Pharmaceutical microbiology Flashcards

Question

Manufacturing a non-sterile ointment and micro is 100 CFU/g. It is gram-negative. What is the source and what do you do?

Answer 1

1. Initial Assessment and Containment * Identify the nature of the organism: Gram-negative bacteria can indicate potential objectionable organisms (e.g., Pseudomonas spp., E. coli). → Send for species-level identification to determine regulatory impact. * Likely sources: o Water system (especially if purified water is used in manufacturing or cleaning) o Leaking equipment, biofilm, insufficient drying o Human contamination – mucosal flora, poor hygiene, or handwashing * Confirm water system status: Is there a validated and qualified system at the site? * Immediate actions: o Quarantine the manufacturing line and the affected batch o Hold any in-process or recently manufactured batches ________________________________________ 2. Start OOS Investigation Phase 1A & 1B (Laboratory Investigation) * Confirm lab technique, analyst competency, method validation * Rule out sampling/handling error Phase 2 (Full Investigation) * Open deviation in the QMS * Conduct impact assessment: o Marketing Authorisation (MA) – Is the specification breached? o GMP impact – breakdown in CCS, human contamination, water quality failure o Product impact – Gram-negative organisms can be endotoxin-producing; assess toxicological risk ________________________________________ 3. Immediate Risk Management * Identify whether other batches are affected: o Check reference samples o Review batch genealogy and material traceability o Look at cleaning validation status and changeover records ________________________________________ 4. Root Cause Analysis (e.g., Ishikawa/Fishbone) a. QMS: * Related deviations, OOS, CAPA, PQR trends b. Personnel: * Capacity issues, gowning logs, training records * Behaviour review (e.g. CCTV for aseptic practices, handwashing) c. Facility & Equipment: * Environmental monitoring trends * Cleaning and maintenance logs * PPM records * Water system trends (e.g. microbial, TOC, conductivity) d. Documents & Records: * Batch Manufacturing Record (BMR) comments, deviations, holds e. Process: * Cleaning validation/verification status * Any change control open or recent? f. QC: * Reference samples, method robustness, trend data g. Supplier/Materials: * Review API/ excipient CoAs * Goods-in inspection data * Supplier quality history or complaints h. Audits: * Review any internal/external audit findings linked to this process or area ________________________________________ 5. CAPA and Further Actions * Implement CAPAs against each confirmed root cause * Conduct effectiveness checks over time (e.g., trending, re-training) * Revalidate water system or cleaning processes if compromised * Consider retraining and requalification of operators if hygiene breach is suspected ________________________________________ 6. Product Disposition and Recall (if applicable) * If released batches are affected: o Recall risk assessment – likely Class 2, Patient-level recall o Convene recall committee, consult with DMRC (Defective Medicines Report Centre) before execution o Prepare and submit recall closing report to DMRC ________________________________________ Viva-ready summary statement: “I would immediately quarantine the batch and initiate an OOS investigation, escalating to deviation and full root cause analysis if no assignable lab cause is found. I would assess the impact on product, GMP, and MA, with special attention to water system integrity and personnel hygiene. If released product is affected, I’d initiate a recall process involving the DMRC. CAPAs would be implemented and monitored for effectiveness to prevent recurrence.”

Answer 2

1. Initial Assessment and Containment * Organism identified: Staphylococcus aureus – a Gram-positive human skin flora, indicating possible operator contamination or compromised hygienic control. * Immediate action: o Quarantine the purified water system — prevent further use for manufacturing, cleaning, or preparation of solutions. o Hold all batches that used water since the last compliant result. ________________________________________ 2. Confirm and Narrow the Source * Review sampling locations to identify contamination origin: o Sample after each key system component (e.g. RO membrane, UV light, storage tank, loop). o Sample all user points and return loop to define scope. * Check last sanitisation record, any maintenance activities, and sampling technique (possible operator error). ________________________________________ 3. OOS Investigation (per SOP) * Phase 1A – Lab-based investigation: o Confirm method validity, analyst technique, equipment calibration. * Phase 1B – Hypothesis testing: o Retest original sample (if permitted), re-sample same point. * If no assignable cause → Phase 2: o Full investigation into the water system. ________________________________________ 4. Phase 2 Deviation and Impact Assessment * Open a deviation in the QMS. * Conduct impact assessment: o Regulatory: Any breach of MA or MIA requirements? o GMP: Was contaminated water used for cleaning, rinsing, or product contact? o Product safety: Assess any released or in-process product for risk (use of water in final product, rinsing, cleaning). o Review EM, cleaning, and equipment logs. ________________________________________ 5. Immediate Technical Actions * Perform flush and sanitisation of the system (chemical or thermal, per SOP). * Daily monitoring at all user points and loop return until system is proven back under control. * Ensure no manufacturing resumes until microbiological compliance is restored and approved by QA/QP. ________________________________________ 6. Root Cause Analysis (RCA) * Use tools such as Ishikawa (Fishbone) to explore: o Personnel: Gowning practices, training, hand hygiene, sampling error o Equipment/facility: Dead legs, biofilm, pressure imbalances, maintenance issues o Process: Was sanitisation effective? Was system in validated state? o Documentation: Deviations, logbooks, change controls ________________________________________ 7. CAPA and Requalification * Implement corrective and preventive actions based on root cause. * Requalify water system if needed (e.g. 3 consecutive compliant days across all points). * Perform effectiveness check over time. ________________________________________ 8. Decision on Product Impact * If product was manufactured with potentially contaminated water: o Trace impacted batches o Review risk to product quality o Quarantine or recall if sterility or safety is compromised (escalate to DMRC if necessary) ________________________________________ Viva-ready summary: “On being informed of S. aureus in the purified water system, I would immediately quarantine the system and hold affected batches. I’d confirm the source by targeted sampling, then open an OOS and deviation. If a true OOS is confirmed, I’d assess GMP and product impact, flush and sanitise the system, and conduct RCA and CAPA. Requalification and effectiveness checks would be required before returning the system to use.”

Answer 3

1. Immediate Containment * Quarantine the water system: prevent use in manufacturing, cleaning, or formulation until the system is under control. 2. Temporary Contingency Plan * Liaise with Production, QA, and Procurement: o Arrange temporary supply of Purified Water or WFI from a qualified external supplier. o Ensure supplier holds appropriate GMP certifications (e.g. WDA, MIA, GDP compliance). o Review and approve supplier CoA and transport conditions. 3. Sanitisation of the Water System * Execute sanitisation as per SOP: o Flush the system with increased velocity o Thermal sanitisation: raise temperature to ≥65–80°C (continuous or periodic hot water) o Chemical sanitisation options:  Ozone (highly effective, low residue)  Peracetic acid, hydrogen peroxide, or chlorine dioxide (based on system compatibility) o Ensure neutralisation and flushing post-chemical sanitisation ________________________________________ 4. Monitoring and Requalification * Perform daily microbiological, TOC, and conductivity testing: o Sample after each major component (e.g. RO, EDI, storage tank, distribution loop) o Include all user points and return loop * System must pass consecutive tests (e.g. 3–7 days) before release for GMP use ________________________________________ 5. QMS and Risk Management * Open a deviation and assess impact on GMP and product: o Was contaminated water used for cleaning, rinsing, or product contact? o Hold and assess impacted batches * Conduct RCA using Ishikawa: o Personnel, equipment, sanitisation failure, biofilm formation, documentation * Implement CAPA and perform effectiveness checks

Answer 4

Based on SOP/validation but: o Ozone – highly effective, no chemical residue o Hydrogen peroxide o Peracetic acid o Sodium hypochlorite (used with caution due to corrosiveness)

Answer 5

Purified water is used for: * Final rinse of equipment for non-sterile manufacturing * Formulation of non-parenteral products (e.g. oral solutions, topical) * Initial cleaning of equipment used for sterile production (not final rinse) Purified water spec: TOC - <500ppb Conductivity - <5.1 uS/cm at 25 degree) Nitrates - <0.2 ppm Endotoxin - N/A Micro - <100CFU/ml

