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Objectives: To further increase awareness about the microbial contaminants found on our bodies and in the environment. To discuss behaviors to minimize.

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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 SPORES Why are they a A MAJOR CHALLENGE!!!! Formed in response to harmful environment: Heat Drought chemicals In 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 contamination Equipment, 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 personnel David Carlberg

6 CONTROLLING contamination Facility design
Personnel AND visitors wear gowns appropriately Personnel 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 classification What 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 ceiling Exhaust vents on floor Drains in aseptic processing areas are inappropriate Airlocks and interlocking doors to control air balance Seamless and rounded floor to wall junctions Readily accessible corners Floors, walls, and ceilings constructed of smooth hard surfaces that can be easily cleaned Limited equipment, fixtures and personnel Layout of equipment to optimize comfort and movement of operators HEPA or ULPA? Ultra low penetration air HEPA 99.97% ULPA % of particles greater than 0.12um A 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 Air Minimum particle collection efficiency: 99.97% for 0.3µm diameter particles. Disposable Filter made of pleated borosilicate glass microfiber                                                Pleated Sheets of filter material increases surface area of filter A 2x2 ft 7.5in deep mini pleat filter really contains 280 ft2 of filter area Separator: often made of aluminum Frame: often made of metal Gaskets: on edges to prevent leaks when installed Available in a variety of depths Most micro organisms are bigger than 0.3um Viruses 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 pollutants Equipment should not disrupt the airflow Turbulant and stagnant air should be prevented Leak Testing at installation and minimum of 2x/year in aseptic processing area Efficiency Testing to determine rating of the filter Should be able to retain at least 99.97% of particulates greater than 0.3um in diameter

10 Facilities: Cleanroom Classification
Class 10,000 cleanroom Class 100 cleanroom

11 Facilities: Cleanroom Classification
Lonza Color Code: Good to have the room next to the aseptic area with the same classification What 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 fpm = feet per minute 1-100 has unidirectional airflow 1, ,000 has mixed air flow

12 Facilities: Pressure Differentials
Used to maintain airflow in the direction of higher cleanliness to adjacent less clean areas A 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 differential If room next to aseptic area is unclassified, it’s best to have a pressure differential of at least 12.5 Pa What room classification do you have next to your aseptic area? What is your oressure differential

13 Facilities: Air Lock Permits 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 airlock Before 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 Cleanroom Fiber-shedding materials such as cardboard and paper Cardboard 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 surfaces Cleaning refers to the physical removal of soil, organic debris, and par-ticulates from a surface Cleanest 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 feet ISO Class 5 and Class 6 room: 600 square feet Additionally, 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 solution 3. Mop floors using a lint free polyester mops attached to stainless steel handles Water 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 day There 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 shampooing Avoid getting sunburned Avoid cosmetics such as face powder, hair sprays, perfumes and aftershave Clothing should be clean, nonfrayed and nonlinting Avoid smoking The 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 fragments Sunburn will peal and slough off Illness with increased nasal flow, coughing and sneezing increases particles in air Assign to desk duty. Review cGMPs Smoking 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 mirror Cover skin and hair (face-masks, hoods, beard/moustache covers, protective goggles, and elastic gloves Overlap gown components (gloves overlap sleeves) Face mask must cover nose and mouth Badges kept inside gowns Do 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 areas If gloves become compromised (for example if you touch your face) change them If gloves become compromised change them Example Check gloves for rips and tears on a frequent basis Minimize handling the outside of the gloves with bare hands Ensure gloves cover skin of wrist and overlap gown If torn, replace immediately Sanitize gloves often

21 Qualifying Personnel Assess after gowning/gloving
Microbiological surface sampling of several locations Glove fingers Facemask Forearm Chest Periodic requalification is necessary Obtain 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 tested Do not sanitize gloves prior to surface sampling

22 Biological Safety Cabinets
Small benchtop sized class 100 clean area Isolators have sealed glove ports or half suits that provide complete separation between the product and personnel Isolators may be closed or open (mouxe hole for material to exit). Air is prevented from entering by continuously having an outward flow of filtered air Even 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 cleanroom Disadvantage: physical constraints and limited flexibility. Advance planning of movments

23 Vertical Laminar Flow Area

24 ISOPROPYL ALCOHOL Powerful disinfectant and antiseptic
Mode of action: denatures proteins, dissolves lipids and can lead to cell membrane disintegration Effectively kills bacteria and fungi Disinfectant: A chemical used on inanimate objects to kill or inhibit the growth of microorganisms Antiseptic: A chemical used on a living object such as skin and mucous membranes to kill or inhibit the growth of microorganisms What is IPA not killed by IPA? bacterial endospores or encapsulated viruses. Why are aqueous solutions are preferred Water is necessary for protein denaturation Water prevents rapid evaporation, giving the alcohol time to work

