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Chapter 8 Microbiological Control
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Chapter Objectives Explain why microbiological control is important Provide examples of how it is achieved and maintained Describe the various sources of microbial contamination, name specific contaminants and their possible sources Explain the different microbiological cleanliness standards required Define aseptic processing Identify measures taken in controlled and classified environments within clean rooms to prevent microbial contamination Describe the components of an effective environmental monitoring program List quality control practices that are essential in the Microbiology QC laboratory
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Microbiological Control Vital for two main reasons: the majority of biopharmaceutical medicines are designed for parenteral administration and will bypass the body’s natural external defense mechanisms biopharmaceutical drug substances are generally large, complex molecules that are susceptible to degradation from enzymes produced by contaminating microbes
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Bacteria, Fungi and Mycoplasma Found in every environment including food, water, air Crucial to life as we know it Decomposition and recycling of elements for future generations Digestion of food Symbiotic with many organisms Rhizoids with plants Cellulase for termites and ruminants Important in food production Cheese Brewing Saurkraut
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Impact of Bacteria, Fungi & Mycoplasma on Production Contamination and or protein degradation/modification possible at all stages of production Goal in cell culture is to maintain axenogenic or monoseptic (aseptic) cultures that contains only the engineered cell of interest achieved through stringent controls of operating equipment and conditions Contaminating microbes typically overgrow the mammalian cell cultures to out compete for the available nutrients Results in loss of batch and product More difficult to detect in a microbial cell culture but is still of concern due to modifications of product and possible residual contaminating proteins
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Mycoplasma Common contaminant in cell culture Smallest, free-living prokaryote; lack a cell wall 0.2 – 0.3 micron in diameter Obligate parasite, requires cholesterol from host cell Can grow to very high concentrations in mammalian cell cultures, but remain unobservable by light microscopy Viewed by fluorescent staining of the nuclear material usually no overt signs that cultures are contaminated
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Types of Mycoplasma 5 common species M. hyorhinis M. arginini M. orale M. fermentans Acholeplasma laidlaw ii
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Mycoplasma Contamination Contamination includes Changed growth characteristics of cell line Changed membrane antigenicity Changed cell metabolism Chromosomal aberrations Disrupted nucleic acid synthesis Altered transfection rates Increased viral susceptibility Significant safety and regulatory concerns Most common sources of contamination The production cell line itself Raw materials Production personnel environmental
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Mycoplasma – Detection/Monitoring Detection technologies include Growth on special media plates; may require up to a month for adequate growth Fluorescent staining of the nuclear material using Hoechst 33258 dye PCR, primarily of the 16S rDNA sequence Monitoring In the cell culture process – a closed system of cells in a vessel with nutrients incubated for some period of time from days to weeks before harvest Extraction and purification processes have no cells therefore Mycoplasma (and viruses) are not considered problematic
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Bacteria and Molds Bacteria and molds Greatest concern during extraction of protein from cell culture and purification Typically carried by people working with the process Concerns : control of their numbers – can overwhelm the capacity of downstream filtration processes to remove them metabolites/products can be harmful to therapeutic protein or to the patient
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Post-harvest processes Considered an open system of equipment and environment Many opportunities for introduction of bacteria or molds Some equipment / materials are difficult to sterilize Filter membranes Chromatography resins Generally the open environment Bacteria & Mold Post-Harvest Considerations
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Post-Harvest Considerations Cont’d Bacterial structures or products that impact the product Proteolytic enzymes that degrade the product Gram- cell walls releasing endotoxins (pyrogens) potentially capable of being harmful to patients Parenteral delivery (injection) Majority of biopharmaceuticals Must remain free of viable organisms to protect patient from infection
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1970’s – enterococci considered “opportunistic pathogens” contaminated infusion fluids resulted in the deaths of a number recipient patients 2002 – fungal meningitis in a spinal injection killed a 77-year old women; the source was found to be a rare fungal species in the environment where production took place 2012 – an repeat outbreak of fungal meningitis occurred in several patients receiving infusions manufactured in a plant eventually shown to not be properly designed to produce that product nor were they properly monitored Historical Examples
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Prions Misfolded Infectious proteins Prion = Protein Infection No associated nuclear material Replicate by inducing other similar proteins to misfold Accumulation leads to neurological disorders Scrapie (sheep) Creutzfeldt-Jakob disease (CJD, humans) Bovine Spongioform Encephalopathy (“mad cow”) Encompassing classification – Transmissible Spongioform Encephalopathies (TSE) Transmitted by eating nerve tissue (brain, CNS) of infected animals Animal feed containing ground offal
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Prions Relevance Number of animal-derived materials used in production Gelatin Amino acids Fetal Bovine Serum (FBS) Examples of Prion Infections Human Growth Hormone (HGH) derived from pituitary gland was shown to carry TSE from donors with CJD Expedited the use of rDNA to produce CJD-free HGH Mad cow disease in the UK was determined to have been transmitted to farm workers No cases have been documented as being derived from pharmaceuticals