Answer 6

1. Immediate Actions * Quarantine the batch immediately to prevent use or release. * Initiate an OOS investigation following MHRA and internal SOP guidance: o Phase 1a: Lab-based investigation (analyst, equipment, media, method validation, controls). o Phase 1b: Hypothesis testing (repeat testing, re-sample if justified). * Send sample for microbial identification (if not already ID’ed). ________________________________________ 2. Phase 2 Investigation (No Assignable Cause) * If no lab error is identified, escalate to Phase 2 (manufacturing process) and open a deviation. * Conduct a full impact assessment: o GMP impact: Possible breakdown in contamination control, particularly water system. o Product impact: P. aeruginosa is an objectionable pathogen; product is not compliant with Ph. Eur. 5.1.4 and must be rejected. o Assess other batches produced using the same water system, equipment, or during the same time period. ________________________________________ 3. Link to Water System – Critical Investigation * Pseudomonas aeruginosa is commonly associated with purified water. * You mention it was previously found in the water system, but attributed to sampling error. o Thoughts as QP:  That conclusion must be re-evaluated. Repeated detection of P. aeruginosa — even with a suspected sampling error — is a signal requiring escalation.  I would review the original OOS investigation report for the water system.  Check:  Historical water trends  Recent OOS/OOT data  Any PQR signals  Raise a deviation if not already done. * Increase sampling frequency for purified water (per risk-based plan) and test at: o RO outlet o Storage tank o All user points and return loop ________________________________________ 4. Root Cause and CAPA * Initiate root cause analysis (e.g. Ishikawa) to explore: o Water system integrity and sanitisation records o Equipment cleaning validation o Operator hygiene and manufacturing environment * Implement CAPAs: o e.g. requalification of water system, revised sampling procedures, retraining ________________________________________ 5. QP Batch Disposition * As P. aeruginosa is a specified organism with an absence requirement, the batch is not compliant with pharmacopoeial limits. * The batch must be rejected. * If other batches are affected (e.g. shared water lot or equipment), conduct risk assessment and consider recall, notifying DMRC if necessary. ________________________________________

Answer 7

3. Link to Water System – Critical Investigation * Pseudomonas aeruginosa is commonly associated with purified water. * You mention it was previously found in the water system, but attributed to sampling error. o Thoughts as QP:  That conclusion must be re-evaluated. Repeated detection of P. aeruginosa — even with a suspected sampling error — is a signal requiring escalation.  I would review the original OOS investigation report for the water system.  Check:  Historical water trends  Recent OOS/OOT data  Any PQR signals  Raise a deviation if not already done. * Increase sampling frequency for purified water (per risk-based plan) and test at: o RO outlet o Storage tank o All user points and return loop ________________________________________ 4. Root Cause and CAPA * Initiate root cause analysis (e.g. Ishikawa) to explore: o Water system integrity and sanitisation records o Equipment cleaning validation o Operator hygiene and manufacturing environment * Implement CAPAs: o e.g. requalification of water system, revised sampling procedures, retraining ________________________________________ 5. QP Batch Disposition * As P. aeruginosa is a specified organism with an absence requirement, the batch is not compliant with pharmacopoeial limits. * The batch must be rejected. * If other batches are affected (e.g. shared water lot or equipment), conduct risk assessment and consider recall, notifying DMRC if necessary.

Answer 8

How would you validate a purified water system? Reference Guidelines: * EU GMP Annex 15 – Qualification and Validation * WHO TRS 970 Annex 2 – Water for Pharmaceutical Use * Ph. Eur. / BP – Water monographs ________________________________________ 1. Qualification Stages a. Design Qualification (DQ) * Ensure system design meets intended use and GMP requirements * Review pipework, flow rate, sanitisation method, materials (e.g. 316L stainless steel), loop design (no dead legs) b. Installation Qualification (IQ) * Verify all components (pumps, RO membranes, tanks, filters, instruments) are installed as per design specs c. Operational Qualification (OQ) * Confirm all components operate within expected parameters: o Flow rate o Temperature o Conductivity meter calibration o Alarm systems o Sanitisation functions (chemical or thermal) ________________________________________ 2. Performance Qualification (PQ) Should be conducted over 3 defined phases: Phase 1 – Baseline Monitoring (2–4 weeks) * Objective: Understand system performance and refine SOPs * Develop and test: o Sampling strategy (user points, RO outlet, tank, loop return) o Sanitisation SOPs o Water system operation SOPs * Test daily for: o Microbiology o Conductivity o TOC o Nitrates (if applicable) * Water cannot be used for manufacturing in this phase ________________________________________ Phase 2 – Operational Control (2–4 weeks) * Objective: Confirm that defined SOPs consistently maintain water quality * Continue daily monitoring at all sampling points * Water may be used for manufacturing, if QA-approved and risk-assessed ________________________________________ Phase 3 – Long-Term Performance (min. 1 seasonal cycle, often ≥1 month) * Demonstrate performance over time, covering seasonal variation * Sampling frequency can be reduced based on risk assessment * Routine operation, sanitisation, and alert/action limit reviews ________________________________________

Answer 9

Purified Water * Conductivity: ≤ 5.1 µS/cm at 25°C * TOC: ≤ 500 ppb * Microbial limit: ≤ 100 CFU/mL * Nitrates: ≤ 0.2 ppm (if tested) Water for Injection (WFI) * Conductivity: ≤ 1.3 µS/cm at 25°C * TOC: ≤ 500 ppb * Microbial limit: ≤ 10 CFU/100 mL * Endotoxins: ≤ 0.25 IU/mL * Must be produced by distillation or equivalent (e.g. membrane-based) process per Ph. Eur Critical Process Parameters (CPPs) Stage CPPs / Parameters Source water Bioburden, chlorine/chloramines, conductivity Pre-treatment Media filters (particulates), activated carbon (chlorine), softeners (ions) RO system Pressure, membrane integrity, rejection rate, flow rate Deionisation (EDI) Conductivity, anion/cation resin status Storage tank Temperature (typically >65°C for hot systems), sanitisation frequency Distribution loop Flow velocity (≥ 1–2 m/s to prevent biofilm), UV, sampling at user points and return Final monitoring Microbial count, TOC, conductivity, endotoxins (WFI only)

Answer 10

1. Goods-In and Dispensing (Grade C Area) Process: * Raw materials and components (e.g., API, excipients, vials, stoppers) are received, checked, and released by QA before use. * Weighing and dispensing are performed under controlled conditions. CPPs: * Goods-in checks: Visual inspection, COA verification, temperature excursions, quarantine status. * Pre-bioburden of API/excipients: Initial microbial load must be controlled to reduce downstream contamination risk. * Environmental controls (Grade C): Particle and microbiological levels must meet EU GMP Grade C. * Dispensing balance calibration and accuracy: Precision in weight to ensure formulation accuracy. ________________________________________ 2. Material Transfer and Sanitisation Process: * Transfer of raw materials, primary containers, and tools into cleanrooms. CPPs: * Sanitisation procedures: Use of IMS (Industrial Methylated Spirit) or sporicidal agents; validated contact time. * Transfer process: Wiping, spraying, double/triple wrapping systems. * Personnel and material airlocks (PAL/MAL): HEPA-filtered, monitored for pressure differential. ________________________________________ 3. Aseptic Mixing (Grade A in Grade B/C background) Process: * Dissolution or mixing of sterile components or filtered solutions under aseptic conditions. CPPs: * Operator aseptic technique qualification: Media fill pass, gowning, and behavioural compliance. * Mixing parameters: Time, speed, temperature—all validated and monitored. * Equipment sterilisation and integrity: Clean steam, SIP where applicable. * Environmental conditions: Grade A for open product; Grade B background monitored for viable/non-viable particles. ________________________________________ 4. Sterile Filtration Process: * The bulk solution is filtered through a 0.22 µm sterilising-grade filter. CPPs: * Filter integrity testing (pre- and post-use): Bubble point, forward flow test. * Filter material compatibility and validation: No leachables/extractables. * Filtration pressure/time parameters: Validated to prevent filter rupture or bypass. * Holding time before/after filtration: Minimised to reduce microbial risk. ________________________________________ 5. Aseptic Filling and Closure (Grade A Isolator) Process: * Filling of sterile product into pre-sterilised containers (vials, syringes), followed by immediate stoppering or sealing. CPPs: * Isolator integrity: Leak test, decontamination (e.g., H₂O₂ or formaldehyde) validated. * Glove integrity testing: Daily before/after operation. * Grade A conditions: Unidirectional airflow, <1 cfu, <1 particle >0.5 µm. * Filling machine parameters: Volume accuracy, fill weight check, and reject systems. * Stopper and cap placement: Sterilised components, monitored application force. ________________________________________ 6. Terminal Processes (if applicable) Note: If not terminally sterilised, this step is omitted. ________________________________________ 7. Visual Inspection and QC Testing Process: * Finished product is visually inspected for particulates and container defects. * Sterility, endotoxin, and potency (assay) testing performed. CPPs: * Visual inspection qualification: Trained staff, challenge sets, 100% inspection. * QC test methods: Validated per ICH Q2(R2) for accuracy, precision, specificity. * Sample handling: Aseptic sampling for sterility/endotoxin. ________________________________________ 8. Batch Release Process: * QA/QP review of all manufacturing and QC data before certification and release. CPPs (indirect): * Traceability: Batch records, equipment logs, environmental results. * CAPA for any excursions: Deviation investigation process. * QMS oversight: Change control, training records, ongoing qualification status.

Answer 11

* Immediately quarantine the water system and stop its use in production. * Send the contaminated plate for microbial identification (e.g., MALDI-TOF). * Initiate an OOS investigation in accordance with MHRA guidance and your SOP (e.g., P22-D-001). * Proceed with Phase 1a and 1b investigations to rule out lab error or systemic contamination. * Consider risk to product and implement CAPA if contamination is confirmed.

