Micro basics Flashcards

Question

What is the warehouse temperature range?

Answer 1

Change of hot and cold spots / air circulation due to change in location

Answer 2

IQ, OQ, PQ including empty and full chamber temperature mapping - worst case to see hot and cold spots, any areas of concern, where to locate continuous monitoring probes, seasonal variation should also be mapped

Answer 3

Quarantine all stock within your control What stored in the warehouse and whats the temperature required - is that actually a problem? Determination of impact to product as a result of exposure to higher / lower temperature – look at accelerated storage condition data for product. Look at mapping / Trending – duration of excursion and range Call engineering to investigate and resolve excursion ASAP 25/30 degrees c, 65% H and accelerated 40 degrees C, 70% H

Answer 4

Raise a deviation Annex 1 and ISO14644 – Clean room classification limits Has the area classification is maintained – likely to be Grade C/D depending on open or closed system Environmental monitoring will be less frequent due to lower risk What are they blocked with ? Frequency of Change of HEPA filters – What is the preventative maintenance schedule is it sufficient. Potential impact to product – quarantine stock Review EM data, Air Flow and Change rates, Air pressures, - anything that could identify the point of cause. How long has work been ongoing? Filter Integrity Test Smoke Test

Answer 5

Cleanrooms should be monitored during processing, and also outside of production operations. Monitoring should be performed using principles of risk management to determine the locations and frequency of monitoring required. This rationale should be documented within an environmental monitoring programme. This programme should be set up in all sterile manufacturing environments, documenting the state of control of the facility and assessing the effectiveness of cleaning and aseptic processes. One key factor of the programme is the setting of appropriate action and alert limits. Action and alert limits should be controlled and specified in standard operating procedures. Action limits are generally set through regulatory guidelines such as Annex 1, the USP or ISO standards, whereas alert levels are specific to the facility and should be set based on the results of performance qualification (PQ) tests and trends in historical data. There are several different approaches that can be taken to set alert limits. As long as these are justified in the environmental monitoring plan and the contamination control strategy, it does not matter which method is used. Once each alert limit has been set based on the results of environmental monitoring PQ tests, there should be a procedure in place to track and trend these and re-evaluate the values if necessary. Three of the most commonly used approaches to setting alert limits are the standard deviation approach, the cut-off value approach, or the non-parametric tolerance limit approach. The standard deviation approach should only be used where historical data includes high counts, and when data is normally distributed. To calculate these values, a mean and standard deviation should be calculated; alert levels are set at the mean plus 2 standard deviations. However, data collected from cleanrooms often contain many counts of zero, with a dispersion of counts which are not often normally distributed. Therefore, alternative approaches may also be considered. The cut-off value approach takes into account the 95th percentile of historical data and sets the alert limit at this level. The non-parametric tolerance limit approach similarly is used for samples which are not normally distributed. This takes into consideration the 95th percent confidence intervals are used, where 95% of the samples will pass the established alert limit. Environmental monitoring data should be analysed regularly to assess whether any trends can be identified in the data. Routine review of environmental monitoring data provides confidence in the control of a facility and allows rapid identification of any adverse trends which may be forming. A trend may indicate a potential problem with the environmental control systems in place within a facility. A single breach may indicate an issue at the time, however multiple breaches, or trends, may indicate a loss of control which must be addressed. Environmental monitoring trend reports should be generated regularly to assess and identify trends in data. These reports should assess any excursions which had occurred and identify any trends in excursion rate or critical recovery rate over a period of time. Any Out of Trend or Out of Specification results should be investigated. As part of these reports, alert and action limits should be reviewed to confirm that they are appropriate, and these may be altered to reflect changes within the facility. These may require alteration due to process changes or advances in technology. All assessments of excursions and trends, and changes to limits, should be subject to management review, to ensure that appropriate action is being taken and control is being maintained. Procedures should be in place to define the actions to be taken following limit breaches or trend identification. These will often include the raising of investigations to assess the impact of the event and to determine a root cause. CAPA should be implemented to prevent reoccurrence, and assessment of the return to expected operating parameters should be provided. Any abnormally high number of recoveries, or significant excursions, should have specific investigations performed to determine whether this incident is an isolated occurrence or can be correlated with other recoveries. An assessment of prior recovery rates should be made to indicate any unusual patterns which may be developing. Breaching an alert limit may not require immediate investigation and corrective action; however, some form of follow-up action is usually required to assess whether the affected area may be involved in part of an adverse trend, and to ensure that the affected area has returned to within normal operating parameters. Subsequent alert limit breaches may indicate a trend, and these would warrant the same investigation as an action limit breach. If an action limit is breached, immediate investigation should be performed to assess the impact of the excursion, determine a root cause of the excursion, and initiate subsequent CAPA. This investigation may determine that the root cause was laboratory error and there was no true failure. However, if a true failure is identified, further investigation should be performed, with identification of recoveries and review of additional samples all used to attempt to determine the root cause and aid in the assessment of impact. Follow-up monitoring may be performed, and remedial actions such as additional cleaning or retraining may be required. An assessment may also be required for previously filled batches. Limit breaches provide a rough outline of the cleanliness of the monitoring location and can provide confidence that manufacture has occurred in a clean environment. However, they can only provide a snapshot of information, they cannot provide a full picture of the environment. A single viable sample with no growth does not guarantee control, and conversely, a single excursion does not indicate loss of control. Therefore, as well as looking at breaches, it is important to supplement these with additional data, such as trending and contamination rates.

