Revised International Clean room Standard By Piyush Tripathi “Thought to Explore”

Revised International Clean room Standard By Piyush Tripathi

How do we classify Clean room? Clean room guidelines are having lot of loop holes as its still not so absolute to be acceptable as thumb rule. Speaking to industrial experts every individual has different opinion about the clean room standard, Classification and its construction. Few common questions that each will answer differently and as per the conditions prevail. Revised international clean room standards discussion is a constant updates that are punched on web and pharmacopeia as revisions. Major changes and standards discussions and clean room designed for one and later revised to non conformance. To overcome this new this we need to have an approach which is acceptable. Understand the process flow of material from one form the other.

Latest updates show a reasonable change in the classification of the clean room or aseptic condition building. As described in Pharmacopeia it states: A clean room, as defined in USP <797>, is a room in which the concentration of airborne particles is controlled to meet a specified airborne particulate cleanliness class. Microorganisms in the environment are monitored so that a microbial level for air, surface, and personnel gear are not exceeded for a specified cleanliness class.

Depending on the nature of the operation, work is performed in different clean room classes.  

Recommended Action Levels for Microbial Contamination

Process Area	Classification	Particle Count (maximum no. particles 0.5u or larger per m3 air	Air Sample (Cfu per cubic meter {1000 ltrs} of air per plate)	Fingertip Sample (CFU per pair of hands)	Surface Sample (Contact Plate) CFU/plate
Filling Zone	ISO Class 5	3,520	> 1	> 3	> 3
Buffer Zone	ISO Class 7	3,52,000	> 10	N/A	> 5
Entrance	ISO Class 8	35,20,000	> 100	N/A	> 100

Clean rooms and associated controlled environments – Part 1: Classification of air Cleanliness

USP<797> Pharmaceutical Compounding: Sterile Preparations US Pharmacopoeia.

CFU- Colony Forming Units

Filling Zone In ISO Class 5 Area.

Applications	USP<797> Requirement
Glove Cleaning	Routine Application of Sterile 70% IPA
Surface Cleaning	Wiping with residue free disinfecting agent such as Sterile 70% IPA
Dry Wiping & Spill Control	Low Shedding Wipes discard after one use
Isolator & Hood Cleaning	Cleaning and disinfecting surfaces frequently
Disinfecting	Compatible, effective non residual disinfectant solution

For Buffer Zone ISO Class 7 & Entrance ISO Class 8 Areas

Applications	USP<797> Requirement
Hand Washing	Hand cleansing procedure before gowning
Glove Cleaning	Disinfection of contaminated gloved hands
Decontamination of Cleaning supplies for filling Zone	Wipe outer surface with Sterile 70% IPA
Surface Cleaning	Low shedding wipes, discard after one use
Dry Wiping & Spill Control	Low Shedding wipes, discard after one use
Floor Mopping (Small Space)	Non shedding mop hands, preferably discard after one use
Floor Mopping (Large Space)	Non shedding mop hands, preferably discard after one use
Disinfecting	Wipe Supplies removed from cartons with a suitable disinfecting agent. Clean Floor with disinfectant Solution.

Section 211.42 (design and construction features) requires, in part, that aseptic processing operations be performed within specifically defined areas of adequate size.  There shall be separate or defined areas for the firm’s operations to prevent contamination or mix-ups.  Aseptic processing operations must also include, as appropriate, an air supply filtered through high efficiency particulate air (HEPA) filters under positive pressure, as well as systems for monitoring environmental condition, and maintaining any equipment used to control aseptic conditions.

Section 211.46 (ventilation, air filtration, air heating and cooling) states, in part, that equipment for adequate control over air pressure, microorganisms, dust, humidity, and temperature shall be provided when appropriate for the manufacture, processing, packing or holding of a drug product.  This regulation also states that air filtration systems, including pre-filters and particulate matter air filters, shall be used. Appropriate filtration level to be maintained on air supplies to production areas.