Answer 12

* Water testing typically uses R2A agar incubated at 20–25°C for 5–7 days, which supports the growth of slow-growing waterborne microbes. * Phase 1a: Check for obvious lab errors – incorrect SOP followed, wrong agar/plate, new staff error, power failure during incubation, or plate damage. * Phase 1b: Supervisor-led deeper review – inspect records, check expiry of consumables, equipment calibration, incubation conditions, maintenance history, and lab cleaning logs.

Answer 13

* Staphylococcus aureus (ATCC 6538) * Pseudomonas aeruginosa (ATCC 9027) * Candida albicans (ATCC 10231) * Aspergillus brasiliensis (formerly A. niger, ATCC 16404) * Bacillus subtilis (ATCC 6633)

Answer 14

* Microbial ID gives insight into source, pathogenicity, environmental origin, and whether it is spore-forming or biofilm-forming (e.g., Pseudomonas aeruginosa). * Helps assess potential impact on product and patient safety, supports risk assessment, and guides effective CAPA.

Answer 15

* Purified Water (PW): o Appearance (clear, colorless) o Conductivity (typically <1.3 µS/cm at 25°C) o Total Organic Carbon (TOC) (<500 ppb) o Microbial count (e.g., <100 cfu/mL, site-defined limits) * Water for Injection (WFI) (additional checks): o Endotoxins (<0.25 EU/mL) o Nitrates (if non-distillation method used)

Answer 16

a. Identified as pseudomonas species b. Went through OOS investigation steps c. Narrowed to sampling d. Use of flexible hoses – inappropriate storage – coiled in a circle on the wall .

Answer 17

1. Perform a Risk Assessment: * Assess the room layout, equipment positioning, airflow patterns, personnel/material flows, and entry/exit points. * Identify critical zones (e.g. Grade A filling line) and potential contamination risks. * Use smoke studies (visualisation studies) to identify airflow direction, turbulence, and stagnant areas where particles could accumulate. 2. Design the Monitoring Plan: * Non-Viable Particle Monitoring (NVPM): * Based on ISO 14644-1, place continuous particle counters in Grade A zones (e.g., inside isolators or LAF cabinets) near critical operations like filling. * Monitor ≥0.5 µm and ≥5.0 µm particles per Annex 1. * Set alert and action limits based on Grade classification (e.g., Grade A: action limit >3520 particles ≥0.5 µm/m³). * Viable Monitoring (VPM): * Use active air samplers, settle plates, contact plates, and glove prints. * Monitor Grade A at working, Grade B at rest and working, Grades C/D as per risk. * Position settle plates near critical operations, air supply diffusers, material transfer points, and high-touch surfaces. 3. Establish Baseline Data: * Conduct initial monitoring during qualification phases (“at rest” and “in operation”) to establish normal environmental flora and particle levels. 4. Set Alert and Action Limits: * Use collected baseline data to calculate statistical alert limits (e.g., mean + 3σ). * Apply Annex 1 maximum limits as absolute action limits for viable counts (cfu/plate or cfu/m³). * Reassess limits regularly based on trend data and changes to operations or cleaning. 5. Documentation and Trending: * Record all results, deviations, and investigations. * Use trend analysis tools (e.g., Excel or QMS software) to monitor shifts, excursions, and seasonal variation. * Review trends regularly in PQRs or quality meetings.

Answer 18

Endotoxins are lipopolysaccharide components of the outer membrane of gram-negative bacteria. They are pyrogens and must be controlled in injectable products. They can originate from water systems (e.g., WFI), raw materials, or inadequate equipment cleaning. Tips: • Control through validated depyrogenation (dry heat) and routine LAL testing. • Refer to Ph. Eur. 2.6.14 (Bacterial endotoxins).

Answer 19

Model Answer: I would: • Review environmental monitoring, bioburden data, and water system logs. • Investigate potential root causes: equipment cleaning (final rinse with WFI?), operator practices, material contamination. • Evaluate depyrogenation tunnel validation. • Assess impact on product quality and patient safety. • Consider re-testing per GMP rules and initiate deviation. Tips: • Bring up phase 1 (lab) to phase 3 (full-scale) investigation framework. • Acknowledge the sensitivity and specificity of the LAL assay.

Answer 20

Model Answer: • Perform challenge studies using endotoxin spiked vials. • Demonstrate at least 3-log reduction of endotoxin using endotoxin indicators. • Dry heat sterilisation (e.g., oven) may require 6-log reduction of Bacillus spores (e.g., Bacillus atrophaeus) if sterility is also claimed. • Validate parameters: belt speed, temperature profile, and dwell time. Tips: • Know the difference between depyrogenation (endotoxin removal) and sterilisation (microbial kill). • Refer to Annex 1 and PDA Technical Report 3.

Answer 21

Model Answer: • Terminal sterilisation: Product is filled and then sterilised (e.g., by autoclave). Offers higher sterility assurance (SAL ≤ 10⁻⁶). Suitable for heat-stable products. • Aseptic processing: Sterile components are filled under Grade A; sterility maintained without kill step. Relies on process validation (media fills), environmental controls, and filter integrity. Tips: • Parametric release only applies to terminally sterilised products. • Terminal sterilisation is preferred per Annex 1 unless not feasible.

Answer 22

Model Reference: Annex 1 (2022), EMA 2008 Aseptic Process Simulation Guidance • Key elements: • Start with clarifying questions: planned/unplanned, location of failure, number of units • Begin failure investigation: review interventions, environmental monitoring, incubation timeline • Identify potential root cause (e.g. transfer hatch, operator technique) • Perform ID of microorganism • Escalate risk assessment: market impact, CAPA • Tip: Structure response using Phase 1A/1B/2, then CAPA, requalification, and market release impact.

Answer 23

PET is performed to verify that the antimicrobial preservative system in a multi-dose sterile product is effective in inhibiting microbial growth during in-use storage. This ensures patient safety if inadvertent contamination occurs during repeated dosing.

Answer 24

Ph. Eur. lists the following standard test organisms: ESCAPZ: Escherichia coli (for oral preparations); Staphylococcus aureus (gram +); Candida albicans (yeast); Aspergillus brasiliensis (mould); Pseudomonas aeruginosa (gram negative) ; Zygosaccharomyces rouxii for sugar-rich oral preparations.

Answer 25

Microorganisms are cultured: Bacteria: on casein soya bean digest agar at 30–35 °C for 18–24 hours. C. albicans: at 20–25 °C for 48 hours. A. brasiliensis: at 20–25 °C for up to 7 days or until sporulation occurs.

Answer 26

Two criteria: A and B. A (preferred): Demonstrates greater log reduction in microbial count over time. B (alternative): Less stringent, used when A can’t be achieved but still acceptable for patient safety. For example, for bacteria: A: ≥2 log reduction at 6 hr, ≥3 log at 24 hr, no recovery at 28 days.

Answer 27

NR: No Recovery — No viable organisms detected. NI: No Increase — No increase in viable count compared to the previous reading.

Answer 28

No. Single-dose sterile products do not require preservatives as they are intended to be used once and not re-entered. PET is applicable for multi-dose containers where there’s risk of contamination during repeated use.

Answer 29

In a multi-dose sterile product, what would you do if PET results only met criterion B?

Answer 30

Possible causes include: Incompatible preservative (e.g. adsorbed to container or formulation excipients) Incorrect pH or preservative concentration Inaccurate inoculum preparation Inadequate preservative distribution Antimicrobial neutralization during testing

Answer 31

The sterility test is designed to detect the presence of viable contaminating microorganisms in terminally sterilized or aseptically processed products. The test is based on incubation of samples in media that support bacterial and fungal growth under controlled conditions for 14 days, observing for turbidity or visible microbial growth.

Answer 32

Ph. Eur. describes two methods: Membrane Filtration – Preferred for aqueous, alcoholic, or oil-based products. Direct Inoculation – Used when membrane filtration is not suitable (e.g. oily or viscous products). Both methods require incubation in: Fluid Thioglycollate Medium (FTM) at 30–35 °C for anaerobic and aerobic bacteria. Soybean Casein Digest Medium (SCDM/TSB) at 20–25 °C for fungi and aerobic bacteria.

Answer 33

Membrane filtration is preferred when the product can be filtered and does not have antimicrobial properties. It allows better removal of preservatives or inhibitory substances. It is ideal for large volume parenterals, antibiotics, and clear aqueous solutions. Direct inoculation is used when filtration is not feasible due to viscosity, oil content, or filter-clogging properties.

Answer 34

Positive control: Test organisms such as Staphylococcus aureus, Bacillus subtilis, Candida albicans, etc., are inoculated into media to verify media support microbial growth. Negative control: Media without product or inoculum, incubated alongside samples to check for contamination in the environment or reagents. Growth promotion test (GPT) is mandatory prior to test to verify media performance.

Answer 35

Through a bacteriostasis/fungistasis test (also called method suitability): Inoculate known low levels (~10–100 CFU) of test organisms into media containing the product. Demonstrate that the product does not inhibit growth. If inhibition is observed, neutralizers or dilution or filtration is used to eliminate the antimicrobial effect before actual sterility testing.