Answer 6

* Energy – sunlight, inorganics, organics * Nitrogen – gas, ammonia, nitrate/nitrite or a nitrogenous organic compound * Carbon – Carbon dioxide or monoxide, methane or complex organic material * Minerals – Phosphorous, Sulfur, Magnesium, Potassium, Sodium and Calcium * Water * Trace Elements – Iron, Zinc, Cobalt, etc.

Answer 7

* Psychrophiles grow best at temperatures below 10°C or 50°F. * Mesophiles grow best between 20° and 45°C or 68° and 113°F. * Thermophiles grow best between 50°and 80ºC or 122° and 176°F. * Extreme Thermophiles grow best above 80°C or 176°F. * Heat Resistant Organisms are normally mesophiles that have sporulated and; therefore, protected from heat.

Answer 8

* Obligate Aerobic Bacteria ‐ Absolutely require oxygen. * Microaerophilic Bacteria ‐ Require oxygen but uncomfortable at high concentrations. * Facultative Anaerobic Bacteria ‐ Utilise oxygen when available, but can metabolise in the absence of oxygen equally well. * Aerotolerant Anaerobic Bacteria ‐ Show no preference to aerobic respiration if O2 is present. Essentially anaerobic * bacteria. * Obligate Anaerobic Bacteria ‐ Only metabolise anaerobically, cannot survive in the presence of oxygen.

Answer 9

Range of pH for different bacteria is broad, from pH 2-12!

Answer 10

- people, skin * Staph epidermidis can cause lung infections from skin shedding into the air being the microbes method of transportation * Staph Aureus is associated with wound infections, toxic shock syndrome, boils and pneumonia

Answer 11

- spore formers - dirt, environment * Bacillus subtilis can cause eye infections and even meningitis in people with weakened immune systems

Answer 12

- water - pyrogenic response

Answer 13

* Mould from cardboard, building fabric and exposed plaster - spore formers * Yeast

Answer 14

* Mycoplasma has no cell wall so cannot be gram stained. * Mycoplasma can escape sterile filtration. * Associated with Arthritis and Pneumonia in immunocompromised patients

Answer 15

Liquids pose the highest risk then creams, ointments, freeze drier product, tablets and capsules present the lowest risk - the more aqueous the product the more at risk from gram negative organisms

Answer 16

Streaking done to obtain a pure culture of a microbe

Answer 17

all neutral pH approx pH 7

Answer 18

Broad Spectrum General Purpose Media Enhanced Media Selective Media Differential Media Enrichment Media

Answer 19

Highly nutritious media formulated to grow a broad spectrum of microorganisms from low numbers. Typically used for enumeration. * Tryptone Soy Agar * Nutrient Agar

Answer 20

General purpose media supplemented with specific growth requirements in order to allow the growth of fastidious organisms with specific growth requirements. * Chocolate Blood Agar ○ Contains Factors X and Y from blood essential for the growth of Neisseria sp * Milk Agar ○ Contains dried milk powder to enhance the growth of dairy pathogens

Answer 21

Media which contain growth inhibitors which exclude the growth of unwanted organisms but allow the growth of the target organisms. These inhibitors take advantage of differences in biochemistry between different organisms. * Cetrimide Agar ○ Contains Cetrimide, a broad spectrum disinfectant with little activity against pseudomonas species which utilise cetrimide as a carbon source ○ Pseudomonas CFC Agar Contains Cephaloridine (a broad spectrum antibiotic), Fucidin (a gram negative antibiotic) and cetrimide, to select against all organisms except pseudomonads

Answer 22

Media which have been formulated to allow the growth of a number of bacteria but which result in the ability to differentiate between the isolates. These media are often also selective media in order to reduce the target spectrum for differentiation. * Baird Parker Agar ○ Formulated to differentiate between Staphylococcus aureus and other Staphylococci. The medium contains potassium tellurite and lithium chloride which inhibit most coliforms. § Staphylococci metabolise the tellurite to telluride turning the colonies black. The agar contains egg yolk emulsion. § S. aeureus contains lipase which clears the egg yolk around the colony, and lecithinase which precipitates lecithin close to the colony. * MacConkey Agar ○ Contains Bile salts which inhibit all organisms except those which have adapted to the environment found in the mammalian gut. The agar also contains lactose and a pH indicator (neutral red). Any organisms that can tolerate bile and will ferment lactose to lactic acid will be visible as colonies with a yellow halo on a blue agar.

