non-sterile in nature, then a “clean room” may not be a necessity, instead a “clean area” as specifically defined by the processor may be defined. However for sterile processes then the regulatory requirements are clearer, for example, clean rooms are mandatory per Annex 1 of EU and PIC/S GMP’s, similarly the FDA defines clean room requirements for manufacturers of sterile products per the CFR’s (Code of Federal Regulations), specifically 21 CFR 201 and 211, current Good Manufacturing Practice for Finished Pharmaceuticals, 21 CFR 600 to 680 detail additional requirements for biological products. The FDA applied standard 209E (Airborne Particulate Cleanliness Classes in Cleanrooms and Cleanzones) up until 2001 until the standard was obsoleted and the FDA recommended the use of the International Standard ISO 14644 Classification of Air Cleanliness.

ISO 14644 consists of the following parts, under the general title “Cleanrooms and associated controlled environments”:

— Part 1: Classification of air cleanliness

— Part 2: Specifications for testing and monitoring

— Part 3: Test methods

— Part 4: Design, construction and start-up

— Part 5: Operations

— Part 6: Vocabulary

— Part 7: Separative devices (clean air hoods, gloveboxes, isolators and mini-environments)

— Part 8: Classification of airborne molecular contamination

Definition of a “clean area”:

ISO 14644: A room in which the concentration of airborne particles are controlled, and which is constructed & used in a manner to minimize the introduction, generation, and retention of particles inside the room and in which other relevant parameters (e.g., temperature, humidity, and pressure) are controlled as necessary.

As per WHO-GMP: An area with defined environmental control of particulate and microbial contamination, constructed and used in such a way as to reduce the introduction, generation, and retention of contaminants within the area.

As per USP (United States Pharmacopeia) Chapter 1116: A room in which the concentration of airborne particles is controlled to meet a specified airborne particulate Cleanliness Class. In addition, the concentration of microorganisms in the environment is monitored; each Cleanliness Class defined is also assigned a microbial level for air, surface, and personnel gear.

FDA regulations and their implementation:

The FDA demand environmental controls, which need to be considered from the initial clean room planning and development phases. There needs to be effective environmental monitoring, which includes humidity, dust, air pressure, temperature, microorganism controls, air changes per hour, air flow rates. There needs to be in place a system for air filtration. Additionally, there needs to be validated, documented and implemented procedures in place for preventing contamination, and for the cleaning and sanitizing of all surfaces and equipment.

Cleanroom Classification.

The ISO 14644-1:2015 standard details the classification of air cleanliness within clean rooms, in terms of the concentration of airborne particles.

Only particle populations having cumulative distributions based on threshold (lower limit) particle sizes ranging from 0,1 µm to 5 µm are considered for classification purposes.

The use of light scattering (discrete) airborne particle counters (LSAPC) is the basis for determination of the concentration of airborne particles, equal to and greater than the specified sizes, at designated sampling locations.

ISO 14644-1:2015 does not provide for classification of particle populations that are outside the specified lower threshold particle-size range, 0,1 µm to 5 µm.

ISO 14644-1:2015 cannot be used to characterize the physical, chemical, radiological, viable or other nature of airborne particles.

The classifications provided within ISO 14644-1, detail the counts associated with non-viable particles. There is no distinction between the processing of sterile or non-sterile products within a clean room, nor are there requirements for any parameter excluding non-viable particles.

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Reference: ISO 14644-1.
FDA’s Aseptic Processing Guidance: “Guidance for Industry, Sterile Drug Products produced by Aseptic Processing — Current Good Manufacturing Practice” provides a breakdown between the levels of permitted viable and non-viable particles associated within each clean room class.

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Reference: US FDA Aseptic Processing, cGMP’s. Cleanroom classification.
Notes to above table:

a- All classifications based on data measured in the vicinity of exposed materials/articles during periods of activity.

b- ISO 14644-1 designations provide uniform particle concentration values for cleanrooms in multiple industries. An ISO 5 particle concentration is equal to Class 100 and approximately equals EU Grade A (reference follow-on details).

c- Values represent recommended levels of environmental quality. You may find it appropriate to establish alternate microbiological action levels due to the nature of the operation or method of analysis.

d- The additional use of settling plates is optional.

e- Samples from Class 100 (ISO 5) environments should normally yield no microbiological contaminants

European Union GMP guidelines are outlined in document “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)”.

This document relates to the manufacture of sterile medicinal products. The guidance does not lay down detailed methods for determining the microbiological and particulate cleanliness of air, surfaces etc.

In general it states that:

The manufacture of sterile products should be carried out in clean areas entry to which should be through airlocks for personnel and/or for equipment and materials. Clean areas should be maintained to an appropriate cleanliness standard and supplied with air which has passed through filters of an appropriate efficiency.

The various operations of component preparation, product preparation and filling should be carried out in separate areas within the clean area. Manufacturing operations are divided into two categories; firstly those where the product is terminally sterilized, and secondly those which are conducted aseptically at some or all stages.

Clean areas for the manufacture of sterile products are classified according to the required characteristics of the environment. Each manufacturing operation requires an appropriate environmental cleanliness level in the operational state in order to minimize the risks of particulate or microbial contamination of the product or materials being handled.

In order to meet “in operation” conditions these areas should be designed to reach certain specified air-cleanliness levels in the “at rest” occupancy state. The “at-rest” state is the condition where the installation is installed and operating, complete with production equipment but with no operating personnel present. The “in operation” state is the condition where the installation is functioning in the defined operating mode with the specified number of personnel working.