Answer 36

A positive sterility test is considered critical. Investigation must confirm: Integrity of test environment (lab contamination?) Integrity of the product container closure system Operator or method errors If genuine contamination is confirmed, batch must be rejected. A second sterility test is not allowed unless invalid test conditions are clearly demonstrated (per Ph. Eur.).

Answer 37

No. Sterility testing is limited by: -Small sample size (statistical limitation) -Retrospective nature -Potential to miss low-level contamination Sterility assurance is primarily based on: Validated sterilization processes Environmental monitoring Aseptic process simulation (media fill) Sterility test is only one part of the sterility assurance system.

Answer 38

14 days total incubation. FTM: 30–35 °C. SCDM (TSB): 20–25 °C. Media must be clear, sterile, and support growth for the entire period.

Answer 39

SBC CAP Staohylococcus aureus (aerobe) Bacillus subtilis (aerobe) Clostridium sporogenes (anaerobe) Candida albicans (yeast) Aspergilus brasiliensis (mold) Pseudomonas aeruginosa (aerobe)

Answer 40

Sterility Assurance Level (SAL) is a critical microbiological performance metric that expresses the probability of a single viable microorganism surviving the sterilisation process. For pharmaceutical products, a SAL of 10⁻⁶ is generally required — this means there’s a one in a million chance that a non-sterile unit remains after sterilisation.

Answer 41

Moist Heat Sterilisation (Autoclaving): Preferred method according to the EMA and the decision tree in Annex 1. It involves steam under pressure at temperatures typically ≥121°C. It is suitable for aqueous preparations and items that can tolerate moisture and heat, including terminally sterilised injectables and porous loads like surgical instruments or clothing. Microbiological Kill Mechanism: • Latent heat of condensation from saturated steam causes coagulation and denaturation of microbial proteins.

Answer 42

A QP must ensure validation includes both physical and microbiological parameters: Physical validation: • Heat distribution studies with thermocouples. • F₀ calculations: Quantifies the lethality of the cycle; for example, an F₀ of 8 minutes at 110°C is considered a minimum. • D-value and Z-value: Used to calculate microbial kill kinetics and temperature-dependence. Biological indicators (BIs): • Use Geobacillus stearothermophilus spores due to their high resistance to moist heat. • Placed in worst-case locations to demonstrate adequate kill.

Answer 43

Porous Load (e.g. surgical textiles, instruments, rubber stoppers): Cycle type: Vacuum-assisted to ensure air removal and steam penetration. Validation requirements: • Bowie-Dick test (daily or before each cycle) confirms adequate air removal. • Leak rate test: Ensures vacuum integrity. • BI placement between layers or inside packaging. • Steam must be saturated, with <3.5% non-condensable gases and a dryness fraction > 0.95.

Answer 44

Fluid Load (e.g. aqueous solutions in ampoules or vials): Cycle type: Typically uses gravity displacement or pressure-pulsed cycles. • Key consideration: Fluids absorb heat more slowly — longer come-up and holding times. Validation requirements: • Thermocouples placed in the slowest-to-heat locations (e.g., center of largest load units). • Monitoring for superheating, air entrapment, and cooling rates. • Vent filters on chamber must be validated for integrity and steam compatibility.

Answer 45

“Yes, I would be concerned. I would immediately quarantine both affected batches and send the isolates for species-level identification. I would initiate an OOS investigation for the failed batch, and begin an atypical result investigation for the second batch in line with MHRA expectations and our SOP. If no assignable cause is found, I would escalate to Phase 2, open a deviation, and conduct a full impact assessment covering GMP, MA, and patient safety.”

Answer 46

“An atypical result is not an OOS, but it deviates from the established microbial flora or trend for the product or process. It may involve a shift in levels or unexpected species — warranting investigation as part of ongoing process monitoring.”

Answer 47

“I’d review analyst training, equipment logs, media preparation, expiry dates, incubation conditions, recent OOS, and the environmental cleaning records for the incubator and lab. I’d also interview the analyst and supervisor, and cross-check with retained plates if available.”

Answer 48

“Bacillus is commonly environmental and may point to poor cleaning, HVAC failures, or contamination from external personnel. P. aeruginosa is objectionable in topical preparations and often waterborne. Its presence raises concerns about water system integrity and patient risk — particularly for creams applied to broken skin.”

Answer 49

“Controls include pretreatment (media filter, carbon, softener), primary treatment (RO, EDI), and polishing steps (UV, biofilter). The system must be qualified and maintained in a validated state. Acceptance limits per Ph. Eur. for purified water include: Conductivity ≤ 4.3 µS/cm at 20°C (or ≤ 1.3 µS/cm at 25°C), TOC ≤ 500 ppb, Microbial count ≤ 100 CFU/mL. TOC is a key indicator of organic contamination, which may signal biofilm formation in the loop.”

Answer 50

“I’d review historical microbiological trends, CUSUM charts for early signal detection, and also consider Shewhart charts. CUSUM is sensitive to small shifts and helps catch trend changes early. I’d also calculate Cp/Cpk for water system consistency. ‘Normalisation’ in this context means re-establishing control after CAPA — once the system shows stable, acceptable performance again.”

Answer 51

“I’d conduct a full impact assessment: MA: non-compliance due to microbiological OOS, GMP: loss of water system control, Patient safety: possible risk of infection. I would reject any unreleased product and consider recall for released batches — following MHRA’s defective medicines guidance, Class 2 recall likely. I’d notify the DMRC before execution and ensure the water system is quarantined and requalified.”

Answer 52

“Depending on system design, I would sanitise using thermal methods (>65°C) or chemical methods such as ozone, hydrogen peroxide, or peracetic acid. The method must follow validated SOPs, and daily monitoring must confirm restoration of compliance before return to service.”

Answer 53

“If the batch meets finished product specifications, the organism is non-objectionable, and the root cause is unrelated to product manufacturing — I could release the batch with QP oversight. However, CAPA must be implemented, including enhanced cleaning of the lab and increased monitoring.”

Answer 54

“I’d escalate CAPA to include revision of SOPs, cleaning frequency for lab doors, and engineering protocols to ensure microbiological control post-maintenance. I’d also consider environmental requalification of the lab area.”

Answer 55

“My duty as QP is to protect patient safety and ensure compliance. Under Annex 16, a batch cannot be released until the investigation of an unexpected deviation is complete and justified. I would explain this to Sales and escalate to senior leadership if needed.”

Answer 56

• Defined in Annex 17 EU GMP. • Applicable for terminally sterilised products where sterility assurance is demonstrated via validated cycle parameters (time, temperature, pressure). • No need to wait for sterility test results before batch certification. • Requires: • Strong CCS in place. • Consistent validated sterilisation processes. • Regular monitoring & periodic sterility testing for verification.

Answer 57

A substance that causes fever. Example: Endotoxin from Gram-negative bacteria.

Answer 58

Lipopolysaccharide component of Gram-negative bacterial cell wall, pyrogenic.

Answer 59

Dormant, resistant form of bacteria (e.g., Bacillus) for survival in harsh conditions.

Answer 60

Commensal (normal flora), Pathogen (disease-causing), Opportunistic Pathogen (causes disease in immunocompromised).

Answer 61

Bacillus, Staphylococcus aureus.

Answer 62

E. coli, Pseudomonas aeruginosa.

Answer 63

Aspergillus niger (environment), Penicillium (food spoilage).

Answer 64

Animal-origin: Prions, viruses; Vegetable-origin: Yeasts, moulds; Minerals: Spore-formers; Sugars: Yeasts, moulds.

Answer 65

Surface efficacy testing, rotating disinfectants to prevent resistance.

Answer 66

Microorganisms can exist in a wide range of sources, including: * Raw materials: APIs, excipients, especially if not sterilised or adequately controlled (e.g. lactose, cellulose). * Water systems: Particularly in Purified Water and WFI loops, biofilm formation is a known risk. * Operators: The human body is a major source—skin, hair, respiratory tract, and improper gowning/behaviour can introduce viable contamination. * Facility surfaces: Walls, ceilings, and floors—especially at junctions, behind equipment, or in hard-to-clean areas. * Equipment: Especially in areas that are hard to clean, like seals, valves, or dead legs in piping. * Materials: Paper products, cardboard, and carbon-based materials can retain and shed particles and viable organisms. * HVAC systems: Filters and ducts can be microbial reservoirs if not properly maintained.

Answer 67

Yes, microorganisms can be present in raw materials—particularly non-sterile excipients, natural-source materials, and API if not sterile by nature. ⸻ Controls to Minimise Microbial Risk: 1. Supplier Qualification: * Audit suppliers for GMP compliance. * Ensure they have validated cleaning and microbial control processes. * Review microbial specs and historical data. 2. Goods-In Inspection: * Check Certificate of Analysis (CoA) for microbial limits. * Inspect tamper-evident seals and packaging integrity. * Review temperature loggers for excursions during transport. * Check for visible contamination or damage to containers. 3. Storage Controls: * Store under controlled temperature and humidity. * Monitor environmental conditions (e.g., microbial sampling in warehouse). * Use first-expiry-first-out (FEFO) system to reduce degradation risk. 4. QC Testing on Receipt (as required): * Perform microbial limits testing for non-sterile materials (Ph. Eur. 5.1.4 / 5.1.8). * Conduct sterility testing if material is claimed sterile. * Perform ID tests for any OOS or high-risk materials.

