Professional Documents
Culture Documents
Outline of Topics
Microbiology 101 Process Flow Manufacturing Facility Design Sterilization and Depyrogenation Environmental Control and Monitoring Personnel Training Aseptic Process Control and Validation In-Process and End-Product Testing
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Microbiology 101
Bacteria
Bacterial cell envelop most important physical feature. Disinfectants and antibiotics work again cell envelope Gram +: peptidoglycan, teichoic acid Staph, Bacillus, Strep, Clostridia Gram - : peptidoglycan, lipoprotein, lipopolysaccharide layers sources of LPS endotoxin Pseudomonas, E coli, Salmonella, Klebsiella, Serrati Various classifications Gram positive or gram negative Anaerobic or aerobic or facultative Pathogenic or non-pathogenic or opportunisitic Vegetative or spore (Bacillus and Clostridium) Spores--form by vegetative cells in response to environmental shock such as nutrient depletion; hundreds more resistant than vegetative forms; selected as biological indicators to measure effectiveness of sterilization methods
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Cellular forms more like human cells which makes these organisms harder to kill without killing human cells ~ 100 of 1,000 known fungi are pathogenic Candida spp. and some dermatophytes are only known fungi transmitted from person to person Mold spores exist, but much less resistant than bacterial spores
Intracellular parasites; do not need food Very small, thus can pass through even 0.1 micron filters Readily inactivated by heat > 60C Very susceptible to surface disinfectants Environmental detection very costly Sterile facility conditions too harsh for viruses to survive
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Knowing a term called the D-Value for the biological indicator used to validate a sterilization (heat, gas, radiation) process is the basis for sterilization validation.
D stands for decimal reduction time that is the time required for a one log reduction in the microbial population
Various mathematical approaches for calculating the D-value D-value is the basis for calculating required Z and F values
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Degree of contamination depends on particle level Buildings Potential mold contamination; nutrients come from plaster, worry about cracks, inadequate sealings Water Always concerned about Pseudomonas Raw Materials Less concerned these days because little/no natural sources Packaging Mold spores, especially if any paper sources People (!)
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> 1.2 million aerobic bacteria per m2 in head and neck region 0.9 3 million per m2 on hands and arms Much higher numbers of viable anaerobes (Proprionibact. acnes) Fully gowned person sitting in cleanroom releases ~ 15,000 particles per minute Walking person releases ~ 157,000 particles/min Ratio of total particles > 0.5m and viable aerobic organisms = 6006007000 to 1. People release 600-1300 total particles per hour in > 0.5m size range 600with ~ 40 CFU viable aerobic organisms among these Properly gowned cleanroom worker will contribute 10-100 CFU of 10viable aerobic organisms to the environment per hour
-- Berit Reinmueller papers, presentations 9
Process Flow
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5 6 7 8
1 7 10
1 3 5 50
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(expect 0) (expect 0)
3,520,000 100
Flow of Manufacturing
Warehouse (unclassified) Preparation (US: ISO 8 or better; EU: ISO 7 or better) Formulation (US: ISO 8 or better; EU: ISO 7 or better)
Prepare solution in appropriate mixing tank (add ingredients to Water For Injection)
The EMEA requires estab. of buffer area where intermediate air quality separates Grade A and D areas
Aseptically fill formulated bulk solution into primary package and stopper (partial stopper if product is to be freeze-dried
Fully insert stopper, remove from freeze dryer Store finished dosage forms at appropriate temperature (usually 2-8C)
100% inspection
Outlier particles and / or agglomerates Problems with dispersion of certain components Foaming due to dumping of recirculated suspension in the tank Production stops and starts Inadequate flushing of filling tubing Particle settling / separation
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Lack of homogeneity
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FacilitiesGMP Requirements
Section 211.42: Must be separate or defined areas of operation to prevent contamination, and that for aseptic processing there be, as appropriate, an air supply filtered through HEPA filters under positive pressure, and systems for monitoring the environment and maintaining equipment used to control aseptic conditions. Section 211.46: Equipment for adequate control over air pressure, microorganisms, dust, humidity, and temperature be provided where appropriate and that air filtration systems, including prefilters and particulate matter air filters, be used when appropriate on air supplies to production areas. Section 212.42 (proposed GMP for LVP) Walls, floors, ceilings, fixtures, and partitions in controlled environment areas shall Have a smooth, cleanable finish that is impervious to water and to cleaning and sanitizing solutions Be constructed of materials that resist chipping, flaking, oxidizing, or other deterioration.