Some known or assumed Air Classifications chart.

Clean Area Classifications	> 0.5 um particles/Ft3	> 0.5 um particles/m3	Micro biological Limit
100	100	3,500	< 1	<3 span="">
1,000	1000	35,000	< 2	< 7
10,000	10,000	3,50,000	< 5	< 18
1,00,000	1,00,000	35,00,000	< 25	< 88

Section 211.42 states that flow of components, drug products containers, closures, labelling, in-process materials, and drug products through the building or building shall be designed to prevent contamination. HEPA filtered air as appropriate classification and as per area required to be facilitated, as well as floors, walls and ceilings of smooth, non-particle shading surfaces that are easily cleanable are some additional requirements of this section.

Section 211.63 states that equipment shall be of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and maintenance. 

Section 211.65 states that equipment shall be constructed so that surfaces that contact the components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.

Section 211.113 states that appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed.

Sections 211.22 states that, quality control unit shall have the responsibility for approving or rejecting all procedures or specifications impacting on the identity, strength, quality, and purity of the drug product.

Section 211.113(b) addresses the procedures designed to prevent microbiological contamination, stating that written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed.

Section 211.25, Personnel Qualifications requires that each person engaged in manufacture, processing, packing or holding of a drug product shall have education, training and experience, or any combination thereof, to enable that person to perform the assigned functions. Adequate number of qualified personnel to perform and supervise the manufacture, processing, packing or holding of each drug product. Section 211.25 also requires that continuing training in CGMP shall be conducted by qualified individuals on a continuing basis and with sufficient frequency to assure that employees remain familiar with CGMP requirements applicable to them.  The training shall be in the particular operations that the employee performs and in current good manufacturing practice, as they relate to the employee's functions.

Section 211.28, Personnel Responsibilities states, Personnel engaged in the manufacture, processing, packing or holding of a drug product shall wear clean clothing appropriate for the duties they perform. It also states that personnel shall practice good sanitization and health habits and specifies that protective apparel, such as head, face, hand, and arm coverings, shall be worn as necessary to protect drug products from contamination. It also states that any person shown at any time (either by medical examination or supervisory examination) to have an apparent illness or open lesions that may adversely affect the safety or quality of drug products shall be excluded from direct contact with components, drug product containers, closures, in-process materials, and drug products until the condition is corrected or determined by competent medical personnel not to jeopardize the safety or quality of drug products.  All personnel shall be instructed to report to supervisory personnel any health conditions that may have an adverse effect on drug products.

Restrictions on entry into limited access areas: Only personnel authorized by supervisory personnel shall enter those areas of the buildings and facilities designated as limited access areas.

Section 211.42 requires the establishment of a system for monitoring environmental conditions.

Guidelines and Classification of Class and Gradations

WHO GMP	US 209E	US Customary	ISO/TC (209) ISO 14644	EEC GMP
Grade A	M 3.5	Class 100	ISO 5	Grade A
Grade B	M 3.5	Class 100	ISO 5	Grade B
Grade C	M 5.5	Class 10,000	ISO 7	Grade C
Grade D	M 6.5	Class 1,00,000	ISO 8	Grade D

Observations on the Clean Room Standards and revisions:

As summarized above for the process, production, packing and material areas clean room conditions are defined based on the process flow and its identifications. It’s not mandatory to have air locks to segregate or buffer the area, if area restrictions are there we can any time create a buffer area hold for man and material movement change the air present in the area and then move forward. Seems surprising but yes it is a possible solution for areas where space constraints and process limitation does not permit. This is entirely personal view. Why not?

Logic why do we create Air Lock? Answer to this is to obstruct person, media, product to enter another higher or lower classified area and ensure that the same is not contaminating or mixing up with or creating cross contamination. If we ensure that the sufficient amount of buffer is provided air lock and lot of space can be saved.