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Endotoxins What is it? toxin (lipopolysaccharide) Where does it come from? The cell membrane of Gram negative bacteria Which products are tested? Injectable drugs and medical devices which will contact blood or spinal fluid raw materials, water and in process monitoring
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Potent, toxic, very stable and present in many pharmaceutical ingredients and on surfaces that come into contact with the product when formulated for parenteral administration Water soluble, and will pass through 0.2 µm filters Not destroyed by autoclaving and are insoluble in organic solvents Very difficult to eliminate in a final preparation Endotoxin
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Endotoxins Cont’d Released when bacterial cells are disrupted Extremely heat stable – conditions for inactivation are 180°C for 3 hours Pyrogenic (fever- inducing) Lowers blood pressure activates inflammation and coagulation
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Generally accepted endotoxin limit (EL) is defined as acceptable endotoxin load that the body can generally tolerate without experiencing the associated adverse events 5.0 EU/kg for parenteral (intreavenous or intravenous) drugs 0.2 EU/kg for the intrathecal (spinal) route of administration Endotoxin Limits
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Viruses Submicroscopic intracellular infectious agents First visualized by electron microscope Simplest forms consist of genetic material and a protective protein coat Only replicate within host (plant, animal, human, prokaryotes) cells, therefore, not considered living Infectious within a cell culture Potential to alter the metabolism of the cultures cell, changing or eliminating the desired product Contaminant in the product Numerous examples of serious outcomes due to contaminated medicines Transfer to patient leads to deleterious, often lethal, infections
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Examples of Virus Contamination HIV virus in pooled blood products, most notably in injections for hemophiliacs Before a detection test was available, as many as 10,000 hemaphiliacs were unintentionally infected A smaller but significant number of blood transfusion recipients were also infected A single infected donor to the pool will contaminate the entire pool A childhood disease vaccine contaminated with an apparently harmless circovirus Although not known to cause a human disease, vaccinations were suspended until the contaminant was removed Not enough information or testing to determine that there was no adverse affect due to contaminant
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Viral Contamination Issues associated with virus contamination Detection of virus How to test for all known viruses vs medically important viruses Test for the commonly known viruses of concern Discovery of new viruses, or mutated forms of old viruses, make it difficult to maintain a complete battery of detection tests Elimination of viruses Stringent control of all processes and components to ensure the product is virus free Where feasible, inactivation or removal of virus More ultrafiltration techniques are being built into biopharmaceutical processes to eliminate contaminating viruses
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Control During Production Control is a continuing challenge in pharmaceutical production Biopharmaceutical processes use many organic materials that may initially be contaminated before use Critical issue because Majority of biopharmaceuticals are parenteral (injectable) and must not cause infections or inflammatory reactions Biopharmaceuticals are generally large, complex proteins that are susceptible to microbial degradation
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Microbiological Control in Biomanufacturing Manufacture of biopharmaceuticals Begins with closed system, axenic mono-culture Onto low bioburden purification Finish with aseptic finish/fill to produce a sterile dosage During the cycle Various microbial agents can enter in various ways Some may be tolerated, often zero tolerance to microbes Necessary to understand, monitor and control products and impurities (microbes, metabolites, etc) Often difficult to determine the concentration or absence of impurities Control at the environmental level is often preferred
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Manufacturing Process Typically two independent activities Manufacture of a drug substance Manufacture of a drug product May occur in separate facilities (or even countries) Each has its own specific, but different, requirements
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Manufacture of Drug Substance Relatively long, discontinuous set of process steps Product is a solution that is allowed to have a low bioburden; however, still need to exclude extraneous microbes to retain quality Monitored with a Bioburden Control Strategy document Ongoing analysis and understanding of the microbes in the air, water, surfaces and the people is required
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Manufacture of Drug Product Preparation of the individual sterile dosage form units from the drug substance One relatively short, continuous process Need to monitor facility, people and processes to achieve sterility Additional elements to monitor Excipients Preservatives Vials Syringes Stoppers Result: sterile product from a series of non-sterile components
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Manufacturing Control Definitions Sterile: the absence of life. All drug products are required to be sterilized once placed in their final container, this is performed to prevent the provision of a product contaminated with microorganisms to patients aseptic: acting in such a way to prevent the introduction of microorganisms; aseptic processing is used for those drug products that must be sterile but cannot be subject to terminal sterilization due to their heat-labile nature; effectively all biopharmaceutical products are in this category axenic: freedom from foreign organisms. All biopharmaceuticals are intended to be axenic cultures- they only contain the cell line desired, without other foreign organisms.