Answer 23

Very often a target organism may be present in a sample in very low numbers compared with other organisms closely related to it. In order to be able to isolate the target organism its numbers must be increased in relation to other organisms. Enrichment media attempt to do this by enhancing the nutritive qualities appropriate to the target organism while introducing an inhibition to other unwanted organisms. Enrichment media are particularly useful if the target organism is sub‐lethally damaged. * Selenite Cystine Broth ○ Sodium biselenite inhibits organisms other than salmonellae while L‐Cystine and sodium phosphate enhance the growth of salmoneallae. Incubation at 370C for 24 hrs will increase the growth of low numbers of salmonellae sufficiently to be detected on selective agars such as XLD and DCA. * Buffered Peptone Water ○ The peptone in BPW provides all the nutrition and the absence of carbohydrates minimises acid production. The solution is well buffered which ensures a neutral pH. These conditions allow the recovery of low numbers of sub‐lethally damaged organisms without them being over‐run by other more aggressive organisms

Answer 24

* to recover inoculated organisms * to inhibit appropriate organisms * to produce the correct differential responses * to produce characteristic colony morphology

Answer 25

* easy to grow * hard to grow * objectionable * facility specific isolates. No more than 100CFU should be used

Answer 26

* Colour * clarity * pH - pH is important as it can give an idea of the quality of the preparation and give an indication of any acidic degradation

Answer 27

To recover inoculated organisms To inhibit appropriate organisms To produce the correct differential responses To produce characteristic colony morphology

Answer 28

While the manufacturers of the dehydrated media subject their products to extensive testing the preparation of finished media introduces critical variables which require the finished product to undergo rigorous fertility testing after preparation. The criteria under assessment include: 1) The ability of the preparation to recover low numbers of inoculated organisms often referred to as Growth Promotion Testing (GPT). 2) The ability of the preparation to produce characteristic colony morphology. 3) The ability of selective media to inhibit appropriate organisms. 4) The ability of differential media to produce the correct differential responses. Associated with these tests are physical tests including clarity, colour and pH. pH in particular can give a detailed insight into the quality of the preparation as many agar are not buffered and are sensitive to slight changes in pH due to the presence of acidic degradation products.

Answer 29

The fundamental basis of any fertility testing is the quality of the cultures used to assess the medium. Each medium should be tested with a range of organisms. 1) This range should include, easy to grow organisms, difficult to grow organisms and organisms to be inhibited. 2) The range should include typical cultures recommended as these will have been used in the initial development of the agar. 3) The range should include laboratory isolated strains to ensure it is capable of detecting organisms appropriate to the company. These organisms should be maintained under precise conditions to ensure that the genome is consistent and not subject to subculture mutations. In practice this means no more than five passages from the type culture freeze dried vial. To maintain this discipline requires a structured culture maintenance programme. Fertility testing should be undertaken on all batches of agar received, no matter how small, as variations can occur during any preparation and distribution. In fact more care is often taken when preparing large batches rather than small batches because of the effort involved.

Answer 30

However, the media is being tested, in order for the answers obtained to be meaningful the methods used should be examined and the limits of usefulness should be understood. That is the methods should be validated and this includes serial dilution of microbial suspensions and plating out techniques. Serial dilutions can be notoriously inaccurate. Technicians should be tested to confirm that their dilution and plating technique is accurate. Parameters for consideration would be: * Specificity – ability to detect and enumerate a specific organism Precision could be validated by: ○ Repeatability in the ability to obtain consistent results when plating the same dilution in triplicate ○ Reproducibility in the ability to obtain consistent results when plating the same dilution using different analysts * Range – the dilutions range determined to provide accurate and precise counts. * Linearity – the ability to count the organisms directly proportional to concentration of organisms in the dilution. Comparative fertility can introduce error if batches are assessed in isolation. Data should be recorded in a way that can identify adverse trends associated with serial dilution i.e control suspension count, dilution selected, person performing the test.

Answer 31

1. Miles Misra 2. Pour Plate 3. Spread Plate 4. Most Probable Number (MPN) 5. Membrane Filtration 6. Spiral Plating 7. Dip Slide 8. Direct Cell Counting

Answer 32

* Dilute inoculum 1 in 10 to 5-40 cfu/ml * Add 0.05ml drop to agar plate * 4 dilutions giving range 1-10,000 * 4 drops per dilution * Incubate * Pros ○ Accurate Count ○ Reduces number of plates ○ 20 cfu/mL * Cons ○ Calibrated pipette ○ Multiple dilutions ○ Labour intensive

Answer 33

* Add 1-5 ml sample to plate * Add approx. 20 ml molten agar to sample (<45C) * Agitate to disperse sample and cool agar * Incubate * Pros ○ 1-500 bacteria/mL ○ Simple * Cons ○ More plates may be needed for accuracy ○ Observation can be obscured ○ Agar Temperature can be too high

Answer 34

* Dry Plate * 0.1mL sample * Spread with sterile rod * Incubate * Pros ○ simple * Cons ○ Correct dryness * Colony Separation ○ Dilution

Answer 35

* Prepare Both tubes * Inoculate first set * Incubate and read * Pros ○ Very accurate ○ Versatile ○ Selective * Cons ○ Materials ○ Time

Answer 36

* Pass solution through a 0.45 micron rated filter * Place filter on agar plate * Incubate * Pros ○ High resolution ○ Simple ○ No inhibition ○ Low cost * Con ○ Must be liquid

Answer 37

* Prepare sample * Add to plate by machine * Machine dilutes as added * Incubate * Pros ○ Automated ○ Rapid ○ Resolution 100 cfu/mL * Cons ○ Needs liquid ○ Cost of capital equipment Need to use calculation tables