Modern method of aseptic processing are adapting to isolation principals that enables or isolates the processing area and the overall running cost of the process side can be minimized. Viz. processing area is isolated by making cubical which is entire process line is placed and outside area is normal Grade D. this helps to reduce the load of AHU of Grade A and due to lesser area overall running cost of the plant goes down. But selection of such principle is completely individual choice. Designing such area has to go through or counter lot of thumb rule followers who believe that the area should as prescribed in USP, IP, BP or any other pharmacopeia, it’s a matter of individual choices. People do think out of box but are forced to close the box as its one step that would require lot of explaining to do but there is always a first step.

Conclusion to revision in Clean Room Standards

By concentrating more on process requirements area can be designed to its absolute. Exploring possibilities with what we have is always the solution. Man has evolved and has made larger changes than what we are thinking of. Following law does not mean that one process can be done one way. SME organization, Job work organizations and individual local companies most of the time back off and does not explore the possibilities to upgrading their facilities as Law is often misinterpreted and misled. This blog is first “Thought to Explore”

“Thought to Explore”

First Step

Sterility or Sterilization Assurance BY Piyush Tripathi

Sterility or Sterilization Assurance Sterility assurance, a specimen deemed sterile only when there is complete absence of viable microorganisms from it. This absolute definition cannot be applied to an entire lot of finished compendia articles because of limitations in testing. Absolute sterility cannot be practically demonstrated without complete destruction of every finished article. The sterility of a lot purported to be sterile is therefore defined in probabilistic reasoning terms, where likelihood of a contaminated unit or article is acceptably remote. Such a state of sterility assurance can be established only through the use of adequate sterilization cycles and subsequent aseptic processing. The basic principle of sterilizing process with reference to USP is as follows. 1. Establish that the process equipment has capability of operating within the required parameters. 2. Demonstrate that the critical control equipment and instrumentation are capable of operating within the prescribed parameters for the process equipment. 3. Perform replicate cycles representing the required operational range of the equipment and employing actual or simulated product. Demonstrate that the processes have been carried out within the prescribed protocol limits and finally that the probability of microbial survival in the replicate processes completed is not greater than the prescribed limits. 4. Monitor the validated process during routine operation. Periodically as needed, re-qualify and recertify the equipment. 5. Complete the protocols, and document steps (1) through (4) above. Aseptic process or sterilization process requires a complete knowledge of the field of sterilization and clean room technology. It is important to employ appropriate instrumentation and equipment to control critical parameters such as temperature, time, humidity and sterilization monitoring controls in order to comply with currently acceptable and achievable limits in sterilization parameters. All processes are required to be timely revalidated extensively as the original program. An important aspect of validation involves biological indicators. Bacterial spores are the most resistant of all living organisms because of their capacity to withstand external destructive agents. Although the physical or chemical process by which all pathogenic and non-pathogenic microorganisms, including spores, are destroyed is not absolute, supplies, product and equipments are considered sterile when necessary conditions have been met during a sterilization process. Reliable sterilization depends on contact of the sterilizing agent with all surfaces of the product / item to be sterilized. Selection of the agent to achieve sterility depends primarily upon the nature of the product/item to be sterilized. Time required to kill spores in the equipment available for the process then becomes critical. Sterilization Methods: Steam Sterilization Heat destroys microorganisms, but this process is hastened by the addition of moisture. Steam in itself is inadequate for sterilization. Pressure, greater than atmospheric, is necessary to increase the temperature of steam for thermal destruction of microbial life. Death by moist heat in the form of steam under pressure is caused by the denaturation and coagulation of protein or the enzyme-protein system within the cells. These reactions are catalyzed by the presence of water. Steam is water vapour; it is saturated when it