Presentation on theme: "Objectives: To further increase awareness about the microbial contaminants found on our bodies and in the environment. To discuss behaviors to minimize."— Presentation transcript:
1 Objectives:To further increase awareness about the microbial contaminants found on our bodies and in the environment.To discuss behaviors to minimize contamination by personnel.To perform lab exercises to reinforce the concepts learned in class.
2 What is Aseptic Processing What is Aseptic Processing? What processes are most vulnerable to contamination?
3 Types of Contamination What is Contamination? There are two broad categories of contamination; particulate and microbial. Non-viable particulate contamination is defined as a solid or liquid generally between .001 and 1000 microns in size. Examples of particulates are dust, fumes, smoke, fogs, and mists.Viable particulate contamination can be defined as a particle that consists of, or supports one or more live microorganisms. Bacteria, viruses, and spores are all examples of viable particulate contamination.
4 Why are they a A MAJOR CHALLENGE!!!! What are SPORESWhy are they a A MAJOR CHALLENGE!!!!Formed in response to harmful environment:HeatDroughtchemicalsIn response to starvation, B. subtilis differentiates into a dormant stress-resistant cell-type known as a spore. Cells initiate spore formation with an asymmetric cell division that generates a large cell (the mother cell) and a small cell (the prospective spore or forespore). Initially these two cells lie side-by-side but later in development the mother cell engulfs the forespore in a phagocytic-like process to produce a cell-within-a-cell. The mother then nurtures the forespore as it prepares for dormancy. Once the spore is fully mature, the mother cell lyses. We use this elegant morphological process to address basic questions in bacterial differentiation
5 Sources of Contamination Many possible sources of cleanroom contaminationEquipment, structures, surfaces can all generate particles.Incoming production components can introduce contaminants.But people working in cleanrooms generate the most particles.The good news?People can control many of these sources.It has been estimated that 75% of pharmaceutical product failures due to contamination can be traced directly to improper handling of products and containers by personnelDavid Carlberg
6 CONTROLLING contamination Facility design Personnel AND visitors wear gowns appropriatelyPersonnel AND visitors behave appropriately
7 Facility Design: Clean Area Classification Lonza Color Code:Good to have the room next to the aseptic area with the same classificationWhat processes are done in each area?What are your alert levels?Are you doing settling plates?FS209 (federal Standard) 1st established in 1963 and revised a few times. Retired in 2001 and replace with ISO classifications
8 Facilities: General Cleanroom Design HEPA/ULPA filters on ceilingExhaust vents on floorDrains in aseptic processing areas are inappropriateAirlocks and interlocking doors to control air balanceSeamless and rounded floor to wall junctionsReadily accessible cornersFloors, walls, and ceilings constructed of smooth hard surfaces that can be easily cleanedLimited equipment, fixtures and personnelLayout of equipment to optimize comfort and movement of operatorsHEPA or ULPA?Ultra low penetration airHEPA 99.97%ULPA % of particles greater than 0.12umA CLEANROOM CAN BE DEFINED as a high-tech, controlled environment where the concentration of airborne particles, humidity, and temperature are all controlled to parameters specified by the ISO
9 Facilities: HEPA Filters High Efficiency Particulate AirMinimum particle collection efficiency:99.97% for 0.3µm diameter particles.DisposableFilter made of pleated borosilicate glass microfiberPleated Sheets of filter material increases surface area of filterA 2x2 ft 7.5in deep mini pleat filter really contains 280 ft2 of filter areaSeparator: often made of aluminumFrame: often made of metalGaskets: on edges to prevent leaks when installedAvailable in a variety of depthsMost micro organisms are bigger than 0.3umViruses are smaller but they can not survive long without the protective effect of attaching to larger particles such as skin cells and saliva droplets. They will dry out in the air or die from environmental stress such as toxic pollutantsEquipment should not disrupt the airflowTurbulant and stagnant air should be preventedLeak Testing at installation and minimum of 2x/year in aseptic processing areaEfficiency Testing to determine rating of the filterShould be able to retain at least 99.97% of particulates greater than 0.3um in diameter
10 Facilities: Cleanroom Classification Class 10,000 cleanroomClass 100 cleanroom