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Contamination Contamination - presence of any unwanted substance that will affect the purity of a drug product Common sources Air Surfaces Water Components used to manufacture the product Influenced by many factors Materials Degree of human contact Manufacturing environment Quality of tools, fixtures, facilities
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Types of Contamination Two types: Particulate/non-viable contaminants Consist of small bits of matter, called particles Categorized by size and type of particle Viable contaminants Microbial, bacteria, viruses, mycoplasma
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Particulate – Non-viable Contamination Any unwanted component Particulate (non-viable) contamination Matter (particles) of microscopic dimensions Figure: Comparison of one micron-size particulate to one human hair
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Types of Particulate Contamination May be organic or inorganic In gases: aerosol or airborne contamination – 97% are microscopic, 3% are “dust” In liquids: suspension when floating, silt when settled In solids: called included matter Figure: Chart of relative sizes
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Sources of Non-Viable Contamination Common sources of non-viable particulates Cellulose fiber from paper glass particulate from breaking glass vials during filling aluminum particles from capping vials gown fibers hair human dead skin cells (one of the most frequently encountered particulates in the cleanroom)
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Microbial/Viable Contamination Microbial (viable) contamination Bacteria, fungi, viruses Ultimately, all microorganisms are excluded to the fullest extent possible Issue: they reproduce, increasing the contamination problem, creating metabolic products including waste by-products A single bacterium can cause significant problems Figure: Example of bacterial reproduction
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Bacterial Reproduction Time of DayNumber of Bacterial Cells 9:00 a.m.1 9:20 a.m.2 9:40 a.m.4 10:00 a.m.8 10:20 a.m.16 1:00 p.m.4,096 1:20 p.m.8,192 1:40 p.m.16,384 2:00 p.m.32,768 2:20 p.m.65,536 4:40 p.m.8,388,608 5:00 p.m.16,777,216
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Types of Common Microorganisms Bacteria is most common, followed by molds Some examples of commonly encountered microorganisms in cleanrooms MicroorganismExampleSource Gram-positive cocciStaphylococcus species humans Gram-positive cocciMicrococcus specieshumans Gram-positive bacilliBacillus speciessoil
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Sources of Microbial Contamination from Humans Sources of microbial contaminants from humans SourceAmount nose secretionapproximately 10 million microbes/gram Spittleapproximately 100 million microbes/gram scalpapproximately 1 million microbes/cm 2 forehead10,000–100,000 microbes/cm 2 Armpit1–10 million microbes/cm 2 Hands100–1,000 microbes/cm 2
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Sources of Human Contamination Common sources Humans Often the biggest problem, esp. in the clean room Shed hair, skin particles Particles from under finger nails, on hands, on clothes Talking, sneezing, coughing – even with mask Typically, will shed 10 grams of skin particles per day
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Control of Contaminants - Design Facility design Clean room is any room or area where an attempt is made to limit, control and eliminate the amount of airborne contamination Properly designing the facility, controlling the air supply for the environment, sterilizing the manufacturing components, using aseptic gowning, following aseptic techniques, and implementing a cleaning and disinfection program
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Contaminant Control in Clean Room Air supply – require high efficiency particulate air (HEPA) filters Remove 99.97% of the particles suspended in air that are 0.3 microns or larger Number of air changes per hour also controlled Pressure differentials, airlocks Sterilization of components and equipment Steam, dry heat, gamma irradiation, vaporized hydrogen peroxide, filtration SIP for large vessels, preceded by CIP procedures
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Sterility Achieving sterility in final product is extremely important Complicated by the fact that biopharmaceutical molecules cannot be subjected the most common method of generating a sterile product: autoclaving Must be sterilized by using a sterilization filtration process applied to the bulk formulation Filled under aseptic conditions into pre-sterilized individual containers and capped with pre-sterilized closures
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Autoclaving – most common and arguably the most effective Equipment and containers may be autoclaved without concern Some pharmaceuticals may be “terminally sterilized” in their sealed containers as a last step in processing before delivery Biopharmaceuticals tend to be heat-labile molecules and therefore can not be subjected to the high heat of steam sterilization without changing or being denatured Result – most products are sterile filtered as a bulk formulation and then added to a sterile container under aseptic conditions, a process that is highly complex and demanding, with each step one more entry level for contaminants Sterilization Methods
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Control of Contamination - Sterilization Control through Sterilization the act or process, either physical or chemical, which eliminates or inactivates all forms of life, including bacterial endospores Methods vary depending on type of material to be sterilized but include dry heat Wet (steam) heat – 121 o C, 15 psi gamma irradiation ethylene oxide Vaporized Hydrogen Peroxide (VHP) filtration Sterilizers must validated, including their load pattern Ensures that it is functioning properly Ensures that every item is sterile Need to be re-validated on a planned schedule Include preventative maintenance
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Validating a Sterilizing Process Demonstrate that the equipment used for the sterilization process (autoclave, dry heat oven, and VHP) is capable of operating to achieve the desired end result run cycles to demonstrate actual operational conditions ensure that the required parameters of microbial kill (i.e., bioburden reduction) are achieved Biological indicators are typically used for this purpose Biological indicators are a material that is inoculated with a known quantity of microorganisms, typically one that serves as a worst case scenario for the sterilization cycle by being resistant to the sterilization conditions continually monitor the process parameters (e.g., temperature, pressure, etc.) during each sterilization cycle to ensure they are operating within the validated parameters perform continuing validation studies periodically to ensure that the loads are maintained in a validated state
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Sterilization Process Validation Important that all sterilization processes operate with the validated parameters to ensure the sterility of components, especially those that cannot be individually tested for sterility Important to check expiration dates and appearance/integrity of sterilized equipment and components before use; cannot use if expired or compromised Autoclave must have even distribution of temperature to ensure all items in the chamber are sterile; usually achieved by generating a vacuum A cycle is only valid if the correct temperature and pressure is maintained throughout the required period of time Materials that can be autoclaved include some culture media, filters, glassware (bottles, etc), miscellaneous items such as caps, tubing, forceps, etc.