Answer 38

* Buy slides * Dip into solution * Incubate * Pros ○ Simple ○ Rapid ○ Selective * Cons ○ Inaccurate - estimate ○ Resolution 100 cfu/mL

Answer 39

* Sample is put through a measuring tube * Can be stained to differentiate between live and non-viable particulate * Sensor records and counts * Pros ○ Applies to gases and liquids * Cons ○ Staining can react with metabolizing cells ○ Bacterial clumping ○ Only works with low numbers ○ False positives and negatives

Answer 40

Does the organism pose a risk? Is it Pathogenic, an opportunistic pathogen, capable of causing spoilage, have resistant characteristics e.g. bacillus spores heat resistant How do we control / remove? Modify cleaning and disinfection procedure Disinfection of water systems Redesign equipment Changes to operational practices

Answer 41

Obtain a pure culture Note obvious cultural characteristics e.g. morphology Perform highly discriminatory tests Perform barrage of less discriminatory tests Compare and contract data with known characterised organisms

Answer 42

* Probe Technology Polymerase Chain Reaction (PCR) Process Isolate nucleic acid Amplify nucleic acid Detect specific sequences with labeled DNA probes * Targets Chromosomal DNA Ribosomal RNA * Polymerase Chain Reaction (PCR) is currently used to detect mycoplasmas in cell cultures and viruses in human plasma donations.

Answer 43

DEFT Rapid Enumeration Method Process: * Preparing sample solution/suspensions * Filtration through a black filter * Staining of organisms with fluorescent stain (e.g. acridine orange) * Detection of organisms by epifluorescence microscopy * Sensitivity - As low as 10 cfu/mL * Time - minutes Chemiluminescence Rapid Enumeration Method Process: * Absorption of fluorochrome by organisms * Development of chemiluminescence * Detection by laser scanning analysis * Sensitivity - Down to a single organism * Time - 60 minutes

Answer 44

* Substrate utilization * Gas chromatography of fatty acids * PCR / DNA / RNA sequencing

Answer 45

There are steps required Prior to ‘Rapid’ Identification using Substrate Utilization which take time. These steps are: * Obtain pure culture (24h min) * Gram Stain * Some biochemical discrimination? (oxidase etc.) * Preparation of inoculum suspension

Answer 46

Gas Chromatography of Fatty Acids Sample Preparation * Obtain pure culture (24-48h) * Chemically modify fatty acids (2-3h) * Methyl esters required

Answer 47

Intensive environmental monitoring Process simulations Setting routine monitoring plans

Answer 48

There are 13 main stages of the microbiological validation of clean rooms: 1. Train the cleanroom operators - all colleagues working or entering any controlled area must have the proper training 2. Establish satisfactory operation of cleanroom complex air systems and other key equipment; for example, HEPA filters, LAF, sterilizers, etc. 3. Qualify cleanroom for physical parameters 4. Perform microbiological monitoring of the cleanroom surfaces, equipment, corners/crevices… any high traffic or hard to reach place that requires special attention. 5. Clean the room and all surfaces and equipment with the validated cleaning agents and disinfectants specified per written procedures. 6. Once cleaning has been performed per procedure, subject cleanroom to intensive grid microbiological monitoring. * Active & passive air samples * All surfaces The minimum standard for the cleanroom in its unmanned, or static state, must be met before proceeding! This standard CANNOT be less strict than the most current regulatory expectations for the cleanroom in its operational state. 7. Confirm results of microbiological monitoring are in compliance with particulate standards for unmanned rooms. 8. Select specific microbiological monitoring sites for use in an operational state. Selection should be based on: * Hard to clean or disinfect areas, general ‘hot spots’ * Representative surfaces and locations of higher human traffic flow * Areas of product risk or vulnerabilities such as in-feeds, out-feeds and the critical product pathway 9. Take well-trained and suitably gowned operators into the cleanroom and perform physical and microbiological monitoring & sampling of: * Active & passive air * Surfaces * Personnel This monitoring & sampling should take place while personnel perform simulated production activities to collect data from a dynamic environment that reflects the conditions experienced during operation. 10. Use the data collected from step 9. to confirm compliance with regulatory standards for cleanrooms in an operational state. If your results are outside regulatory boundaries, initiate an investigation and repeat steps until the issue is identified, corrected through the CAPA system, and compliant results achieved. 11.Perform process validation by appropriate process simulation - remember sterile media fills for aseptic products. 12. Review the data collected from the cleanroom in its operational state… * Physical environmental results * Microbiological environmental results * Sterile media fill results 13. If all results comply with regulatory standards and requirements… * Establish provisional operational microbiological monitoring sites, frequencies, and limits based on observations and data collected from the validation process

Answer 49

Multidisciplinary team Map area layout and divide into coded grids (Grade A 0.5m2, Grade B, C, D 4m2) Walk the facility and select potential sample locations considering: Personnel flow Number of personnel Sources of contamination Materials flow Proximity to exposed product Proximity to direct product contact parts Complexity of activities or interventions Time taken for activities Ease of cleaning Surface characteristics