11 Facilities: Cleanroom Classification Lonza Color Code:Good to have the room next to the aseptic area with the same classificationWhat processes are done in each area?What are your alert levels?Are you doing settling plates?FS209 (federal Standard) 1st established in 1963 and revised a few times. Retired in 2001 and replace with ISO classificationsfpm = feet per minute1-100 has unidirectional airflow1, ,000 has mixed air flow
12 Facilities: Pressure Differentials Used to maintain airflow in the direction of higher cleanliness to adjacent less clean areasA minimum of Pascals should be maintained between the aseptic area and an adjacent rooms with differing cleanroom classifications (doors open)Even if room adjacent to aseptic room is same classification, still a good idea to maintain this pressure differentialIf room next to aseptic area is unclassified, it’s best to have a pressure differential of at least 12.5 PaWhat room classification do you have next to your aseptic area?What is your oressure differential
13 Facilities: Air LockPermits the passage of objects and people into a cleanroom.Consists of two airtight doors in series which do not open simultaneously.Spray down materials with 70% IPA before placing in the airlockBefore opening either door, the air pressure of the airlock (volume between the doors) is equalized be the same as the environment behind the next door to open. A gradual pressure transition minimizes air temperature
14 Material NOT permitted in a Cleanroom Facilities:Material NOT permitted in a CleanroomFiber-shedding materials such as cardboard and paperCardboard packaging must be removed and items placed into non-cardboard containers.Wood (i.e. wooden pallets)Undesignated charts
15 Facilities: Cleaning Water should be changed FREQUENTLY 1. Vacuum all accessible surfacesCleaning refers to the physical removal of soil, organic debris, and par-ticulates from a surfaceCleanest part of the room to the dirtiest part of the room.Technician should always work towards the exit. A HEPA filtered vacuum,Studies have shown that cleanrooms that use traditional wiping methods left twice as much contaminant in the cleanroom when compared to a vacuuming program with a HEPA vacuum.The second step in the cleaning process is to wipe all surfaces with an approved cleaning solution. The cleaning should be done in a right to left or left to right manner. Never wipe in a circular motion, as this will contaminate areas, which have already been cleaned. The wipe should be inspected and re-folded after each pass to make sure that the wipe is not soiled and a fresh surface of the wipe is always used on a new cleaning pass. Finally, the cleaning chemical should never be sprayed onto the surface that is being cleaned, rather sprayed directly on the wipe.Lint free polyester or PVA mops attached to stainless steel handles are required. Additionally, a stainless steel or auto-claveable plastic two or three bucket mop system is standard equipment in contamination control. The two bucket system allows for the safe isolation of the contaminants that have been mopped from the surface of the clean-room. In other words, the cleanroom technician will not re-mop an area with dirty or contaminated mop water.Mopping should be done utilizing the pull and lift method, where the technician places the mop no more than three feet in front of the body, pulls the mop straight towards the body, lifts the mop and replaces slightly to the side, three feet in front of the body again. The strokes should overlap by at least four inches. This method, similarly to the wiping technique, prevents the dirty mop surface from touching an area that has already been cleaned.It is very important to change the water in the buckets frequently.ISO Class 7 and 8 cleanroom: change after every 1000 square feetISO Class 5 and Class 6 room: 600 square feetAdditionally, the water should be changed whenever a new room is cleaned, regardless of square footage cleaned.A good contamination control program will document the number of water changes as well as the lot number of the cleaning chemical utilized and the length of time the cleaning chemical was allowed to dwell on the surfaces. Additionally, the documentation should note the specific tasks performed during each cleaning service and by whom.All three cleaning methods, HEPA vacuuming, unidirectional wiping, and pull and lift mopping have been validated at the ISO level.2. Wipe surfaces with a cleaning solution3. Mop floors using a lint free polyester mops attached to stainless steel handlesWater should be changed FREQUENTLY
16 Avoid Cleanrooms When Sick!!! There are about a trillion bacteria on your skin.They dine on ~10 billion flakes of skin that you shed every dayThere are trillions more bacteria in the gut, digestive tract and nasal passages.The digestive system has 100 trillion bacteria (400 different kinds!)This is why you should not chew gum out back!!The risk of contaminating product with one of these organisms is increased by chewing gum.Normal talking (saliva) contaminates a 2-3 ft area. There is evidence suggesting that the louder one talks, the more contaminants are released.