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Autoclaving -Advantages and Disadvantages Advantages Consistently highly effective Simplistic Short processing times No toxic residues Disadvantages High temperatures limit materials to those not adversely effected by heat, moisture or pressure Burn hazards for operators
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Sterilization - SIP / CIP / Dry Heat Steam-in-Place (SIP) / Clean-in-Place (CIP) SIP is used for large vessels and tanks that will not fit into an autoclave CIP must be completed before SIP to prevent “baked-on” residuals that would result in cross contamination between uses Both processes, SIP and CIP, must be validated Dry Heat Sterilization Hot air at 160 – 170 C Oven-like chamber Circulated air for even temperature throughout chamber For heat stable but moisture sensitive material, including glassware Validated for time, temperature and load pattern
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Dry Heat - Advantages / Disadvantages Dry Heat Sterilization – advantages – Simplicity – Heat penetration – No toxic residues – No corrosion – Able to inactivate pyrogens (depyrogenate) Dry Heat Sterilization – disadvantages – Long processing times – High temperature limits materials – Burn hazard for operators
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Gamma Irradiation Gamma Irradiation Alternative for moisture and/or heat sensitive material Gamma ray sources include cobalt-60 and cesium-137 Time sensitive dosage delivery High energy waves that disrupt DNA leading to death of any organisms No toxic or radioactive residues Disadvantage – relatively expensive method Gamma irradiators shielding Storage and disposal Safety issues Can be outsourced
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Vaporized Hydrogen Peroxide (VHP) Vaporized Hydrogen Peroxide (VHP) Aerosolized, low temperature Used to kill bacteria (including endospores) on enclosed surfaces Isolators Workstations Safety cabinets Pass-through rooms Advantages Kills a wide variety of microorganisms and spores Low temperature 31% H 2 O 2 can be converted to water vapor and oxygen Disadvantage Only effective on exposed surfaces Toxic so must be used in an enclosed / sealed area
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Filter Sterilization Removes but does not kill microorganisms Sterilize heat-sensitive materials Sterilize gas or liquid by passing through a porous membrane Filters are classified by nominal pore size ratings Will not retain microorganisms smaller than the rated size
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Control with Aseptic Gowning Human body sheds ~ 1 billion dead cells and hair plus oils and moisture each 24 hours ~1000 bacteria-containing cells per minute Can get into the air flow to cause contamination Control by wearing clean, sterilized clothing to create barrier Protects product and critical surfaces Not 100% effective Reduces but does not eliminate shed microorganisms Aseptic gowning to cover all or most of the person
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Aseptic Gowning Full Aseptic Gowning Gloves Gown Mask Hood Goggles Boots Gloves
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Aseptic Gowning Practices Good practices for aseptic gowning: Wash hands with soap and water prior to entering the gowning room Remove cosmetics Trim and clean fingernails Remove watches and jewelry Follow SOP for gowning process Make sure gowning apparel does not make contact with floor, walls or equipment Disinfect gloves with 70% alcohol Inspect gloves and gowning apparel and replaced damaged items Keep talk to a minimum; avoid sneezing and coughing
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Gowning Requirements Gowning requirements by area classification ISO 14644-1 designations provide uniform particle values for cleanrooms in multiple industries. Clean Area Classification (0.5 micron particles/ft 3 ) ISO Designation a Typical AreaGowning Class 1005point of fill, sterility testing or other aseptic manipulation full aseptic gowning, to consist of sterile gown/hood, sterile double gloved, mask, goggles, and boots Class 10,0007background room/area for aseptic manipulation same as Class 100 Class 100,0008personnel and equipment airlocks leading into the cleanroom facility non-shedding one- or two- piece suit gathered at wrists and ankles; hair, beard, and shoe covers Controlled Unclassified NAaccess and exit to/from classified areas; packaging areas employer-issued uniform plus non-shedding smock, hair, beard, and shoe covers
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Working in a Cleanroom Follow high standards of personal hygiene and cleanliness No respiratory or gastrointestinal infections No open skin lesions or other skin conditions (excess shedding) Ability to gown properly and operate in a slow, deliberate manner Keep talk to a minimum avoid sneezing and coughing Ability to work in clean room conditions for long periods of time
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Working in a Cleanroom Cont’d Entering a cleanroom via an airlock requires the following behaviors All equipment must be disinfected in the airlock prior to entering an aseptic area and before an aseptically-gowned person contacts it Opening and closing of airlock doors should be kept to a minimum, they should be opened only to perform necessary activities Both doors of an airlock should never be opened simultaneously
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Clean Room Conduct All equipment must be disinfected in the airlock and before an aspetically gowned person contacts it A gowned person and an un-gowned person should never be in the airlock at the same time Do not open an airlock door to talk to anyone Never open both doors of an airlock at the same time
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Control through Aseptic Techniques Control through aseptic techniques Focus on procedures executed with the mindset of reducing risk and contamination to the lowest possible level Operator technique is the single most important constant in maintaining aseptic processes due to proximity to the product or culture Extrinsic factors include sterile material, environment, and equipment
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Control of Contamination through Aseptic Technique in BSC Aseptic techniques Critical surfaces must not come in contact with anything that is potentially contaminated Laminar flow hoods and rooms are Class 100 environments No body part, equipment, objects or contaminated air should come between the HEPA filter and the critical site – first air rule
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Control of Contaminants in Clean Room Working in a laminar flow hood or room Verify proper operation of hood and certification Perform all aseptic manipulations at least six inches from the front edge and sides of hood Disinfect all equipment prior to transferring to hood Disinfect gloves when entering hood and frequently while working Do not touch critical surfaces Avoid excess turbulence and particulate shedding Minimize number of people and equipment under hood Limit talking Work in an unhurried manner