Answer 68

There are several methods to count bacteria, depending on the context (e.g. viable vs. total count, rapid vs. compendial). Key methods include: ⸻ 1. Plate Count Method (Viable Count) * Description: Serial dilution of sample, plated on agar, incubated, and colonies counted as colony-forming units (cfu). * Use: Compendial method for microbial enumeration (e.g., raw materials, water, environmental monitoring). * Limitations: Only counts viable, culturable organisms. ⸻ 2. Membrane Filtration (for low bioburden samples like water) * Description: Sample is filtered through a sterile membrane, which is then placed on agar and incubated. * Use: Water testing, large volume samples. * Advantage: More sensitive for dilute samples. ⸻ 3. Most Probable Number (MPN) * Description: Statistical estimation based on dilution series and presence/absence of bacterial growth in multiple tubes. * Use: Useful when bacteria are stressed or slow-growing. * Common in: Water, food microbiology. ⸻ 4. ATP Bioluminescence (Rapid Method) * Description: Measures adenosine triphosphate (ATP) from microbial cells using a luciferase-based light reaction. * Use: Rapid hygiene monitoring (e.g., cleanroom surfaces, equipment). * Limitation: Detects both live and dead cells — not specific to viable count. ⸻ 5. Flow Cytometry (Rapid Method) * Description: Uses lasers to count and characterise single bacterial cells in fluid based on size and fluorescence. * Advantage: Can give total and viable cell counts using viability dyes. * Application: Biotech, ATMPs, process monitoring. ⸻ 6. Turbidity (Optical Density, OD600) * Description: Measures cloudiness of a culture using a spectrophotometer. * Use: Quick estimate of bacterial concentration. * Limitation: Cannot distinguish live vs. dead cells, not used for final QC.

Answer 69

1. Sampling Locations: * Sampling should cover: * Each user point (e.g., outlets used for manufacturing or cleaning) * Upstream and downstream of critical components, such as: * Raw water inlet * Pre-treatment units (e.g., softeners, carbon filters) * Reverse Osmosis (RO) units * Electrodeionisation (EDI) * Final filters * Storage tanks * Return loop (including farthest point) This ensures a full picture of system performance and contamination risk. ⸻ 2. Good Sampling Practices: * Use clean, sterile, depyrogenated containers appropriate to the test (e.g., endotoxin-free vials for endotoxin testing). * Perform flushing before sampling to avoid false results due to stagnation. * Use aseptic technique during sample collection. * Label samples with date, time, location, operator ID. * Transport samples quickly to the lab under controlled conditions (e.g., chilled if required). * Ensure sampling is performed by trained personnel following a written SOP. Frequency - based on risk assessment such as volume used or criticality to process: Daily - conductivity,

Answer 70

Disinfection destruction of microorganisms to reduce them (not sterilize). Not necessarily killing all microorganisms but reducing them to an acceptable level for a defined purpose.  Sanitization being the process of cleaning and disinfection in a single process (the agent used usually has surfactant properties making it surface active for cleaning) often associated with CIP processes.

Answer 71

My Approach: 1. Initiate Change Control * Raise a Change Control (CC) within the Pharmaceutical Quality System (PQS) to document all qualification activities for the new sterile suite. * Ensure cross-functional approvals and maintain traceability throughout the process. 2. Regulatory Framework * Conduct the qualification in accordance with: * ISO 14644-1: Classification of air cleanliness by particle concentration. * ISO 14644-2: Specifications for testing and monitoring to prove continued compliance. * ISO 14644-3: Test methods for cleanrooms. * EU GMP Annex 1 (2022): Guidelines for the manufacture of sterile medicinal products. 3. Risk Assessment and Sampling Strategy * Perform a risk assessment to define EM sampling locations, considering: * Room layout and floor plan. * Personnel and material flow to identify high-risk zones. * Equipment positioning and potential areas of turbulent airflow. * Conduct smoke studies (airflow visualization) to identify: * Unidirectional airflow patterns in Grade A zones. * Areas where particles may settle or be retained. 4. Selection of Sampling Methods and Locations * Non-Viable Particle Monitoring (NVPM): * Use light-scattering particle counters to monitor airborne particles ≥0.5 µm. * Place counters in critical zones, such as near filling lines and aseptic connections. * Viable Particle Monitoring (VPM): * Utilize active air samplers, settle plates, contact plates, and glove prints. * Position sampling devices in areas with high product exposure risk. 5. Room Classification and Limits * Align these classifications with EU GMP Annex 1 Grades A-D as appropriate. 6. Qualification States * Conduct EM qualification in three states: * As-built: Empty and operational cleanroom. * At-rest: Installed equipment but no personnel. * In-operation: Normal production conditions with personnel. 7. Establish Alert and Action Limits * Collect baseline data during qualification phases. * Calculate alert and action limits using statistical methods (e.g., mean ± 3 standard deviations). * Regularly review and adjust limits based on trend analysis and process changes. 8. Documentation and Reporting * Compile a comprehensive qualification report including: * Test results and data analysis. * Sampling locations and rationale. * Deviations and corrective actions. * Obtain QA/QP approval before commencing GMP manufacturing.

Answer 72

‐ high traffic areas, areas with poor airflow, sinks Regulatory References: * ISO 14644 – For cleanroom classification and monitoring principles. * EU GMP Annex 1 (2022) – For general cleanroom principles. * EU GMP Annex 9 – For non-sterile product manufacturing and hygiene control. ⸻ Objectives of the EM Programme: * To ensure the environment is maintained in a state of control, particularly in areas where the product is exposed. * To define viable and non-viable monitoring locations, sampling frequencies, and alert/action limits based on risk assessment. * To establish a baseline for microbial and particulate levels, allowing ongoing trend analysis and timely intervention. ⸻ Key Considerations and Setup Process: 1. Risk Assessment * Perform a risk assessment considering: * Floor plan and room classification (e.g., typically Grade C or D for non-sterile creams). * Equipment positioning and movement patterns. * Personnel and material flow, especially during weighing, mixing, and filling. * Smoke studies to identify airflow patterns, turbulence, and areas of potential particle accumulation. 2. Room Classification * Classify rooms based on ISO 14644-1 particle limits and Annex 9 guidance: * For open product exposure, aim for controlled but not sterile environments (e.g., Grade C/D). * Ensure environmental conditions are appropriate to minimise contamination risk. 3. Selection of Monitoring Methods * Non-viable particle monitoring using particle counters (≥0.5 µm and ≥5.0 µm). * Viable monitoring using: * Settle plates (passive air sampling). * Contact plates (surfaces, equipment, operator gloves). * Active air samplers, if justified by risk. 4. Monitoring States * Conduct EM qualification and baseline studies in three states: * As-built: Room ready for use but empty. * At rest: Equipment installed but no personnel present. * In operation: During actual manufacturing with personnel and materials. 5. Alert and Action Limits * Establish baseline data from initial monitoring runs. * Apply statistical analysis (e.g., mean ± 3 standard deviations) to define alert limits. * Action limits should be based on regulatory thresholds and risk assessment. * Plot on a control chart for ongoing trend analysis and early warning of deviations. ⸻ What You’re Looking For: * Early detection of environmental contamination trends (e.g., increasing viable counts). * Identification of deviations that could compromise product quality. * Ensuring the environment remains within validated and compliant limits to protect the product and patients.

Answer 73

Note: Finger dabs are not routinely required for Grades C and D. ⸻ Agar Media Used: * Tryptic Soy Agar (TSA) – general-purpose medium for bacteria. * Sabouraud Dextrose Agar (SDA) – selective for fungi (yeasts and moulds). These plates must pass growth promotion testing (GPT) using compendial organisms (e.g., S. aureus, C. albicans, A. brasiliensis). ⸻ Additional Notes: * Grade A requires continuous non-viable monitoring and frequent viable sampling near critical operations (e.g. filling). * Settle plates typically exposed for up to 4 hours. * Contact plates used on surfaces (benches, walls, equipment). * Glove (finger dab) sampling required for aseptic operators at end of operation in Grade A/B.

Answer 74

Potential contamination of the product: Compression is typically a non-sterile process, but microbial control is still important — particularly in products for immunocompromised, paediatric, or inhaled/oral suspensions. Environmental monitoring (EM) trending indicates increasing bioburden. If alert/action levels are breached, this may signal a breakdown in cleaning, HVAC, or personnel practices. Failure to investigate and control may lead to product recall, regulatory non-compliance, or MHRA inspection finding.