contains a maximum amount of water vapour. Direct saturated steam contact is the basis of the steam process. Steam, for a specified time at required temperature, must penetrate every fibber and reach every surface of items to be sterilized. When steam enters the sterilizer chamber under pressure, it condenses upon contact with cold items. This condensation liberates heat, simultaneously heating and wetting all items in the load, thereby providing the two requisites: moisture and heat. No living thing can survive direct exposure to saturated steam at 250 F (120 C) longer than 15 minutes. As temperature is increased, time may be decreased. A minimum temperature-time relationship must be maintained throughout all portions of load to accomplish effective sterilization. Exposure time depends upon size and contents of load, and temperature within the sterilizer. At the end of the cycle, re-evaporation of water condensate must effectively dry contents of the load to maintain sterility. Ethylene Oxide Sterilization Ethylene oxide is used to sterilize products/items that are heat or moisture sensitive. Ethylene oxide (EO) is a chemical agent that kills microorganisms, including spores, by interfering with the normal metabolism of protein and reproductive, processes, (alkylation) resulting in death of cells. Used in the gaseous state, EO gas must have direct contact with microorganisms on or in products/items to be sterilized. Because EO is highly flammable and explosive in air, it must be used in an explosion-proof sterilizing chamber in a controlled environment. When handled properly, EO is a reliable and safe agent for sterilization, but toxic emissions and residues of EO present hazards to personnel and patients. Also, it takes longer than steam sterilization, typically, 16-18 hrs. to complete the sterilization cycle. EO gas sterilization is dependent upon four parameters: EO gas concentration, temperature, humidity, and exposure time. Each parameter may be varied. Consequently, EO sterilization is a complex multi-parameter process. Each parameter affects the other dependent parameters. Others Sterilization Processes: Dry Heat Sterilization Dry heat in the form of hot air is used primarily to sterilize anhydrous oils, petroleum products, and bulk powders that steam and ethylene oxide gas cannot penetrate. Death of microbial life by dry heat is a physical oxidation or slow burning process of coagulating the protein in cells. In the absence of moisture, higher temperatures are required than when moisture is present because microorganisms are destroyed through a very slow process of heat absorption by conduction. Microwaves The nonionizing radiation of microwaves produces hyperthermic conditions that disrupt life processes. This heating action affects water molecules and interferes with cell membranes. Microwave sterilization uses low-pressure steam with the nonionizing radiation to produce localized heat that kills microorganisms. The temperature is lower than conventional steam, and the cycle faster, as short as 30 seconds. Metal instruments can be sterilized if placed under a partial vacuum in a glass container. Small tabletop units may be useful for flash sterilizing a single or small number of instruments, when technology is developed for widespread use. Formaldehyde Gas Formaldehyde kills microorganisms by coagulation of protein in cells. Used as a fumigant in gaseous form, formaldehyde sterilization is complex and less efficacious than other methods of sterilization. It should only be used if steam under pressure will damage the item to be sterilized and ethylene oxide and glutaraldehyde are not available. Its use for sterilization has been almost abandoned in the United States, Canada, and Australia. The method dates back to 1820, and it is still used in Europe and Asia with is also slowly abandoning the process. Hydrogen Peroxide Plasma Hydrogen peroxide is activated to create a reactive plasma or vapor. Plasma is a state of matter distinguishable from solid, liquid, or gas. It can be produced through the action of either a strong electric or magnetic field, somewhat like a neon light. The cloud of plasma created consists of ions, electrons, and neutral atomic particles that produce a visible glow. Free radicals of the hydrogen peroxide in the cloud interact with the cell membranes, enzymes, or nucleic acids to disrupt life functions of microorganisms. The plasma and vapor phases of hydrogen peroxide are highly sporicidal even at low concentrations and temperature. Ozone Gas Ozone, a form of oxygen, sterilizes by oxidation, a process that destroys organic and inorganic matter. It penetrates membrane of cells causing them to explode. Ozone is an unstable gas, but can be easily generated from oxygen. A generator converts oxygen, from a source within the hospital, to ozone. A 6 to 12 percent concentration of ozone continuously flows through the chamber. Penetration of ozone may be controlled by vacuum