Answer 50

Slit to agar Sieve sampler Gelatin pad sampler Centrifugal sampler

Answer 51

Air is drawn into a "slit orifice", under vacuum onto a revolving agar plate. The plate is slowly rotated below the slit so that collected organisms are spread across the agar surface. Strengths of the slit to agar method are: * Manufacturers stated efficiency is 98% * The unit provides a time-count relationship, and it is * Portable Weaknesses are that it is * Not suited for high concentrations of organisms, and * Some units cumbersome

Answer 52

The Sieve Sampler is where air is drawn in and accelerated through holes in a circular sieve plate to impact particles onto the underlying agar. At a constant sampling rate, the air velocity and impaction speed of entrained particles is a function of sieve plate diameter. Strengths of the Sieve Sampler are the * Manufacturers stated efficiency of 95 – 99%, and the * Multistage configurations capable of separating particles into size ranges A weakness is that if multiple particles enter through the same hole in the sieve, they will appear as one cfu rather than however many came through that location.

Answer 53

The Air Gelatin Pad Sampler operates by drawing air through a gelatin filter pad, which can then be dissolved in sterile diluent for plating out. Alternatively, the pad can be transferred directly onto agar for culturing where it eventually dissolves into the surface of the agar plate. Strengths of the Gelatin Pad Sampler: * They have a very high flow rates which allows a large volume of air to be sampled, and * The Gelatin pad can be placed in the sampling head either directly in front or at the end of an extension A weakness of this sampler type is that the Gelatin pads are hard to manipulate and prone to desiccation.

Answer 54

Centrifugal Active Air Sampler. Air is drawn into an open-ended cylinder housing and impacts onto rotating impeller blades (∼ 4096 prm). Following impact the air is forced radially outwards onto a plastic strip coated with agar. The strengths are: * Cheapest method available * Most portable * Battery operated The weaknesses are: * Air sampling rate is assumed to be 40l/min. which may only apply to particles greater than 4µm. Although the RCS model is considered 100% efficient for particles >16µ diameter it is not considered accurate for particles <4.0µ * The sampler causes significant turbulence

Answer 55

Petri dishes filled with medium, usually tryptone soya agar, are placed in predetermined validated areas within the aseptic core and clean rooms to monitor the environment by allowing any airborne particulate and bacteria to “settle” onto the plate. Advantages: * Monitoring can take place throughout the period of production * Very little intervention is required into critical areas * Settle plates cause very little turbulence * Settle plates are very convenient… nothing could be simpler and cheaper! Disadvantages: * Settle plates will not detect rapid changes * Volume of air sampled is not known * Method is not quantitative * Counts are often reported as cfu per plate. This method of reporting disguises the fact that results could be reported as cfus per surface area of plate per hour * Settle plates could dry out, causing microbial death. However, Work by Whyte and Niven demonstrated that a 13% reduction in agar water content had very little detrimental effect on the viability of deposited microorganisms. This water loss is equivalent to 24 hours exposure in still room air and 6 hours in unidirectional flow.

Answer 56

Surfaces are most commonly sampled for microbial contamination by the use of Contact plates, prepared so that they have a convex sampling surface which facilitates pressing in the agar onto the surface of interest. This method can be used for all surfaces including fingertip and gown sampling; however, it should be emphasised that a surface sampled in this fashion will carry small amounts of the sampling medium which will support the multiplication of any micro-organisms present and post sampling disinfection and cleaning must be carried out on hard sampled surfaces. Operators perform personnel sampling just before they leave the aseptic core and any residue on gowning is professionally sanitized between uses. The use of sterile swabs avoids this difficulty but may not offer the same convenience and efficiency of capture as Contact agars.

Answer 57

Selecting EM Sites for Monitoring For general monitoring for effectiveness of housekeeping, maintenance and operator discipline, sites should be selected which can adequately provide data on such parameters including: * Air sampling (usually by active methods) * Hard surface sampling (walls, floors, worktops etc) * Liquid sampling (e.g. disinfectants, cleaning fluids) Sampling locations should give good coverage of the whole clean room and associated areas including: * Changing rooms * Air locks * Transfer hatches * Preparation area And be chosen to include “worst case” locations: * High traffic areas such as personnel routes * Areas where air flows may be low * Adjacent to air extract returns * Step over barriers in changing rooms * Sinks * Door handles

Answer 58

* ISO Class 5 or better: Each shift (if a Class 5 hood is used to control non-viable particulates only, then no microbiological testing is required * Isolator systems: active air sampling: once per day * Isolator systems: surface monitoring: at the end of each campaign * Aseptic processing area adjacent to ISO Class 5 e.g. Class 7: Each operating shift * Other support areas in aseptic processing (ISP Class 8): Once per week Non-Aseptic Processing * Oral solid and liquid filled capsules - quarterly * Liquid Orals, topicals, rectals - monthly * Vaginal suppositories , nasal sprays - weekly * Inhalation aerosols - daily