17 Personnel: Hygiene Avoid cleanrooms when ill Frequent bathing and shampooingAvoid getting sunburnedAvoid cosmetics such as face powder, hair sprays, perfumes and aftershaveClothing should be clean, nonfrayed and nonlintingAvoid smokingThe entire epidermis or outer layer of skin is replaced approximately every four days. Oils from the skin are shed; hair is shed. And whatever is on a person’s skin or hair is ultimately deposited into the cleanroom.Keep hair and skin clean to minimize shedding of skin fragmentsSunburn will peal and slough offIllness with increased nasal flow, coughing and sneezing increases particles in airAssign to desk duty. Review cGMPsSmoking immediately before entering cleanroom is especially bad. Continue to exhale thousands of smoke particles long after the last puff
18 INCORRECT 1 2 3 4 Follow the gowning procedure Check your gowned self in the mirrorCover skin and hair (face-masks, hoods, beard/moustache covers, protective goggles, and elastic glovesOverlap gown components (gloves overlap sleeves)Face mask must cover nose and mouthBadges kept inside gownsDo not unzip gown to get tools or anything out of pockets
20 Personnel: Gloves When are gloves worn? What compromises gloves? How often should gloves be examined and sanitized?.Gloves should be worn in critical areasIf gloves become compromised (for example if you touch your face) change themIf gloves become compromised change themExampleCheck gloves for rips and tears on a frequent basisMinimize handling the outside of the gloves with bare handsEnsure gloves cover skin of wrist and overlap gownIf torn, replace immediatelySanitize gloves often
21 Qualifying Personnel Assess after gowning/gloving Microbiological surface sampling of several locationsGlove fingersFacemaskForearmChestPeriodic requalification is necessaryObtain surface samples of each operator’s gloves on a daily basis or in association with each lot.Other strategically selected locations of the gown should also be testedDo not sanitize gloves prior to surface sampling
22 Biological Safety Cabinets Small benchtop sizedclass 100 clean areaIsolators have sealed glove portsor half suits that providecomplete separationbetween the productand personnelIsolators may be closed or open (mouxe hole for material to exit). Air is prevented from entering by continuously having an outward flow of filtered airEven in isolators personnel intervention should be minimized because of glove failures (rips and pinholes)Advantage: place in an ISO 6 or 7 cleanroom and avoid having to build q class 5 cleanroom (which costs big bucks!)Easier to decontaminate, monitor and provide greater sterility than in a conventional cleanroomDisadvantage: physical constraints and limited flexibility. Advance planning of movments
24 ISOPROPYL ALCOHOL Powerful disinfectant and antiseptic Mode of action: denatures proteins, dissolves lipids and can lead to cell membrane disintegrationEffectively kills bacteria and fungiDisinfectant: A chemical used on inanimate objects to kill or inhibit the growth of microorganismsAntiseptic: A chemical used on a living object such as skin and mucous membranes to kill or inhibit the growth of microorganismsWhat is IPA not killed by IPA? bacterial endospores or encapsulated viruses.Why are aqueous solutions are preferredWater is necessary for protein denaturationWater prevents rapid evaporation, giving the alcohol time to work
25 Personnel: Behaviour Minimize movement: Work slowly and purposefully Particles >= 0.3µm emitted per minutePersonnel ActivitySnap SmockMembrane CoverallNo Movement100,00010Light Movement500,00050Heavy Movement1,000,000100Change Position2,500,000250Slow Walk5,000,000500Our skin is a rich source of microbes. Adults have between 11,000 (forearm ) and 1.5 million (scalp) bacteria per square cm of skinDust is 90% skin flakesTypical bacteria:Propionobacterium acnesStaphylococcus epidermidisStaph aureusOnly trained operators should enter aseptic processing areaIf not wearing special garments, can release 10 million dead skin fragments and other particles evrey minutes most of which would be carrying microorganismsNote: Light/heavy movement refer to partial body movements (motioning with arm, tapping toes, etc.). Change of position refers to whole body motion (standing up, sitting down, etc.).Austin Contamination IndexSource: Encyclopedia of Clean Rooms, Bio-Cleanrooms and Aseptic Areas, Dr. Philip Austin, PE, 2000