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Reducing the Probability of Contamination Environment The environment must be clean, with HEPA-filtered air circulating The aseptic area should be cleaned before and after use; cleaning after use reduces the risk of cross-contamination in a multi-product suite and reduces the microbial load on working surfaces Equipment and materials Ensure that equipment and materials are cleanable and fit for purpose, i.e., use equipment that is designed for cleanrooms;
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Particle Generation Estimated particles (0.3 micron and larger) generated by various activities Particles EmittedActivity 100,000motionless—sitting/standing 500,000upper body motion 1 millionupper body and minor leg motion 2.5 millionsitting to standing or vice versa 5–10 millionwalking >2.0mph
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Viable and Non-viable Particles If specifications require measurement of viable and non-viable particulates during your operations, the following experiments can be performed : Viable particulates: surface monitoring plates may be used to measure viable contaminants on personnel and equipment/surfaces and air viable devices may be used to measure viable air contaminants Non-viable particulates: a particulate measuring device can also be used to measure the total of both viable and non-viable particles
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Contamination Control – Cleaning and Disinfecting Control through cleaning and disinfecting Cleanrooms do not have self-cleanup capabilities to offset any contamination Most contaminants settle to the floor or other horizontal surfaces Introduced into the air by air currents or activity A one micron anthrax spore requires ~20 minutes to settle or move laterally one meter Contamination needs to be removed from these surfaces by frequent cleaning and disinfection Cleaning is applying a detergent (along with the physical removal of particles and microorganisms from surfaces) by mopping, wiping, or brushing Disinfection is the elimination of most recognized disease-causing or harmful microorganisms but not necessarily all microbial forms Methods include UV radiation, boiling water, steam or, typically, chemicals
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Contamination Control – Cleaning Purpose is to remove contaminants and residues that interfere with effective disinfection Four step surface cleaning process Scrub the surface with a mop or wipe and use a detergent solution Rinse the surface before the surface dries Collect any remaining liquid on the surface by wiping or vacuuming Allow the surface to dry, then disinfect
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Contamination Control – Disinfecting Disinfection Chemical agent/s used on surfaces that destroy disease-causing microbes A disinfectant will typically contain both an antimicrobial and a detergent The pH of a disinfectant may limit the growth of a contaminant if 10 No single disinfecting agent or procedure is adequate for all purposes Especially, endospores, which require special procedures Effectiveness of a disinfectant depends upon: Disinfectant concentration Length of exposure to the disinfectant Amount of organic matter (e.g., soil, blood) present Nature and amount of microorganisms on the surface Material to be disinfected
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Classification of Disinfectants Chemical disinfectants are classified based on their antimicrobial compound alcohols – 70% IPA aldehydes halogens - sodium hypochlorite peroxygens - peracetic acid phenolics surface active agents Further classified by microbial target Bacteriocidal Fungicidal Virucidal Sporicidal Bacteriostatic Solutions must be sterile for use in class 100 and 10,000 areas
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Summary of Disinfectants and Efficacy Disinfectant Category Gram- Positive Bacteria Gram- Negative Bacteria EndosporesFungi Alcohols3302 Aldehydes4444 Halogens3312 Peroxygens4444 Phenolics3213 Surface Active Agents 3102 Summary of disinfectant categories and their efficacy 4 = most efficacious in killing 1 = least efficacious in killing 0 = not effective in killing
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Disinfecting - Best Practices Best practices Rotate types of disinfectants Usually alternate between two different disinfectants each cycle Rationale – what resistant microbe one does not eliminate, the other will All equipment and containers should be disinfected in the equipment airlock Using sterile 70% IPA, wipe from top to bottom and back to front From cleanest to most contaminated All components being used in a laminar flow hood should also be wiped with 70% IPA Frequently apply 70% IPA to gloved hands while working in the laminar flow hood
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Disinfectant Usage Surface disinfectant usage on surfaces in various area classifications PA: Peracetic Acid (Peroxygen) Hypochlorite: Halogen IPA: Isopropyl Alcohol NA: Not Applicable Area Classification CeilingsWallsFloorsPlastic CurtainsCritical Work Surfaces Class 100phenolic and monthly sporicide PA or hypochlorite and monthly sporicide all followed by IPA wipe PA or hypochlorite and monthly sporicide all followed by IPA wipe Class 10,000as above NA Class 100,000phenolic NA
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Frequency of Usage/Application Frequency of application for disinfectants The frequency of disinfectant use should be determined by environmental monitoring results Area Classification CeilingsWallsFloorsPlastic Curtains Critical Work Surfaces Class 100daily to weekly mopping daily to weekly mopping or spraying daily mopping daily mopping, wiping, or spraying daily wiping or spraying Class 10,000weekly mopping weekly mopping or spraying daily mopping NA Class 100,000monthly mopping monthly mopping or spraying daily mopping NA
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Environmental Monitoring Program Mandated by cGMP – for facilities, personnel and process utilities Pre-req’s HEPA filter certification program Clean room cleaning and disinfection program Clean room qualification/requalification program Air flow pattern visualization program SOP’s – provide a written description of the program specifics for monitoring and testing of classified areas, personnel and utility systems Also include alert and action levels Personnel training program – documented training!
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Environmental Monitoring Include scheduled monitoring of: Airborne viable/microbial and non-viable particulate levels Microbial contamination on personnel, work surfaces, floors, walls and equipment Microbial contamination of clean utilities Pressure differentials Direction of air flow Temperature humidity
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Water and clean steam monitoring Air monitoring- non-viable Air monitoring – viable Microbial surface testing using RODAC plates Gown and fingertip RODAC testing Environmental monitoring includes…..