Answer 59

* Alert Level is defined as that level which implies that the normal level of operation, with its normal acceptable variation, may have been exceeded. * Exceeding the alert level requires documented follow up but not necessarily corrective actions. * Action Level is defined as the level which when exceeded requires immediate documented follow-up and corrective actions. ISO 14698 also requires the establishment of a Target level which is the level that indicates the normal, controlled performance of a location. When the Target level is breached, the Action level is triggered. They even supply a calculation to determine the relationship between the Target level and the Action level. Action Level = Target Level + 3(√Target Level) * If the Alert level is exceeded, tell production immediately and document room temperature, humidity, differential pressures and air changes per hour. Record organism(s). * If the Action level is exceeded, document the control measure, as if an alert limit was exceeded, contain any impacted product, investigate and determine root cause, if possible, and perform corrective action. Since the operators are the major source of clean room contaminants, be sure to look at: ○ Levels of procedural compliance ○ Levels of aseptic practice ○ Exit monitoring results (any adverse trends) ○ Finger plating results ○ Workload increase? ○ Equipment problems ○ Staffing levels ○ Motivation ○ Management issues ○ Talk to the operating teams in a nonthreatening manner * The Quality decision makers will need to determine if the impacted batches can be released. Failing a batch on environmental monitoring results is always going to be an empirical decision based upon all of the data available. Considerations to be taken are: ○ Was Grade A put at risk? ○ What was the extent of non-compliances? ○ How atypical was the result? ○ How dangerous was the contaminant? ○ How severe was the breakdown in the control measures? ○ What interventions were being undertaken? * The decision to release a batch must be fully justified. If the results indicate a widespread breakdown in environmental controls, then release may be difficult. A robust environmental monitoring program will provide an early warning of a break-down in control measures if it is managed correctly.

Answer 60

“Moving Average” and “CUmulative SUM” graph technique, known as CUSUM

Answer 61

The moving average technique will take away large peaks and indicate changes in the underlying population average. However, the graph must be used carefully as an inappropriate averaging period can mask information rather than making it clearer. Inspectors are very suspicious of moving average graphs as they can be used to hide poor performance.

Answer 62

* A very powerful technique to spot changes in underlying trends is the CUmulative SUM control chart. This technique takes into consideration the normal performance of the data set and cumulatively sums the deviations from it. If the deviations are equally positive and negative the graph will be under control. If however there is a shift from the norm then this will be exaggerated and the graph will indicate the change very quickly.

Answer 63

* Failure Mode and Effect Analysis or FMEA is a technique which has been used successfully to assess risk associated with activities and operations. Although its roots are in engineering, FMEA has been successfully used for risk assessment in numerous industries including pharmaceutical manufacturing. * FMEA involves defining a failure mode and its effect, then analyzing it using the following criteria… ○ What is the Severity of the consequence of failure (S) ○ What is the probability of Occurrence of the failure? (O) What is the probability of Detection of the failure should it occur? (D)

Answer 64

Incubate at 30-35C for 3days for TAMC Incubate at 20-25C for 5days for TYMC

Answer 65

Typically bought in sterilised (gamma irradation) and ready to use. Growth Promotion Testing performed to show media supports growth TAMC (aerobic) on TSA (Tryptic Soy Agar) TYMC (yeast/mould) on SAB/SDA (Sabouraud Dextrose Agar) Specific medias for specific m/orgs, e.g. MacConkey – Ecoli R2A – Water Testing

Answer 66

Available Water pH Temperature Nutrients

Answer 67

Critical for microbial growth, tablets ~ 0.3-0.4 Aw Bacteria typically require ~0.9 Aw Moulds and Yeasts ~0.6 Aw Liquid products much higher risk of microbial growth

Answer 68

Membrane Filtration: Solution filtered through membrane, membrane then put on agar and incubated. (Direct Inoculation) Total Plate: Direct inoculation of sample on plate. Pour Plate: Add sample, then add molten agar, agitate, allow to set and incubate Most Probable Number Method: least accurate method, but used for certain products with very low bioburden. Series of dilutions. Method used based on nature of product & required limit of m/organism. Need pure culture to perform identification. ID methods include API/Vitek.

Answer 69

Rods (bacillus) or cocci (round). Cocci arrange in clusters/chains. Gram positive (purple) or negative (pink) Gram + cell wall. Source of spores, e.g. Gram + bacillus. Gram - no cell wall. Source of endotoxins/pyrogens. Also have ‘slime’ layer that can forms biofilms in water systems. Spores = bacteria in hibernation until temp and environment suitable. Difficult to remove

Answer 70

Preservative Efficacy Test (PET) or Antimicrobial Efficacy Test (AET) – Pharm Eur 5.1.3 Performed to demonstrate suitable levels of antimicrobial preservative to prevent growth / limit microbial contamination during normal conditions of storage & use. Efficacy of antimicrobial preservative may be increased or decreased by API/formulation/container and closure used. The item under test should be inoculated in its final container. Inoculate (with approx. 106) and incubate at 20-25. Test a sample to see if there’s any growth. Incubate again and test again. Test at 7, 14, (21) and 28 days (based on product). Looking for a 1-3 log reduction by day 14 in the no. of viable orgs from what was inoculated and no sub. increase. Typical Organisms to be inoculated: (CAPS) Candida albicans (yeast) Aspergillus brasiliensis (mould) Pseudomonas aeruginosa (Gram negative bacteria) Staphylococcus Aureus (Gram positive bacteria)

Answer 71

Chemical Alcohols Hydroxybenzoates (methyl) Phenols

Answer 72

Gram positive rod shaped bacteria Typically found in the environment (soil, atmosphere, cardboard) Possible BI contaminant!! SPORE FORMING