44 ENVIRONMENTAL MONITORING “In aseptic processing, one of the most important laboratory controls is the environmental monitoring program”Allows potential routes of contamination to be promptly identifiedAllows for implenetation of correctios before conamintation occursThe results of the environmental tests will validate the efficiency of the contamination control program. Results outside the acceptable limits may suggest more frequent cleaning, more stringent gowning requirements, less foot-allowable human traffic inside the cleanroom, or a need for an improved air filtration or air flow.Until there is no human traffic in a cleanroom, there will always be the need for a contamination control program. The contamination control goal is to keep the outside world from entering the cleanroom, and to efficiently remove whatever does enter the room. All contamination control programs will depend on the population of the room, the amount of air and air filtration, and the level of cleanliness that is desired. With proper training, cleaning equipment, and cleaning methodology, the desired level of cleanliness can be achieved and maintained to keep the outside world out.Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice,FDA, September 2004
45 Microbial Monitoring Methods Environmental Monitoring: Surface MonitoringTouch or Contact platesRODAC PlatesSwabsMicrobial Monitoring MethodsEnvironmental IsolatesThe air is monitored for airborne viables and total particulates (can be viable or non-viable)The surfaces of the facility are monitored with RODACs (Replicate organism detection and counting). Fingertips are also monitored in this wayEvery quarter isolates from each area are identified and trended.Each time there is an excursion we identify the organism.Industry standards call for the following testing:• Viable and nonviable airborne sampling under dynamic conditions • Surface sampling before and after processing • Surface sampling of critical or problematic areas • Personnel sampling of gloves and gowns after exposure to aseptic processesOverexposure or exposure to high air flow of settling plates can cause them to dry out slightly (desiccate) and inhibits recovery of micro organisms
46 Environmental Monitoring: Particulate Air Monitoring
47 Environmental Monitoring: Viable Microbial Air Monitoring RCS: RotorCentrifugal SamplerActive Air MonitoringRCS PlusPassive Air MonitoringSettling PlatesActive Air MonitoringImpingers: collect into liquid (not as popular but advantage is you can aliquote the samplesImpaction solid or semisolid surfaceCentrifugalMembrane (or gelatin) samplersCAUTION: Choose an instrument that can be clean and sterilized and does not disrupt unidirectional airflowMost are portable, but should not hold when counting. Microbial contamination from the operator can create a biased estimate of the airborne levels in the facilityCells are experience extreme stress when passing through. As air passes through they can dehydrate.Passive Air Monitoring (qualitative or semi quantitative)Settling Plates – petri dishes with nutrient growth mediumGreat for evaluating small areas whereunidirectional flow is disrupted around equipment or air velocity is reducedNot subject to stressValidation requires evaluating media exposure conditionsSurface Monitoring : clean after contact to remove the nutrient rich residueSubject to significant sampling errors due to manner in which sample is collectedNeed standardization through full documentationIf disinfectant is present ,need a neutralizer in the agar. (tween80 or lecithintouch or contact plates: weights available to exert a uniform and standardized pressureSwabs: great for irregularly shaped surfaces or hard to reach areasOverexposure or exposure to high air flow of settling plates can cause them to dry out slightly (desiccate) and inhibits recovery of micro organisms
48 Environmental Monitoring: Trending Data Why is it important?Evaluate the disinfection efficiencyAre certain microbes migrating into the aseptic processing area from a lesser controller area?Long and short termData by:ShiftRoomOperatorProductContainer typeFilling lineSamplingTesting personnelEvery quarter isolates from each area are identified and trended.Each time there is an excursion we identify the organism.