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Testing Performed by QC Microbiology Environmental monitoring Utility testing Sterility testing Microbial content testing Bioburden Microbial Limit Bacterial Endotoxin (LAL) Microbial identification Antimicrobial Effectiveness Testing Cleaning validation Media fills
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Air Monitoring-Non Viable Contaminants Non-viable particulate monitoring Provides real time data on the environment Airborne particle counters Measure particles in a number of size ranges; particles of 0.5 microns or greater are generally recognized as indicators of environmental contamination Optical particle counting – vacuum pump pulls air into the sensor; particles in the air sample pass through the optical detection view where a laser light source is concentrated; particles scatter the laser light, which is focused onto a photo diode; the photo diode detects and converts the light signal to electrical impulses; height of impulse is directly proportional to particle size
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Design of Environmental Monitoring Strategy No monitoring can provide a high level of confidence without an overall cleanroom systems management Adherence to cGMP Facility design control Effective supervision Sound corrective action steps Proper employee training A documented sampling program that Describes the procedures and methods for sampling in a cleanroom Identifies the sampling sites, frequency and number of samples Describes the method of analysis and interpretation of results
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Sample Site Selection Air, surface, personnel and utilities that are representative of locations that: Come in contact with exposed product and/or components Are in close proximity to exposed product Are areas of high personnel and equipment flow Contribute to the particulate and microbial levels within an area (e.g., personnel, equipment, etc.) Represent the most difficult or inaccessible areas to clean or disinfect Collectively represent the systems performance over time for a quality product Sites may be selected based on analysis of risk, smoke studies, and or data from the Performance Qualification
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Sample-Site Collection Considerations Consideration should be allowed for extent of exposure or contact at each location, choosing sites with the greatest opportunity for contaminating the product May not be able to sample at the most critical contact sites Sample may increase the risk of contamination Routine sampling sites include non-product contact sites Floors Walls Doors Ceilings May not sample areas with low probability of contamination during processing
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Recommended Critical Sampling Locations (Laminar Flow) TestsTest LocationRationale Critical Sites (Laminar Flow) Microbial Air Particulate Air Sample within approximately one foot of the critical process point or container opening. Sample is to monitor the air where there is increased activity at a location that is close to / representative of the air in contact with the open product. Surface Contact Plate Sample within approximately one foot of the product container opening or critical aseptic process step. Samples are to monitor the critical surfaces where there is increased activity at locations that represent surfaces close to or in contact with exposed product or components. Personnel Contact Plate Sample gown in two locations (chest, forearm) and gloved fingertips of each hand. Samples are to reflect the parts of the gowned and gloved person that are in closest proximity to the exposed product. Examples of critical test-site locations and rationale
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Recommended Routine Sampling Locations (Non-Laminar Flow) TestsTest LocationRationale Routine Sites (Non-Laminar Flow) Microbial Air Particulate Air Sample in a central location in the room or otherwise representative location. Sample is to reflect general area/room conditions which are representative of room conditions. Surface Contact Plate Sample on a representative wall, curtain (outside), door push plate/handle/push button, floor surface, or other frequently- utilized surfaces as determined by the sampling plan. Sample is to reflect the general condition of the area surfaces. Personnel Contact Plate Sample gown in 2 locations (chest, forearm) and gloved fingertips of each hand. Routine personnel testing is to ensure that gowning is performed properly and that glove disinfection is effective. Examples of routine test-site locations and rationale
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Sampling Frequency Vary by room classification and nature of operation Per process or critical site testing Microbial and particulate testing accompanying manufacturing Process: a set of independent steps or manipulations conducted within the same set-up or procedure Begins with aseptic set-up and concludes with product completion Routine monitoring Not performed during processing Minimum – weekly Ensures that operations, cleaning and HVAC continue to operate and perform satisfactorily and consistently
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Environmental Testing for Class 100 areas Example of critical site test requirements and frequency of testing for Class 100 areas Environmental TestClass 100 – Critical Site Testing Frequency Microbial Air (active)once per process (minimum of 1m 3 of air) Microbial Air (passive)one settling plate per designated location Surface Contact Plateminimum of one site per process Product Contact Surface Contact Plate (Sterile Filling Only) minimum of four product contact surface sites to be performed during filling prior to the breakdown of a line at the end of a fill; to include all component bowls and representative fill needles Personnel Contact Platethe fingertips of both gloved hands and the chest and forearm of personnel who perform aseptic operations or testing during processing Particulate Airminimum of once per hour per process in LFH (minimum of 1m 3 of air)
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Environmental Testing and Response Categories Interpretation of results and response Passing: within acceptable, i.e., established, levels. Failures are called environmental test excursions and require a response Alert level: test excursion above the established norm for the site. Usually based on historical statistical data and re-evaluated yearly. Set at 10% - 50% of the action level Action level: result reaches or exceeds area classification as established by industry or regulatory guidelines. Indicates a possible problem that requires a corrective action be taken. An investigation is launched to determine the impact on the product and the root cause (Root Cause Analysis)
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Environmental Testing and Classification Examples
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Industry/Regulatory Guidance for Environmental Monitoring Environmental TestClass 100Class 10,000Class 100,000 Particulate Air ( 0.5 microns/m 3 ) > 3,520> 352,000> 3,520,000 Particulate Air ( 5 microns/m 3 ) > 29 > 2000> 20,000 Active Microbial Air (Colony-Forming Unit or CFU/m 3 ) ≥ 1 > 10> 100 Passive Microbial Air (CFU)≥ 1 N/A Surface Contact Plate (CFU) 1 > 5> 25 Floor Contact Plate (CFU)> 3> 10N/A Compressed Gas Particulate ( 0.5 microns/m 3 ) > 3,520> 352,000> 3,520,000 Compressed Gas Microbial (CFU/m 3 )≥ 1 > 10> 100 Example of industry/regulatory guidelines for environmental testing