in the chamber, or enhanced by adding humidity. At completion of exposure time, oxygen is allowed to flow through chamber to purge the ozone. Cycle time may be up to 60 minutes depending on the size of the chamber or load. Chemical Solutions Liquid chemical agents registered by the EPA as sterilants provide an alternative method for sterilizing heat sensitive items if a gas or plasma sterilizer is not available, or the aeration period makes ethylene oxide sterilization impractical. To sterilize items, they must be immersed in a solution for the required time specified by the manufacturer to be sporicidal. All chemical solutions have advantages and disadvantages; each sterilant has specific assets and limitations. These chemicals are: peracetic acid, glutaraldehyde, and formaldehyde. Ionizing Radiation Some products commercially available are sterilized by irradiation. It is the most effective sterilization method but is limited for commercial use only. Ionizing radiation produces ions by knocking electrons out of atoms. These electrons are knocked out so violently that they strike an adjacent atom and either attach themselves to it, or dislodge an electron from the second atom. The ionic energy that results becomes converted to thermal and chemical energy. This energy causes the death of microorganisms by disruption of the DNA molecule, thus preventing cellular division and propagation of biologic life. The principal sources of ionizing radiation are beta particles and gamma rays. Beta particles, free electrons, are transmitted through a high-voltage electron beam from a linear accelerator. These high-energy free electrons will penetrate into matter before being stopped by collisions with other atoms. Thus, their usefulness in sterilizing an object is limited by density and thickness of the object and by the energy of the electrons. They produce their effect by ionizing the atoms they hit, producing secondary electrons that, in turn, produce lethal effects on microorganisms. Cobalt 60 is a radioactive isotope capable of disintegrating to produce gamma rays. Gamma rays are electromagnetic waves. They have the capability of penetrating to a much greater distance than beta rays before losing their energy from collision. Because they travel with the speed of light, they must pass through a thickness measuring several feet before making sufficient collisions to lose all of their energy. Cobalt 60 is the most commonly used source for irradiation sterilization. The product is exposed to radiation for 10 to 20 hours, depending on the strength of the source. Establishing Sterility Assurance A typical Validation program, as outlined below is one designed for steam autoclave, but the principles are applicable to other sterilization procedures. The installation qualification stage to establish the controls and other instrumentation check properly designed and calibrated or not. Documentation should be on file demonstrating the quality of the required inputs or feeds Utilities Viz. Steam, Water and Air. The operational qualification confirms that the empty chamber functions within the parameters of temperature at all of the key locations prescribed. It is appropriate to develop heat profile records, i.e. simultaneous temperatures in the chamber employing multiple temperature sensing devices. A typical acceptable range of temperature in the empty chamber if + 1o when the chamber temperature is not less than 121o. The confirmatory stage of the validation is actual sterilization of product or material. This determination requires the employment of temperature sensing devices inserted into samples of the articles, as well as either samples of the articles to which appropriate concentrations of suitable test microorganisms have been added. The effectiveness of heat delivery or penetration into the actual articles and the time of the exposure are the two main factors that determine the level or lethality of the sterilization process. The final stage of validation program requires the documentation of the supporting data developed in executing the program. It is generally accepted that terminally sterilized injectable articles or critical devices purporting to be sterile, when processed in the autoclave, attain a 10–6 microbial survivor probability, i.e., assurance of less than 1 chance in 1 million that viable microorganisms are present in the sterilized article or dosage form. With heat-stable articles, the approach often is to considerably exceed the critical time necessary to achieve the 10–6 microbial survivor probability (overkill). However, with an article where extensive heat exposure may have a damaging effect, it may not be feasible to employ this overkill approach. In this latter instance, the development of the sterilization cycle depends heavily on knowledge of the microbial burden of the product, based on examination, over a suitable time period, of a substantial number of lots of the presterilized product.