Answer 75

What are these organisms? Micrococcus luteus: Gram-positive coccus Source: Human skin flora Often a sign of operator hygiene failure (e.g. hand contamination, gowning breach) Bacillus subtilis: Gram-positive, spore-forming rod Source: Environmental (soil, dust, air, packaging materials) Spores can survive standard cleaning — indicates cleaning may be inadequate

Answer 76

Generally not for healthy individuals, but: May pose risk to immunocompromised, elderly, or infants Bacillus subtilis spores may persist in product and degrade API or excipients

Answer 77

Cleanroom classification and EM limits: ISO 14644-1 (cleanroom particle classification) EU GMP Annex 1 (2022) — Section 9 (environmental monitoring)

Answer 78

Microbial limits for tablets: Ph. Eur. 5.1.4 TAMC ≤ 10³ CFU/g TYMC ≤ 10² CFU/g Absence of E. coli 1g or 1ml

Answer 79

1. Initiate OOS/OOT Investigation Follow site SOP in line with MHRA OOS/OOT guidance Phase 1: rule out sampling, analyst, or incubation error Phase 2: if confirmed, escalate to deviation and perform full impact assessment 2. Quarantine Affected Areas and Batches Quarantine the compression room and manufacturing line Quarantine any affected or unreleased batches Review other batches made in the same room since last cleanroom qualification 3. Assess Impact on: Marketing Authorisation (MA) — Are limits breached? GMP compliance — Cleaning, HVAC, gowning, material flow failures? Product quality and patient risk — Immunosuppressed population? Shelf-life impact? 4. Perform Root Cause Analysis and CAPA Fishbone/Ishikawa covering: Personnel (training, behaviour) Cleaning (logs, disinfectant efficacy, frequency) HVAC (filter status, air change rates) Equipment (material ingress, packaging dust) Implement full cleaning and disinfection using sporicidal agent if needed Review and revise EM frequency or alert/action levels if needed. You may only release if: Batch is within MA spec Root cause identified Impact assessment confirms no risk to product Deviation and CAPA are documented and approved

Answer 80

Initial QP Concerns: Even though one test passed: This is an OOS, because a confirmed initial failure cannot be invalidated by a passing retest. Must treat in line with MHRA OOS guidance (Inspectorate blog and GMP Chapter 1). The failure involves moulds, which are objectionable in creams (especially for broken skin/immunocompromised use).

Answer 81

tep 1: Open a formal OOS investigation Phase 1A – Analyst/lab error check: Was the correct SOP followed? Were media and plates within expiry? Any visible anomalies (cracks, dryness, over-incubation)? Analyst training and qualification Phase 1B – Supervisor review: Sampling and labelling process Cleaning records for incubator and lab Cross-contamination risks If no assignable lab cause is identified → Phase 2 (manufacturing investigation + deviation).

Answer 82

Described in Ph. Eur. 5.1.3 / BP Volume 5 The product is inoculated with defined microorganisms Log reduction is measured over a time period (e.g. Day 0, 14, 28) Acceptance criteria vary by product category (A/B/C) Must demonstrate effectiveness over entire shelf life

Answer 83

Staphylococcus aureus (Gram-positive cocci) Escherichia coli (Gram-negative rod) Pseudomonas aeruginosa (Gram-negative rod, waterborne) Candida albicans (yeast) Aspergillus brasiliensis (mould)

Answer 84

over the product shelf life

Answer 85

This now indicates a trend or systemic issue, not an isolated incident. QP actions: Check if both batches were part of the same manufacturing campaign Open OOS investigation — if no assignable lab cause, escalate to Phase 2 and deviation Conduct full risk and impact assessment: Impact on GMP, product, MA, and patient safety Quarantine any other affected batches Immediate actions: Deep clean manufacturing area Repeat environmental monitoring Consider additional sampling (water, air, personnel) Category Key Points to Investigate QMS Recent deviations, PQR, trend reports Personnel Training, hygiene, gowning logs, handwashing compliance Equipment Maintenance, HEPA filters, surface residues Facility EM trends, HVAC, water ingress, external work Documents Batch record, cleaning logs, change control Materials Paper use, cardboard boxes in clean area Process Preservative content, mixing times, process hold times Cleaning Agent efficacy, rotation, frequency, dirty-hold times Supplier Excipient/API micro spec and goods-in checks Audit Any findings related to cleaning, EM, or PET CAPA: Improve cleaning frequency and agent rotation Retrain operators Requalify EM programme Possibly revise PET strategy Check preservative concentration vs. formulation target

Answer 86

A media fill (aseptic process simulation, APS) failure is a critical GMP event and must be investigated thoroughly to determine potential impact on product sterility assurance and release decisions. ⸻ Immediate Actions: 1. Confirm the details of the failure: • Number and identification of contaminated vials • Location of vials in the batch (e.g. line positions, timestamps) • Whether they are from a single operator qualification, requalification, or routine process simulation 2. Initiate a structured investigation: Investigate both laboratory and manufacturing causes: ⸻ Phase 1A – Micro lab assessment: • Confirm media growth promotion test passed • Confirm incubation conditions (temperature, time, orientation) • Examine vials for cracks, leaks, improper seals • Conduct Gram staining or microbial identification to determine organism type (e.g. skin flora vs environmental contaminant) Phase 1B – Process/line assessment: • Review: • Filling line documentation and intervention logs • Operator training and gowning records • Environmental monitoring (viable and non-viable) • Video recordings (if applicable) for the contamination window • Check if failure correlates with: • A specific intervention • Operator error • A breach in aseptic technique ⸻ Determine Scope and Impact: • Was this a routine qualification run? → If yes, review if previous commercial batches used the same set-up/operator. → May trigger a recall or batch review if linked. • Was it an operator qualification? → Impact may be limited to qualification; retesting with root cause mitigation may be possible. • Is it an isolated case or part of a trend? → Trend analysis over past media fills and EM data must be conducted. ⸻ Follow-up Actions: • Quarantine potentially impacted product batches • Notify QA and senior leadership • Consider regulatory reporting (e.g. MHRA if impacting released products or sterility assurance) • Plan for repeat media fill with enhanced controls • Review Contamination Control Strategy (CCS) and aseptic technique SOPs • Implement CAPA: retraining, procedural updates, equipment changes as needed ⸻ Key phrases for viva success: • “Criticality of sterility assurance” • “Root cause determination: intrinsic vs extrinsic” • “Phase 1A/1B structure” • “Evaluate impact on past and future batches” • “Always assess operator technique, EM data, and procedural compliance” • “Repeat media fill only after CAPA” “Was this a routine process simulation or part of an operator qualification? This determines whether the impact is potentially product-wide or operator-specific.”

Answer 87

Routine Qualification Operator Qualification Purpose To qualify the overall aseptic manufacturing process To qualify the aseptic technique of an individual operator Frequency Performed every 6 months (or more frequently if needed) for each aseptic process line Performed before an operator can work independently in aseptic processing; requalification typically annually Scope of assessment Validates entire process including equipment setup, cleanroom controls, interventions, and environmental state Focuses on gowning, interventions, and aseptic handling of one specific operator Failure impact Potential impact on commercial batches manufactured since last successful media fill â€” may trigger quarantine or recall Failure is usually isolated to the operator; no direct impact on product unless that operator has worked on released batches Team involved Manufacturing, QA, QP, Validation, Engineering, QC Operator, QA, line supervisor, microbiology Investigation focus Process integrity, facility, equipment, EM, gowning, interventions Individual performance, training, gowning technique Example outcome May require repeat full media fill + batch impact review Operator retraining and requalification before further aseptic work

Answer 88

TSB is used in media fills because it is a broad-spectrum growth medium that supports the growth of aerobic and facultative anaerobic microorganisms, including most common environmental and operator-related contaminants. It is clear, making it easy to detect turbidity, and is suitable for dual incubation at 20–25°C and 30–35°C, ensuring the detection of a wide range of organisms under aseptic manufacturing conditions. FTM, on the other hand, is primarily used in sterility testing, as it supports the growth of anaerobic bacteria, which are less relevant in media fills. FTM is slightly turbid, which can make detection of microbial growth more difficult in simulation studies. In summary: • TSB = media fills (detect likely contaminants in aseptic areas) • FTM = sterility testing (detect strict anaerobes)

Answer 89

Aspect Routine Qualification Operator Qualification Purpose To qualify the overall aseptic manufacturing process To qualify the aseptic technique of an individual operator Frequency Performed every 6 months (or more frequently if needed) for each aseptic process line Performed before an operator can work independently in aseptic processing; requalification typically annually Scope of assessment Validates entire process including equipment setup, cleanroom controls, interventions, and environmental state Focuses on gowning, interventions, and aseptic handling of one specific operator Failure impact Potential impact on commercial batches manufactured since last successful media fill — may trigger quarantine or recall Failure is usually isolated to the operator; no direct impact on product unless that operator has worked on released batches Team involved Manufacturing, QA, QP, Validation, Engineering, QC Operator, QA, line supervisor, microbiology Investigation focus Process integrity, facility, equipment, EM, gowning, interventions Individual performance, training, gowning technique Example outcome May require repeat full media fill + batch impact review Operator retraining and requalification before further aseptic work