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Facility Layout
Sterile Block design
Control P among various areas Proper directional flow of air, materials and people movement Avoidance of clean to dirty crossovers Minimize critical area work force
Mechanical Area General Area Clean Area Aseptic Adjacent and Aseptic Area
Floors: Epoxy terrazo, urethane, Mipolam (solid vinyl) on concrete Walls: Mipolam, cement plaster, Kydex (acrylic/PVC) shields Ceilings: Mipolam, lighting and fixtures recessed Curtains: Vinyl or lexan
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Modular Construction
e.g. www.pharmadule.com
Basic materials of construction much like a normal building (Concrete, Steel, Gypsum) Each module built separately, then entire set of modules put together (Sweden) Entire setup tested to ensure that everything works properly with customer approval (Sweden) Modules disassembled (equipment, utilities, etc remain within each module), shipped to customer site, reassembled Everything re-qualified 12-18 months average from start of design to commissioning and qualification
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Air Handling Systems for Cleanroom Control, Sterile Pharmaceutical Products, Avis, ed., 1996, p. 57.
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Methods of Sterilization
Thermal Moist heat (saturated steam under pressure) Dry heat Chemical Gaseous Radiation Bright Light Filtration
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Thermal Sterilization
Lethality depends on Degree of heat Duration of exposure Humidity Lethal mechanism is coagulation of protein in the cell Moist heat is more effective than dry heat e.g. Clostridium botulinum destruction @ 121C Moist heat: 10 minutes Dry heat: 120 minutes Basic principle: Raising the boiling point of water from 100C at atmospheric pressure to 121C at 15psig above atm.At 121C, saturated steam, when hitting a cooler surface, will condense, releasing up to 500+ cals/g degree. By comparison, dry heat at the same temperature will only release 1 calorie/g degree
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Steam Sterilization
Primary applicationssterilizes Equipmenttanks, filling nozzles, aseptic attachments, lyo chambers Process tubing Filters and apparatuses Rubber closures Final products, if possible Basic cycle Preconditioning--must remove air, then heat Sterilization Drying--removal of air and release of pressure
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Steam Sterilization
Key Factors Steam must reach innermost recess of material being sterilized Biggest challengeremoval of all sources of air For solutions in containers, wall of container must be heated to raise temperature of solution inside the container to generate steam There MUST be a source of water for steam to be generated under temperature/pressure Not suitable for anhydrous oils, powders or any system that is enclosed and dry inside Lag time and cycle based on nature of materials to be sterilized and configuration of load At least 7 different designs of steam sterilizers, differentiated by the post-sterilization phase.
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Use either regular batch ovens or tunnel sterilizers Most effective method to depyrogenate Not used for drug product sterilization, but for sterilization of processing itemsglass, stainless steel
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Radiation Sterilization
Damages nucleoproteins of microbes Few variables: dose and time Types Gamma radiation Cobalt 60 high energy photons Accelerated electrons Beta particles (electron beam) Ionizing radiations Less penetrative than gamma Ultraviolet light
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Radiation Sterilization
Big concern: formation of radiolytic by-products (e.g. *OH) which in turn can cause damage to ingredients and surfaces Typical dose: 25 kGray (2.5 mRad) Sterilizes plastic devices, gowns, potentially drug products Significant growthnearly 50% of sterilization market 12 D overkill approach always used Bacillus pumulis is B.I.
Typical D values = 1.7-2.0 kGray Typical dose = 25 kGray Can use as low as 2 to 8 kGray
Gamma sterilization still method of choice; E beam sterilization being re-evaluated carefully, especially for finished products
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Gaseous Sterilization
Sterilization gases: Formaldehyde Ethylene oxide Propylene oxide Beta-propiolactone Ozone Peracetic ccid Vapor hydrogen peroxide Chlorine dioxide
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Ethylene Oxide
Very potent and highly penetrating gas Alkylating agent used for years and for which standardized efficacy measurements exist Carcinogenic agent Standard mixture: 12 EtO:88 Freon Profound environmental issues! Alternative diluent: CO2 Main issue: Residuals--ethylene glycol and ethylene chlorhydrin Must have specifications on residue levels Typically 1 mcg/mL or g for EtO 50 mcg/mL or g for EtCh OSHA and EPA have tried to curb use because of residues
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Filtration
Objectives
Clarification Sterilization (majority of SVPs sterilized by filtration)
Filter types
Different polymers and porosities
Filter integrity
Bubble point Diffusion test
Controversial issues
Non sterile filtrates through 0.2m filters!
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Pyrogens/Endotoxins
Pyrogen = Pyro (Greek = Fire) + gen (Greek = beginning). Fever producing, metabolic by-pdts of microbial growth and death. Bacterial pyrogens are called Endotoxins. Gram negative bacteria produce more potent endotoxins than gram + bacteria and fungi. Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not present in cell-free bacterial filtrates Stable to at least 175C; steam sterilization ineffective Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm) so endotoxins can easily pass through 0.22m filters Sources: Water (main), raw matls, equipment, process environment, people, and protein expression systems if using gram negative bac.