51 Metabolic Based Assays QC Micro: Identifying MicrobesMetabolic Based AssaysVitekBiolog offers three systems for reading and interpreting MicroPlate identification results. This is a picture of the Biolog MicroStation, our automated system. The other two systems are visual read systems. They are called MicroLog 1 and MicroLog 2. These systems require a computer. The operator manually keys in positive and negative results from the MicroPlate. The MicroLog 1 System is a computerized identification notebook. It has no customizable features. The MicroLog 2 performs identifications but also allows the user to save unknown organisms in customized databases. If you recall the slide that referred to the number of organisms that are outside Biolog’s and other databases, it is very likely that microbiologists will find organisms that are not yet identified. With the MicroLog System you can save unknown patterns for future work. This is a unique and powerful feature of our system. Finally, with the MicroStation, we include an automated reader. The MicroStation Reader standardizes the reading of the MicroPlates, eliminating operator to operator variability. This system also allows the user to customize databases.550 bacteria and yeastlike strains in databaseAPI 20 testsVitek 64 tests
52 Metabolic Based Assays QC Micro: Identifying MicrobesMetabolic Based Assays
53 Reduction of Tetrazolium Violet QC Micro: Identifying MicrobesMetabolic Based AssaysReduction of Tetrazolium VioletStaphylococcus xylosus
54 Metabolic Based Assays QC Micro: Identifying MicrobesMetabolic Based Assays“ID BOX” = ID Result Six possible results:Species IDGenus CallNo IDToo Few Positive MatchesToo Few PositivesToo Many Borderlines
55 Genotype Based Assay: MicroSeq QC Micro: Identifying MicrobesGenotype Based Assay: MicroSeq
56 Genotype Based Assay: MicroSeq QC Micro: Identifying MicrobesGenotype Based Assay: MicroSeqPCR: Polymerase Chain Reaction
57 Genotype Based Assay: MicroSeq QC Micro: Identifying MicrobesGenotype Based Assay: MicroSeqSEQUENCE THE DATA
59 Sepsis: Progression of Symptoms FeverDecreased Blood PressureRapid Breathing and Heart RateSkin LesionsSpontaneous Blood ClottingOrgan FailureDeathActivated Protein CInhibits Factor Va and VIIIa in the blood clotting cascadeApproved only for adults with severe sepsisEstimated 4 day treatment cost of $6,800Removing EndotoxinsFiltrationChromatraphySize: MWCO of 10kDCharge: Positively charged bead or filterHydrophobicitySize is a problem becuae most therapeutic proteins are >10kDSingle Endotoxin molecule is 10-20kd but can aggregate to 1MDaDivalent cations like Ca++ and Mg++ stabilizes aggregatesDetergents break up aggregates
64 QC Micro: LAL Assay TYPES of LAL ASSAYS Gel Clot – a clot forms and stays intact at the bottom of the assay tube when it is invertedTurbidimetric - increase in turbidity.Chromogenic - addition of a peptide thatwhen cleaved during the assay turns adistinct color.Endpoint - Measure turbidity/absorbanceafter a definitive time period.Kinetic - Measure rate of increased turbidity/absorbance.Cambrex sells PyroGene,a Recombinant Factor CrFC is activated by endotoxinActivated rFC cleaves a synthetic peptidereleasing a fluorogenic moleculeAfter a 1 hour incubation the amount of fluorescenceis measured at excitation/emissionwavelengths 380/440nm
65 This product was funded by a grant awarded under the President’s High Growth Job Training Initiative as implemented by the U.S. Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual organizational, non-commercial use only.