Answer 90

Environmental Monitoring (EM) ensures that cleanroom environments remain under microbiological and particulate control, especially for aseptically prepared or sterile products. It is applied to viable and non-viable particles in both “at rest” and “in operation” states. ⸻ Step-by-Step Order of EM Establishment and Execution 1. Risk Assessment • Performed before EM starts to define critical and non-critical areas. • Based on: • Process and product risk • Facility layout and material/personnel flow • Equipment design and intervention types 2. Smoke Studies (Airflow Visualization) • Conducted during facility qualification to visualise unidirectional airflow, turbulence, and dead zones. • Helps identify: • Critical Control Points (CCPs) • Sample locations for viable and non-viable monitoring • Operator influence on airflow • Used to justify EM sampling points. 3. Define Sampling Locations & Frequency • Based on risk assessment + smoke study data. • Includes: • Air sampling (active/passive) • Surface sampling • Operator garments (e.g. glove prints) • Equipment surfaces • Frequency depends on room grade and activity. 4. Baseline Microbial Flora Establishment • Performed during facility qualification and initial runs. • Includes: • At rest state first → verifies background cleanliness and air handling • In operation state next → simulates routine operation with people and equipment • Done to: • Understand “normal” flora • Set realistic alert/action limits 5. Define Alert and Action Limits • Based on baseline data from at rest/in operation studies. • Typically set statistically: • Alert: mean + 2 SD • Action: mean + 3 SD • Limits must align with Annex 1 and ISO 14644-1 expectations for room grade. 6. Routine Environmental Monitoring • Begins after facility qualification and media fill qualification. • Includes: • Non-viable: continuous particle counters (especially Grade A) • Viable: settle plates, active air sampling, surface/contact plates • Done: • At rest: before process begins • In operation: during aseptic processing, filling, interventions 7. Trending and Continuous Improvement • Regular trending of EM data (monthly/quarterly/annual). • Any alert/action limit excursions trigger deviation investigations. • Re-evaluate limits and sampling strategy if trends shift. • Supports the Contamination Control Strategy (CCS) under Annex 1 (2022). ⸻ Quick Recap of Order: 1. Risk assessment 2. Smoke studies (airflow visualisation) 3. Define EM locations 4. Establish baseline flora: At rest → In operation 5. Set alert/action limits 6. Start routine EM 7. Trending & continual improvement

Answer 91

Model Answer: Yes, validation is the documented evidence that a process consistently produces a product meeting its predefined specifications. The three types are: • Prospective validation is performed before routine production. It involves executing a validation protocol under predefined conditions to confirm process performance. For example, media fill runs done before starting a new aseptic fill line. This is the expected approach for aseptic processes, as it provides assurance before patient exposure. • Concurrent validation occurs during actual production runs. It’s used when prospective validation isn’t practical, such as when introducing a new strength of an established product or during initial commercial supply post-approval. Real-time data is reviewed before batch release, and QP must assess the risk. It is permissible but requires strong justification, especially in sterile manufacturing. • Retrospective validation relies on analysing historical batch data to demonstrate process consistency. It’s not acceptable for aseptic processes, due to the inherent risk to sterility assurance and lack of real-time controls. It’s only used in exceptional scenarios, such as during a pandemic. In summary, for aseptic manufacturing, prospective validation is the standard, supported by media fills, equipment qualifications, and EM trends prior to release.

Answer 92

Model Answer: Before certifying any product from a new aseptic filling line, I would expect a minimum of three consecutive successful media fill runs per shift and process condition (e.g., container size, fill duration) – in line with Annex 1 (2022), section 9.28, and PIC/S aseptic validation guidance. If one of the three runs fails, I would initiate a root cause investigation immediately. The decision on whether to repeat all three runs or continue depends on the nature and timing of the failure: • If the failure was in the first or last run, I would likely need to repeat all three. • If the second failed, and the root cause is clearly isolated and corrected, then the last successful run may count as the first of a new series. However, the critical requirement is to demonstrate three consecutive successful runs post-CAPA implementation. The QP cannot certify any batches until this is achieved, and the CAPA must be closed and verified effective before starting the re-validation.

Answer 93

Model Answer: Concurrent validation can be considered in sterile manufacturing, but only under justified circumstances and with a well-documented risk assessment. Acceptable situations may include: • When introducing a new strength or minor formulation change to a product with a well-characterised process. • When there’s an urgent clinical or supply need, such as for an IMP or compassionate use product. • During technology transfer where the process is established at another site and there’s sufficient data to support similarity. Even in these cases, the QP has to assess the risk of certifying each batch, ensuring that all in-process controls, sterility assurance measures, and early stability data are acceptable. The process must be monitored closely in real time, and the concurrent batches must meet all specifications. The final validation report should confirm process consistency before switching to routine production. Concurrent validation is not a shortcut, and prospective validation should follow when feasible.

Answer 94

Model Answer: No, retrospective validation is not acceptable for aseptic processes under current GMP expectations (Annex 1, PIC/S, MHRA). This is because it involves relying on historical data, without real-time process controls or media fills, and poses a high risk to sterility assurance. However, in exceptional cases, such as emergency supply during public health crises (e.g., COVID-19), regulators may permit limited retrospective validation to support rapid product deployment. Even then, this must be accompanied by robust justification, risk mitigation, and regulatory dialogue. But in routine aseptic manufacturing, retrospective validation is no longer considered an acceptable practice.

Answer 95

Parameter Purified Water (EP/BP) WFI (EP/BP) Conductivity <5.1 ÂµS/cm at 25Â°C <1.3 ÂµS/cm at 25Â°C TOC <500 ppb <500 ppb Microbial Limit â‰¤100 CFU/mL â‰¤10 CFU/100 mL Endotoxins Not required â‰¤0.25 EU/mL Production RO + deionization Distillation (or membrane + UF)*

Answer 96

Validation of an autoclave is critical as sterilisation efficacy cannot be verified solely by testing the product. The process must be validated to demonstrate it consistently delivers sterilisation conditions across all load types. The validation lifecycle follows the stages of Installation Qualification (IQ), Operational Qualification (OQ), and Performance Qualification (PQ), in alignment with EU GMP Annex 15 and HTM 2010 Part 3. 1. Installation Qualification (IQ): * Verify that the autoclave has been installed per manufacturer’s specifications. * Check all utilities (steam supply, compressed air, water, drainage) meet design specifications. * Ensure correct calibration certificates are provided for critical instruments (temperature and pressure gauges, timers). * Electrical safety tests (earth continuity, insulation resistance). * Document equipment details (model, serial number, software version). * Ensure compliance with Pressure Systems Safety Regulations (PSSR). Reference: Annex 15 Section 3; HTM 2010 Part 3, Section 3.0. ________________________________________ 2. Operational Qualification (OQ): * Test the automatic control system (cycle initiation, door interlock functions). * Perform Bowie-Dick tests (for steam penetration in air removal systems). * Conduct vacuum leak tests to confirm chamber integrity. * Verify calibration of temperature and pressure sensors using independent calibrated devices. * Conduct empty chamber mapping using thermocouples placed throughout the chamber to confirm uniformity (per HTM 2010 Part 3). * Test alarms and safety systems. Reference: Annex 15 Section 4; HTM 2010 Part 3, Sections 4 & 5. ________________________________________ 3. Performance Qualification (PQ): * Define representative load configurations (minimum, maximum, worst-case loads). * Place thermocouples and biological indicators (e.g., Geobacillus stearothermophilus spores) at the most challenging locations (cold spots) within the load. * Run sterilisation cycles and ensure: o Sterilisation conditions (e.g., 121°C for 15 mins) are consistently met across all sensors. o Biological indicators demonstrate a ≥6 log reduction in spore count. * Perform 3 consecutive successful runs for each load type to demonstrate reproducibility. Reference: Annex 15 Section 5; HTM 2010 Part 3, Chapter 8 (Performance Qualification). ________________________________________ 4. Requalification: * Conduct annual requalification or after significant changes (e.g., repairs, software updates, new load types). * Follow similar procedures as PQ, including thermometric tests and biological indicator validation. Reference: Annex 15 Section 10; HTM 2010 Part 3, Section 2.39. ________________________________________ Additional Considerations: * Steam quality tests: Non-condensable gas, dryness fraction, and superheat must be assessed (per HTM 2010 Part 3, Chapter 9). * Documentation: A Validation Report summarising all stages, test results, deviations, and approvals (including QP/QA sign-off for GMP operations). ________________________________________ Summary Reference Framework: * EU GMP Annex 15: Qualification and Validation. * EU GMP Annex 1 (for sterile environments). * HTM 2010 Part 3: Validation and Verification of Sterilizers.