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Microbial Identification for All Alerts Microbial Identification Must be determined for all alert and action level results Aids in evaluating impact on product, source Microbes deemed “objectionable”: by FDA include Burkholderia cepacia Escherichia coli – fecal contamination indicator Pseudomonas aeruginosa Salmonella species – pathogens Shigella species Staphylococcus aureus – generally, poor production hygiene Parenteral pharmaceuticals must be sterile, therefore, all microbes are objectionable
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Environmental Excursion Investigations Environmental excursion investigations Required for all action level and recurring alert level findings Assess environmental controls during production Include time, location and conditions during sampling Alert / action results of particulate & viable counts included in report Activities in progress during the sampling Previous cleaning disinfecting Any potential causal events The investigation should attempt to determine the probable cause Many other elements may go into an excursion investigation including past maintenance and activities, trending data, data from other sites, identification of isolated microbes, physical inspection of the site, personnel input and corrective actions being taken
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Corrective Actions Corrective action examples Restricting activities that may increase the bioburden or the total number of microorganisms detected in or on an article Increasing the area ventilation, room pressure, or quality of air delivered (e.g., HEPA filtration) Testing of process HEPA filters and correcting leaks Increasing or altering cleaning/disinfecting procedures and using a sporicidal agent Conducting additional personnel training to reduce practices that may add bioburden Increasing gowning requirements for the area Restricting improper personnel flow from less clean areas improving facility surfaces
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Data Trending Trending environmental monitoring test results There is a need to overcome the delay factor in getting viable test outcomes Prevent filling and package of contaminated product Can decrease costly activities by establishing shift and trending data for the environment Hold until new test data is obtained based on previous testing Developed through regular evaluation of critical areas with graphical representations of results Shift is the tendency for the most recent six-month period to have either higher or lower results than the previous six-month period Statistically significant change Trends only follow the most recent six-month period Shows upward or downward movement of test results Also statistically significant trend
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Testing Methodology Test methodology Airborne counters Non-viable particles Viable microbes on growth media Most be disinfected before use if moved between locations Ideally, should be dedicated to a particular space Direct contact plates and settle plates For surfaces and timed air sampling, respectively Microbial growth media Sampling is done during dynamic “in operation” activities Ensures environment is under control during processing Static sampling is usually only Performance Qualification test
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Air Monitoring Microbial air monitoring Airborne microbes have a higher potential for contamination vs surface microbes Standard unit of measurement is the colony forming unit (CFU) Require 5 day incubation for growth Active air monitoring Performed with equipment ; an air vacuum to draw a specific volume of environmental air sample across a media plate Report as CFU/m 3 (cubic meter) of air in 100 and 10000 level CFU/ft 3 (cubic foot) in 100,000 level Air samplers must be calibrated annually
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Passive Air Monitoring Passive microbial air monitoring Uses settling, or exposure, plates Media plates are opened to the air and dust and microbes are allowed to settle onto the media Exposure time varies but should not exceed 4 hours Reported as CFU/4 hours or some other time period
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Non-viable Monitoring Non-viable particulate monitoring Generally, basis of clean room classification vs viable Real time data, no incubation required Difficult to correlate particle count to viable without direct comparison of both results from tests done at the same time >0.5 micron particle size indicates environment contamination Expressed as the number of particles >0.5microns and >5.0 microns per cubic meter or cubic foot
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Surface and Equipment Microbial Testing Surface and equipment microbial monitoring Methods need to be qualified using standard laboratory procedures Contact impression plate and swabbing techniques yield direct microbial monitoring of surfaces RODAC plates Replicate organism detection and count Media extends above wall of plate base and will make direct contact with a surface Results are reported as CFU/plate Figure: RODAC plate with bacterial growth colonies
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Surface and Equipment Microbial Monitoring (Continued) Surface and equipment microbial monitoring Swabbing Used on irregularly-shaped surfaces not suitable for contact plates Pre-moisten with sterile culture media Sample ~4 in 2 (square inches) while rolling the swab to contact all surfaces Swabs generally are submitted to the microbiology lab for processing All tested surfaces (plate or swab) must be disinfected following sampling Microbial monitoring of gowned personnel People are the greatest source of contamination in a cleanroom Need to be trained in proper aseptic gowning and techniques Must pass initial qualification testing following training Personnel monitoring ensures both
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Personnel Microbial Monitoring Microbial monitoring of gowned personnel Periodic gown and fingertip microbial testing on all personnel Contact plates used on chest, forearms and fingertips of both gloved hands Figure: Microbial personnel / gloved-fingertip RODAC testing Figure: Microbial personnel / chest RODAC testing
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Process Utility Monitoring Initial Qualification (IQ) of a process utility system ensures that the actual performance is consistent with the required performance All process utilities that have contact with the product must be monitored Compressed gas Clean steam Water for injection (WFI) – produced from filtering and distilling potable water
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High-Purity Water Monitoring Same methods for clean steam Tap water contains up to 500 CFUs/ml – not high quality for manufacturing Water standards are set by the USP for water used to manufacture pharmaceuticals and clean equipment = Water For Injection (WFI) Samples taken periodically from holding tank and distribution system Test assess microbial quality – colony count, indicator microorganisms and endotoxin
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Water System Sampling Samples need to be collected in a manner consistent with the manufacturing process Sample from flush cycle if there is one, do not flush for a sample if there is no flush cycle in the manufacturing process Sample from hoses rather than point of use, if appropriate Samples should be refrigerated at 2 o – 8 o C until processed Minimum quantity is 200 ml Use multiple containers for samples used in multiple tests Appropriate safety measures need to be in place for hot water sampling