The “in operation” and “at rest” states should be defined for each clean room or suite of clean rooms.

For the manufacture of sterile medicinal products 4 grades can be distinguished.

Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections. Normally such conditions are provided by a laminar air flow work station. Laminar air flow systems should provide a homogeneous air speed in a range of 0.36 – 0.54 m/s (guidance value) at the working position in open clean room applications. The maintenance of laminarity should be demonstrated and validated. A uni-directional air flow and lower velocities may be used in closed isolators and glove boxes.

Grade B: For aseptic preparation and filling, this is the background environment for the grade A zone.

Grade C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products.

Clean rooms and clean air devices should be classified in accordance with EN ISO 146441. Cleanroom classification should be clearly differentiated from operational process environmental monitoring. The maximum permitted airborne particle concentration for each grade is given in the following table.

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For cleanroom classification purposes in Grade A zones, a minimum sample volume of 1m³ should be taken per sample location. For Grade A, the airborne particle classification is ISO 4 dictated by the limit for particles ≥5.0 µm. For Grade B (at rest) the airborne particle classification is ISO 5 for both considered particle sizes. For Grade C (at rest & in operation) the airborne particle classification is ISO 7 and ISO 8 respectively. For Grade D (at rest) the airborne particle classification is ISO 8. For classification purposes EN/ISO 14644-1 methodology defines both the minimum number of sample locations and the sample size based on the class limit of the largest considered particle size and the method of evaluation of the data collected.

Portable particle counters with a short length of sample tubing should be used for classification purposes because of the relatively higher rate of precipitation of particles ≥5.0µm in remote sampling systems with long lengths of tubing. Isokinetic sample heads shall be used in unidirectional airflow systems.

“In operation” classification may be demonstrated during normal operations, simulated operations or during media fills as worst-case simulation is required for this. EN ISO 14644-2 provides information on testing to demonstrate continued compliance with the assigned cleanliness classifications.

Key environmental design aspects to consider in achieving and maintaining desired cleanroom classification.

Air Change Rates:
The airflow pattern and air change rate in a cleanroom largely determines the class of cleanliness that can be maintained during a given operation. Non-unidirectional flow cleanrooms rely on air dilution as will as a general ceiling to floor airflow pattern to continuously remove contaminants generated within the room. Unidirectional flow is more effective in continuously sweeping particles from the air due to the piston effect created by the uniform air velocity. The desired air change rate is determined based on the cleanliness class of the room and the density of operations expected in the room, for example a Class 10,000 cleanroom typically requires 40-60 air changes per hour. In unidirectional flow cleanrooms, the air change rate is generally not used as the measure of airflow but rather the average cleanroom air velocity is the specified criterion.

Air Pressure:
A pressure differential should be maintained between adjacent areas, with the cleaner area having the higher pressure. This will prevent infiltration of external contamination through leaks and during the opening and closing of personnel doors. A minimum overpressure between clean areas of 5 Pa is recommended. The pressure between a clean area and adjacent unclean area should be 12-14 Pa (.05 in. W. C.). Where several cleanrooms of varying levels of cleanliness are joined as one complex, a positive pressure hierarchy of cleanliness levels should be maintained, including airlocks and gowning rooms. Note that for certain processes it may be desirable to have a negative pressure relative to surrounding ambient in one or more rooms when containment is a major concern. A “room-with-in-a-room” may have to be designed to achieve this negative pressure yet still meet the needs of clean operation.

Temperature:
The comfort of those working in the clean room needs to be carefully considered and general practice is to aim for a nominal temperature range of 19C+/- 2 which will usually provide a comfortable environment for people wearing a typical lab coat. Where a full “bunny suit” or protective attire is to be worn room temperatures below 18C may be preferable. If the temperature is to be controlled in response to process concerns the value and tolerance should be specified early in the design phase to insure that budgeting is accurate.

Humidity:
Humidity requirements for comfort are in the range of 30-60%RH. If process concerns suggest another value it should be specified as soon as possible in the design process. Bio-pharmaceutical materials sensitive to humidity variations or excessively high or low values may require stringent controls.

Potential sources of contamination in a clean room.

There are an infinite potential list of contamination sources, however, all contamination can generally be broken down into five basis categories, namely the people, facilities, equipment, product and fluids.

The individuals present in a cleanroom will generate particle contamination (even without any motion), contamination can arise from hair loss, clothing debris e.g. fibres, cosmetics & perfumes, loss of skin tissue or transmission of oil from skin surfaces, etc..

The facilities if not properly specified and installed can act as a contamination due to emission from the construction materials, flaking of paint or coatings, vapours being exhausted, leaks in pipework’s, etc..

Equipment will generate contamination due to particles being created from wear and tear, vibrations can create sources of contamination within the sphere of the vibration effects, the lubricants applied to equipment can generate emissions, etc..

Fluids used within the cleanroom can introduce bacteria, organic materials, moisture, plasticizers can outgas, chemical cleaners can leave residues, etc..

The product being processed within the cleanroom can generate particles, flakes or other forms of debris depending on the nature of the product itself, etc..

Product and Process Validation.

The Validation Master Plan. Performing a Gap Analysis. Validation in the Design Process. Etc..
How to ensure Validation efforts are aligned with potential Risks.
How to ensure that Validation activity is appropriately documented, reviewed and aligned with requirements.
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