Answer 73

Gram positive cocci bacteria Prevalent skin organism

Answer 74

Yeast Cause spoilage of products Can produce moderately resistant spores

Answer 75

Gram negative rod shaped bacteria Commonly found in the environment in water and soil. Indicative of standing water. Human and plant pathogen, largely opportunistic in nature which means infection tends to occur alongside existing illness or disease. Typically contaminates WATER SYSTEMS. The organism tends to infect immunocompromised patients, essential to check for absence of Pseudomonas in finished product and water systems. Can form Endotoxins – can be major issue if in product. Can cause serious complications in patients with CF. Infections often life threatening. Die off quickly in absence of moisture and temperatures over 45C.

Answer 76

Gram positive cocci (round) bacteria Prevalent skin organism with an estimated 25% of people being long-term carriers. Can come from healthy people but also associated with wound infections and boils. Due to its presence on the skin, it can be a major cause of contamination of pharmaceutical product due to improper handling or poor aseptic technique. Certain strains are a particular problem as they have developed antibiotic resistance. High TVC's in product or water samples is indicative of bad practice by operators. Whilst commonly found and usually harmless on the skin, once it is introduced into the bloodstream it can cause life threatening illness. S. aureus is a major cause of sepsis when introduced into the body via implants, surgical incisions and injectable medicines.

Answer 77

Mould Environmental contaminant Common cleanroom contaminant Produce SPORES (not as resistant as bacterial spores)

Answer 78

Performed to check agar used within the laboratory before use. Inoculate agar with a low level inoculum (<100 cfu) of known test organisms The test organisms must be regularly replaced in line with expiry dates Looking for a recovery of 50-200 cfu Based on organisms appropriate to your facility

Answer 79

Micro methods must be validated to demonstrate they are capable of consistently performing as expected. Demonstrate that the expected organisms can grow on the agar used Demonstrate that selective media only grows the organisms of interest Demonstrate that growth is reflective of the inoculation level Demonstrate different batches of agar, different analysts etc. do not impact the results (robustness) Pharmacopoeial methods are available for testing of microorganisms therefore full method validation is not required if using these methods. However a verification exercise should be performed to demonstrate your lab will find microorganisms if present in your products. Documented in protocol and report.

Answer 80

Control microbial contamination during use – not to cover for poor GMP Broad spectrum of antimicrobial activity Compatible with API, excipients and container closure system Effective at required pH Safe to use (toxicity profile), physically undetectable and fast acting Chemically stable (natural/synthetic) & active against m/orgs in all conditions

Answer 81

Type of pyrogen and cause fever Released from outer membrane of Gr- bacteria when cell dies Must be removed/inactivated in sterile products Inactivation: Dry Heat (depyrog. process) Removal: Filtration/Rinsing with endotoxin free water Tests: Rabbit (develops fever) / LAL test (horseshoe crab extract - physical clotting reaction) / Recombinant Factor C reagent (fluorescence).

Answer 82

Absence of all viable m/orgs Targeting SAL of 1x10-6 Tested after product has been sterilised Required sample size in 2.6.1

Answer 83

PCR: Isolate and amplify nucleic acid and detect specific sequences with labelled DNA probes. Currently used for virus detection. ATP bioluminescence: Detects ATP in living organisms by using the energy from ATP to produce light. Can be used to count levels of m/orgs present. Fluorescent labelling: Sample is labelled with a fluorescent stain which is detected by microscopy. Does not distinguish between viable/non-viable cells. Electrical resistance: Sample is inoculated into growth media and incubated. Measure of impedance (current) increases due to m/org numbers. MALDI-TOF: Mass spec method for ID, does not require prior Gram staining prior to ID. Very quick. Need pure sample. Need library for comparison.

Answer 84

316L Stainless steel Turbulent flow 1-3m/sec No dead legs (3 x i.d. of pipe) Sloped pipes Pipes completely drainable Smooth internal surfaces Heat (>65oC)/ UV /chemical sanitisation (Hydrogen Peroxide) Hose Control!! Tanks: spray ball, all surfaces constantly wetted, self draining, bursting disc, low level sensors Bursting disc, pressure relief Biofilm – remove by increasing flow rate

Answer 85

CPPs - flow, temperature, low level sensor in tank to prevent tank fully draining, continuous flow CQAs - Micro quality, conductivity, TOC, endotoxin

Answer 86

URS: Water quality required? Source Water? Seasonal changes? Daily & Max demands? Circulating temp? Total number of user points? Hot/cold user points? Sanitisation

Answer 87

Potable to Purified by RO Dump control to drain water if in-line testing fails spec Sanitisation by Ozone Alarms

Answer 88

Links to ‘towns’ water Water Pre-treatment? Storage vessels (MOC, capacity, location) Circulating/re-circulating system Design of user points (use of hoses?) Pumps Locations of valves/drains Links with Utilities/services Pipe slopes In-line monitoring Sanitisation

Answer 89

IQ: Ensure system is installed as per Design Spec OQ: Ensure system can produce Purified Water & all elements are working as installed