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Water System Monitoring Microbial colony counts performed by filtration of the water sample through 0.45 micron filter and incubation of the filter on low-nutrient agar for 5 days at 30 o – 35 o C If counts reach alert or action level, microbes must be identified Selective media is used to screen for particular objectionable organism Pseudomonas aeruginosa Burkholderia cepacia Coliforms Any WFI test fails must be investigated to determine product quality Possible quarantine or product discard
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Environmental Monitoring – Media Fills Simulation of an aseptic process that uses microbial growth-promoting culture media in the place of actual product To demonstrate the capability of a specific aseptic process to produce sterile drug products To qualify or certify aseptic processing personnel To comply with regulatory requirements Should be performed under actual full normal conditions for a production process Containers are incubated for 14 days Contamination leads to investigation and ID of isolated microorganisms Must be able to achieve successful media fills before processing product
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Microbial Monitoring and Identification of Microorganisms Start with a pure culture isolated by streak culture Gram stain for classification and morphology Phenotypic methods Observable physical properties Metabolic reactions Genotypic methods Some DNA characterization Often 16S ribosome gene sequence May use RNA characterization ID both genus and species level for isolates from the 100 and 10,000 level areas Mold is also identified at genus and species level by macro- and microscopic methods Changes in cleanroom flora should be investigated for breaches
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Environmental Monitoring and Data Utilization Utilization of information derived from environmental monitoring Can generating massive amounts of data weekly, based on size of facility Interpreted by the QC department Gathered and converted into “knowledge” which needs to managed and communicated To personnel at various levels within the organization To the right personnel at the right level of detail to endure productive actions To determine if the facility is in control or has regained control if once lost Patterns are based on all data collected from all aspects of the processes, personnel, utilities, facilities and any other sources Knowledge Management (KM) involves acquiring, analyzing, storing, and disseminating information
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Knowledge Management Methodology Utilization of information derived from environmental monitoring Figure: Knowledge Management Methodology
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Data Utilization and ICH Q10 Utilization of information derived from environmental monitoring ICH Q10 (International Committee on Harmonization) “Product and process knowledge should be managed from development through the commercial life of the product up to and including product discontinuation. For example, development activities using scientific approaches provide knowledge for product and process understanding.” KM is “...a systematic approach to acquiring, analyzing, storing, and disseminating information related to products, manufacturing processes, and components. Sources of knowledge include, but are not limited to, prior knowledge (public domain or internally documented); pharmaceutical development studies; technology transfer activities; process validation studies over the product lifecycle; manufacturing experience; innovation; continual improvement; and change management activities.”
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Data Utilization Considerations Utilization of information derived from environmental monitoring Questions to be considered for effective Knowledge management What are the data? What do the data mean (i.e., what knowledge can we obtain from the data)? How is this knowledge owned by the area management? How are those who work in the area kept informed of the requisite knowledge? How does one monitor the effectiveness of the actions undertaken and have effective investigations in this area?
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Quality Control, Microbial Quality Control Practices in the Microbiology Laboratory Plan to ensure reliable, reproducible and accurate test results Guidelines are available from government agencies and professional organizations Includes end-user assessment of commercially obtained material Quality Control of Microbiological Culture Media Culture media has the potential to impact every aspect of laboratory operations Three primary aspects the control of the culture media preparation and storage conditions the physical and chemical characterization of culture media growth promotion testing
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Control of Media Preparation – In-house Prep Control of the culture media preparation and storage conditions Areas of QC for media prepared in-house Raw material storage conditions and expiration dates Compounding of the media to prevent errors Weighing Water measurement Improper mixing of ingredients Heating to melt agar before sterilization without damaging heat-labile components Validated autoclave cycle (15 psi, 121 o C, 15 minutes minimum) Need to be able to audit each stage of preparation
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Control of Media Preparation – Commercially Prepared Control of the culture media preparation and storage Areas of QC for commercially prepared media Little control of QC Check for physical and chemical properties and growth promotion Store under controlled conditions through the expiration date Temperature Humidity Light exposure where applicable Dehydration Steps to prevent dehydration of prepared media Discard if the media becomes dehydrated
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Quality Control of Media Control of physical and chemical characteristics Screen for acceptability before more labor intense testing Examine visually for clarity and color Turbidity could result from precipitating components OK to use if the precipitate re-dissolves at incubation temperature Failure should result in discarding the media Check containers for cracks or leaks Growth promoting testing Test with compendial and other stock microorganisms to ensure it supports growth Miles and Misra (surface variable count) method
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Quality Control – Maintenance of Stock Cultures Stock maintained to provide known strains of microbes for compendial tests Confirmation of identity of all microorganisms received must be the first step in building a stock culture supply, even if received from a reputable source Ensure pure cultures (Homogeneity) by streak isolation plating of the culture Stocks should include representatives of Typical morphology Physiological and biochemical characteristics Typical isolates found within the facility Provide culture media to maintaining stable and viable stocks for long periods No excessive growth or metabolic activity Storage at proper temperature Complex methods such as lyophilization (freeze drying) for indefinite storage
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Quality Control – Barrier Isolation Intrinsic need to separate people (high contamination risk) from aseptic processing Barrier isolators are 100 level, airtight enclosures of impervious material Operations are performed from the outside By remote controlled machinery Individuals wearing half body suits of rubber arm length gloves Increasing popularity within the aseptic processing industries
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