25 Personnel: Behaviour Minimize movement: Work slowly and purposefully
Particles >= 0.3µm emitted per minute Personnel Activity Snap Smock Membrane Coverall No Movement 100,000 10 Light Movement 500,000 50 Heavy Movement 1,000,000 100 Change Position 2,500,000 250 Slow Walk 5,000,000 500 Our skin is a rich source of microbes. Adults have between 11,000 (forearm ) and 1.5 million (scalp) bacteria per square cm of skin Dust is 90% skin flakes Typical bacteria: Propionobacterium acnes Staphylococcus epidermidis Staph aureus Only trained operators should enter aseptic processing area If not wearing special garments, can release 10 million dead skin fragments and other particles evrey minutes most of which would be carrying microorganisms Note: 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



















“In aseptic processing, one of the most important laboratory controls is the environmental monitoring program” Allows potential routes of contamination to be promptly identified Allows for implenetation of correctios before conamintation occurs The 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 Monitoring Touch or Contact plates RODAC Plates Swabs Microbial Monitoring Methods Environmental Isolates The 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 way Every 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 processes Overexposure 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: Rotor Centrifugal Sampler Active Air Monitoring RCS Plus Passive Air Monitoring Settling Plates Active Air Monitoring Impingers: collect into liquid (not as popular but advantage is you can aliquote the samples Impaction solid or semisolid surface Centrifugal Membrane (or gelatin) samplers CAUTION: Choose an instrument that can be clean and sterilized and does not disrupt unidirectional airflow Most are portable, but should not hold when counting. Microbial contamination from the operator can create a biased estimate of the airborne levels in the facility Cells 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 medium Great for evaluating small areas whereunidirectional flow is disrupted around equipment or air velocity is reduced Not subject to stress Validation requires evaluating media exposure conditions Surface Monitoring : clean after contact to remove the nutrient rich residue Subject to significant sampling errors due to manner in which sample is collectedNeed standardization through full documentation If disinfectant is present ,need a neutralizer in the agar. (tween80 or lecithin touch or contact plates: weights available to exert a uniform and standardized pressure Swabs: great for irregularly shaped surfaces or hard to reach areas Overexposure 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 efficiency Are certain microbes migrating into the aseptic processing area from a lesser controller area? Long and short term Data by: Shift Room Operator Product Container type Filling line Sampling Testing personnel Every quarter isolates from each area are identified and trended. Each time there is an excursion we identify the organism.

49 QC Micro: Identifying Microbes

50 QC Micro: Identifying Microbes
Gram Stain

51 Metabolic Based Assays
QC Micro: Identifying Microbes Metabolic Based Assays Vitek Biolog 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 database API 20 tests Vitek 64 tests

52 Metabolic Based Assays
QC Micro: Identifying Microbes Metabolic Based Assays

53 Reduction of Tetrazolium Violet
QC Micro: Identifying Microbes Metabolic Based Assays Reduction of Tetrazolium Violet Staphylococcus xylosus

54 Metabolic Based Assays
QC Micro: Identifying Microbes Metabolic Based Assays “ID BOX” = ID Result  Six possible results: Species ID Genus Call No ID Too Few Positive Matches Too Few Positives Too Many Borderlines

55 Genotype Based Assay: MicroSeq
QC Micro: Identifying Microbes Genotype Based Assay: MicroSeq

56 Genotype Based Assay: MicroSeq
QC Micro: Identifying Microbes Genotype Based Assay: MicroSeq PCR: Polymerase Chain Reaction

57 Genotype Based Assay: MicroSeq
QC Micro: Identifying Microbes Genotype Based Assay: MicroSeq SEQUENCE THE DATA


59 Sepsis: Progression of Symptoms
Fever Decreased Blood Pressure Rapid Breathing and Heart Rate Skin Lesions Spontaneous Blood Clotting Organ Failure Death Activated Protein CInhibits Factor Va and VIIIa in the blood clotting cascade Approved only for adults with severe sepsis Estimated 4 day treatment cost of $6,800 Removing Endotoxins Filtration Chromatraphy Size: MWCO of 10kD Charge: Positively charged bead or filter Hydrophobicity Size is a problem becuae most therapeutic proteins are >10kD Single Endotoxin molecule is 10-20kd but can aggregate to 1MDa Divalent cations like Ca++ and Mg++ stabilizes aggregates Detergents break up aggregates

60 QC Micro: LAL Assay

61 QC Micro: LAL Assay Tumor Necrosis Factor Endotoxin MACROPHAGE
Interleukins (1,6,8) Endotoxin MACROPHAGE


63 QC Micro: LAL Assay The Lysate

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 inverted Turbidimetric - increase in turbidity. Chromogenic - addition of a peptide that when cleaved during the assay turns a distinct color. Endpoint - Measure turbidity/absorbance after a definitive time period. Kinetic - Measure rate of increased turbidity/absorbance. Cambrex sells PyroGene, a Recombinant Factor C rFC is activated by endotoxin Activated rFC cleaves a synthetic peptide releasing a fluorogenic molecule After a 1 hour incubation the amount of fluorescence is measured at excitation/emission wavelengths 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.

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