Answer 90

PQ: Confirm the system reliably produces water to the URS/FS Protocol to define & when system can be used under quarantine & when fully released Must be run over the course of a year to account for seasonal variations. Phase 1: 14 days intensive monitoring - Water not suitable for use Sample all points daily & test all parameters (including in-going) Sample points inc. each stage of processing, storage, distribution and points of use Use Pharm Eur acceptance criteria System should operate continuously with no deviations/failures Develop & finalise sanitisation Write SOP for operation, sanitisation, maintenance, trouble shooting Use data generated to develop internal Alert Levels Phase 2: 14 - 20 days – System may be used under quarantine (if in protocol & QA Approved & passes all criteria) Generally Sample all points daily & test all parameters (including in-going), but may rotate sample points (QRM). Use Pharm Eur acceptance criteria Finalise internal alert/action levels Phase 3: 1 year – System can be used if Phase 1 & Phase 2 Pass (if in protocol & QA Approved & passes all criteria) Reduced frequency based on QRM Rotate user points and parameters Monitor seasonal variation

Answer 91

Environmental Monitoring – PDA Guidance (See slide 59) Non-viable particles: Continuous counting in Grade A, recommend continuous counting for Grade B Number of sampling sites taken from ISO14644 Viable Particles (Micro): Active Air (air sampler) Passive (Settle plates) Usually TSA Exposed to atmosphere will collect particles & micro Contact plates Surfaces Finger dabs Exit monitoring: Contact plates taken from locations on operators gowning. TRENDING CRITCAL in environmental monitoring. Set Alert & Action limits Setting up a Environmental monitoring based on: QRM. Look at smoke studies – support QRM. Worst case areas. Facility / Eqp / Processes Process inputs Risk assessment periodically reviewed for effectiveness

Answer 92

Legislation ISO14644/Annex 1 provides guidance on requirements for particulates and microbial contamination based on room grade Guidance on EM is provided in PDA guidance (steriles) Non sterile facilities unclassified - typically monitor to Grade D/C Purpose: Provide supporting data to demonstrate contamination control measures are sufficient, e.g. cleaning, training, material control etc. Ensure background levels are acceptable for the process being performed. Documented EM programme that includes: QRA to determine sample locations and frequency based on risks in your facility. Considerations include expected microbes (process/raw materials), process design, product/personnel flow, process risk areas. Defined schedule for routine monitoring covering all sampling locations: Sample locations chosen based on ‘worst-case’ locations Suggested frequency non-sterile OSD: quarterly Suggested frequency liquids: monthly Suggested frequency steriles: Daily/weekly Batch/Process specific monitoring to support batch disposition Approved sampling diagrams and instructions EM procedure for storage/transfer/receipt/testing/OOS/OOT investigation Trained personnel Data is used to establish action (typically Annex 1 limits) and alert limits EM data must be trended and reported (CUSUM) Water monitoring typically monitored as separate system, not part of EM

Answer 93

Performed to confirm effectiveness of cleaning for product contact eqp. Non-product contact eqp dependant on risk (must be documented rationale). May take some time to complete CV  validation with verification after each batch acceptable approach until sufficient data to consider cleaning method validated Visual check importance, but cannot be only criteria used  demonstrate API removal Factors which impact the effectiveness of cleaning should be assessed, e.g. rinse times. Use worst case situations for basis of validation studies. Limits for carryover should be tox based, with limits used justified in risk assessment. CHMP Guideline on setting HBEL in shared facilities Is compound toxic: PDEs calculated using NOAEL and safety factors Non EU: 1/1000 therapeutic dose still an expectation therefore many companies calculate both and apply tightest limit Limits to include removal of cleaning agents. Worst case product approach used  Justify Micro & Endotoxin risk considered Clean & dirty hold times and maximum campaign length times Cleaning procedures to detail location & rationale for swab points & define acceptance criteria Swab recovery from all cleaning methods validated  demonstrate recovery from all PC materials Manual cleaning must be check for effectiveness. If can’t clean, product dedicate with other controls. Initial new product assessment: Potency, toxicity, solubility, allergenic ingredients, cleanability

Answer 94

Reducing bioburden within a system/piece of equipment by flushing, e.g. water system sanitisation Sanitising agent must be removed from system. Effectiveness of flushing must be validated and monitored.

Answer 95

Using disinfect to kill/inactivating microorganisms on surfaces Must have an established documented disinfection programme Surfaces must be CLEAN before disinfection Rotate disinfectants to prevent bacterial resistance/report to changes in environment E.g. phenolic compounds, alcohols, chlorine compounds (sporicidal) Programme should include regular use of a sporicidal following QRM Disinfectants must be purchased from approved supplier Disinfectants must be validated to show they are effective at killing targeted microbes

Answer 96

Temperature: Higher temps more effective Application: Physical methods of application more effective Concentration: Level that will be effective Time: Must be in contact with surface for sufficient time

Answer 97

Established time period which materials can be held under specified conditions Hold time studies establish these time periods, use QRM Documented using protocol / report / approved sampling plan Typical tests based on stage of material: E.g. phy props, appearance, assay, blend uniformity, micro, dissolution/profile, rel subs, LOD, viscosity, friability etc. Test using validated stability indicating methods

Micro basics Flashcards

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