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Depyrogenation Methods
Endotoxin is removed or destroyed, not killed Removal by rinsing (dilution), distillation, RO, ultrafiltration (10,000 nmwl filters), electrostatic or hydrophobic attraction, activated carbon, affinity chromatography Destroyed/inactivated by
Acid and base hydrolysis Oxidation--Use of hydrogen peroxide Alkylation--Use of ethylene oxide (very questionable) Dry heat Moist heat--At least 3 hours exposure Ionizing radiation--Must use fairly high doses
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Environmental Monitoring
Roomsurfaces and air People Utilities Water Compressed gases Clean steam HEPA filters Filling nozzles after media fill Performance qualification Air Monitoring Nutrient agar plates Slit samplers Electronic counters
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Environmental Monitoring
Shift-by-shift monitoring of air and all surfaces Written procedures to include List of locations to be sampled and when Duration of sampling Surface area and air volume sizes Established frequencies Limits Critical surface sampling may be performed at conclusion of process Air and surface samples should be taken at actual working level/surface Daily surface samples of each aseptic operators gown and finger pads, at random intervals Personnel monitoring program considered a separate procedure from air and surface EM to accommodate different types of follow-up actions (e.g. increased scrutiny, retraining, requalification)
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Environmental Monitoring
Low level contamination not always detected. Because of existence of false negatives, consecutive growth results should not be considered the only type of adverse trend. Look for increased evidence of contamination over a given period in comparison to that normally detected Establishing limits and trends Limits established based on relationship of location to operation Both alert and action limits Individual results exceeding alert limits should focus on trend data and deviation records and actions Individual results exceeding action limits will prompt more thorough documented inquiry Must have SOPs describing review, ID, and response to trends by QC unit and regular updating responsible mgmt. Routinely generate trend reports as function of location, shift, lot, room, operator, or other search parameters Investigate atypical microbes found
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Personnel Training
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Characteristics of sterile dosage forms I Freedom from microbial, pyrogenic, and particulate contamination The problem of contamination I Sources of contamination I Basic microbiology I Implications and consequences of a contaminated product Testing I Objective I Hands-ongowning, media fill, broth test I Remedial What FDA looks for when auditing training
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Between media fills, regular training updates supplemented by routine evaluations by supervisory personnel of each operators conformance to written procedures and basic aseptic techniques during actual operations
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I I I I
Mechanisms used to evaluate training I Media fills and/or broth tests I Sampling gowning surfaces I Documentation of observations of operators in actual practice I Written tests I Frequency of monitoring and evaluation Appearance and actions of operator during processing Observation of hand washing and sanitation Retraining procedures (failed plate counts, improper actions) Degree of supervision
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Inappropriate techniques were observed within aseptic areas Different degrees of proper aseptic gowning were widely observed Not all personnel observed in the aseptic areas were wearing goggles as required Operator observed leaning over the accumulator for no apparent reason Exaggerated movements (dancing) was observed Plexi-panels were open on both sides of critical area; operators could talk to one another Too may people located within aseptic areas One operator noted to run up to the filling line, arms waving A group of five operators congregated inside the Class 100 critical area Too much leaning over exposed vials observed Operator appeared to be touching sterile tweezers while hand stoppering Operator went into critical area three times without sanitizing their hands Operator not correctly using tweezers to remove overturned bottles on accumulator Cleaning/Sanitizing of aseptic areas not unidirectional Head covers did not always cover the face Beard covers did not always cover beard
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Periodic re-qualification
Assure validity of initial qualification Typically done semi-annually
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In-Process Potency Preferably by UV, sometimes need HPLC pH Optical density (for suspensions) Density Osmolality (occasionally) End-Product Appearance, other possible physical tests Assay pH Sterility Endotoxin Particulate matter Lyo productsrecon time, residual moisture Dispersed systemsparticle size, dispersibility, etc Syringes/cartridgesfunctionality tests
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I I I I
Guidelines for Processing Aseptic Drug Products, FDA, 2004 Current Practices in the Validation of Aseptic Processing--2001, Technical Report No. 36, Parenteral Drug Association Validation of Aseptic Pharmaceutical Processes, FJ Carleton and JP Agalloco, eds, Informa USA, Inc., 2007 RJ Harwood, JB Portnoff, and EW Sunbery, The Processing of Small Volume Parenterals and Related Sterile Products, in Pharmaceutical Dosage Forms: Parenteral Medications, Vol 2, 2nd ed, KE Avis, HA Lieberman and L Lachman, eds, Marcel Dekker, NY, 1992. MJ Groves and R Murty, eds., Aseptic Manufacturing II, Interpharm Press, Buffalo Grove, IL, 1995. MJ Groves, WP Olson, and MH Anisfeld, eds., Sterile Pharmceutical Manufacturing, Vols 1&2, Interpharm Press, Buffalo Grove, IL, 1991. Development and Manufacture of Protein Pharmaceuticals, SL Nail and MJ Akers, eds., Kluwers-Wolters, New York, 2002. Articles in PDA Newsletter, PDA J PST, BioPharm, trade magazines
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Thank you!
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