Answer 97

1. F₀ Value (Sterilisation Equivalent Time) * Definition: The F₀ value represents the equivalent time (in minutes) at 121°C that would deliver the same microbial lethality as the actual sterilisation process conducted at varying temperatures. * Why important: It allows comparison of different sterilisation cycles (even if not run at exactly 121°C) by converting them into an equivalent time at 121°C. This ensures consistent microbial kill across processes. * Formula: F₀ = \int 10^{(T - 121)/z} dt Where: o T = temperature at any time point (°C), o z = temperature coefficient (commonly 10°C for moist heat), o dt = time increment. * In Practice: A sterilisation cycle must achieve a minimum F₀ of 12 minutes for critical items (this gives a 12 log reduction of spores like Geobacillus stearothermophilus). ________________________________________ 2. D Value (Decimal Reduction Time) * Definition: The D value is the time required at a specific temperature to achieve a 1 log₁₀ (90%) reduction in the microbial population. * Example: If the D121°C of G. stearothermophilus spores is 1.5 minutes, it means every 1.5 minutes at 121°C reduces the viable spores by 90%. * Use: It quantifies the resistance of microorganisms (usually spores) to a sterilisation process. * Viva tip: D values are microbe-specific and are often provided on biological indicator (BI) certificates. Z Value (Temperature Coefficient) * Definition: The Z value is the temperature increase required to achieve a 10-fold (1 log₁₀) reduction in the D value. * In other words: It tells you how sensitive the microorganism is to changes in temperature. A lower Z value means the organism is more sensitive to temperature changes; a higher Z value means more heat-resistant. ________________________________________ Example (for moist heat sterilisation): * For Geobacillus stearothermophilus (the typical BI for steam sterilisation): o Z value = 10°C This means increasing the temperature by 10°C will reduce the D value by a factor of 10 (e.g., D121°C = 1.5 min, so D131°C = 0.15 min). ________________________________________ Why is Z value important? * It’s used in F₀ and A₀ calculations to adjust for different temperatures in the sterilisation cycle. * It helps describe the thermal resistance profile of the target microorganism or endotoxin (for depyrogenation).

Answer 98

What is MALDI-TOF? MALDI-TOF is a mass spectrometry-based technique used to identify microorganisms (bacteria, yeasts, and fungi) by analysing their protein profiles — mainly ribosomal proteins. How Does It Work? Sample preparation: A colony from an agar plate is spotted onto a target plate and overlaid with a matrix solution (usually an organic acid). Laser pulse: The sample is hit with a laser, causing desorption and ionization of proteins. Ion flight: The ions are accelerated through a vacuum tube toward a detector. The time it takes for ions to reach the detector (Time-of-Flight) depends on their mass-to-charge ratio (m/z). Pattern generation: The resulting spectrum is a unique “fingerprint” of the organism’s protein profile. Database matching: The fingerprint is compared to a library of known organisms to identify the microorganism. Why Is It Used in GMP? Rapid: Identification in minutes instead of 24–72 hours (as in biochemical/API-based methods). Accurate: High precision, especially for common environmental isolates. Cost-effective: Once purchased, cheaper than repeated biochemical kits. Regulatory-accepted: Used in many MHRA/EMA-compliant labs under a validated state. Example in QP Context “During my site visit, I observed the microbiology team using MALDI-TOF for rapid identification of environmental isolates. This improved turnaround time and supported timely investigation of EM excursions. I understood its advantage in reducing reliance on subjective biochemical tests and supporting faster batch disposition.”

Answer 99

o Instead of giving a long verbal explanation, I drew a diagram to illustrate my understanding. This visual below is something I took from an online webinar. Viva Question (Paraphrased from Transcript): “What does the Contamination Control Strategy (CCS) mean to you? How would you put it into practice?” * This was presented as a new question for the candidate. * The assessors did not want a speech, but rather practical application. * The candidate drew a visual diagram representing the CCS cycle. ________________________________________ Candidate’s Key Points (Extracted): * Used the PIC/S/ECA-style cycle diagram with key pillars: o Identify critical control points (CCPs) o Validate and implement controls o Establish monitoring and effectiveness checks o Implement CAPA o Drive continuous improvement * Emphasised that all 16 elements of Annex 1 Section 2.5 should go through this cycle. ________________________________________ Model Answer (Structured for QP Viva): “The Contamination Control Strategy (CCS) is a living, site-wide document that brings together all controls and rationales for preventing microbial, particulate, chemical, and cross-contamination throughout the facility. It is a GMP requirement under revised Annex 1 (2022), specifically Section 2.5.” ________________________________________ Structure: 1. Purpose and Scope “The CCS must demonstrate how all risks to product quality and patient safety are prevented and controlled, spanning facility design, process controls, equipment, utilities, cleaning, disinfection, gowning, and personnel training.” 2. Foundation in QRM “The CCS must be based on formal Quality Risk Management (QRM) principles in line with ICH Q9. It involves identifying Critical Control Points (CCPs) and ensuring they are adequately controlled, monitored, and continuously improved.” 3. CCS Cycle in Practice “In practice, I follow a structured cycle: * Identify: Determine all contamination risks and critical points. * Control: Implement technical/procedural controls (e.g., HVAC, RABS, operator gowning). * Monitor: Establish alert/action limits and environmental monitoring. * Review: Trend EM data, audit findings, deviations. * Improve: Apply CAPA and feed into periodic review and QMS updates.” (Optionally, draw the PIC/S CCS lifecycle diagram showing: CCP → Risk → Control → Monitor → CAPA → Review) 4. Annex 1 - 16 Elements “The CCS must address the 16 specific areas listed in Annex 1 Section 2.5, including facility and equipment design, personnel, utilities, cleaning/disinfection, process validation, and more. Each area should be risk-assessed and justified with supporting data and cross-referenced documents (e.g., SOPs, VMP, HVAC qualification).” 5. Governance and Review “The CCS must be: * Governed by senior QA and production leadership, * Reviewed periodically (e.g. annually or after significant changes), * Updated to reflect ongoing improvements, audit findings, and changes in regulatory expectations.” 6. Application as a QP “As a QP, I would expect the CCS to be a key reference document to support batch certification, especially in aseptic or high-risk products. It helps me verify that the facility maintains a robust and proactive contamination prevention framework.” ________________________________________ Key References for Viva: * EU GMP Annex 1 (2022): Section 2.5 (16 CCS elements) * ICH Q9: Quality Risk Management * PIC/S Aide-memoire on CCS (optional reference if assessors ask for more detail)

Answer 100

o I explained the zoning of cleanrooms, selection of EM locations, alert/action limits, sampling frequency, and data trending. They asked several sub-questions, which I answered confidently. Candidate’s Key Talking Points (Extracted): 1. Regulatory References: o Cited ISO 14644 for cleanroom classification and airborne particle limits. o Referred to Annex 1 of EudraLex Volume 4. o Also mentioned Annex 9 (WHO) for non-sterile products. 2. Objectives of EM Programme: o Establish routine monitoring of viable and non-viable contaminants. o Define alert/action limits. o Verify that the manufacturing environment remains under control. o Provide early warning of microbial excursions to protect product quality. 3. Determination of Sampling Locations: o Conduct risk assessment considering:  Personnel flow  Material flow  Equipment layout  Airflow direction (e.g., smoke studies)  High-touch areas (e.g., compounding tanks, filling heads) o Use of floor plans to mark sample points. o Emphasised worst-case locations (e.g., near open product, operator gown contact zones). 4. Cleanroom Classification: o Non-sterile cream manufacturing likely operates in Grade C or D, depending on open handling steps. o Mentioned potential classification of:  Grade C for product exposure during filling.  Grade D for background compounding areas. 5. Types of Monitoring: o Viable monitoring: Settle plates, contact plates, finger dabs. o Non-viable monitoring: Continuous or intermittent particle counters. ________________________________________ Model Answer (Structured for QP Viva): “When setting up an environmental monitoring (EM) programme for a cream manufacturing suite, I would begin by identifying the objectives: to verify that the environment remains in a state of microbiological and particulate control, and to provide early warning of excursions that could compromise product quality.” Regulatory Basis: “I would follow ISO 14644 for cleanroom classification and Annex 1 of EudraLex Volume 4, even for non-sterile products, as it outlines principles of contamination control. For non-sterile creams, I would also refer to Annex 9 (WHO) and BP guidance on microbial limits.” Risk-Based Site Selection: “Sampling sites are determined by a formal risk assessment, taking into account: * Personnel and material flow, * Equipment location and airflow, * Interventions and product exposure, * Results of smoke studies to confirm airflow direction. I would select worst-case locations for monitoring — for example, close to open containers, compounding tanks, filling heads, and operator working positions. Sampling points would be marked on floor plans and justified.” Cleanroom Classification: “For a cream product, the typical setup might be: * Grade C for product filling under open conditions. * Grade D for background compounding areas. These classifications may vary based on risk of contamination and whether the cream contains preservatives.” Monitoring Types and Frequencies: “I would include both: * Viable monitoring: settle plates, contact plates, swabs, and finger dabs. * Non-viable particle monitoring: using particle counters to monitor airborne particulates. Monitoring would occur at rest and in operation, and I would define alert and action limits based on historical data and classification guidance.” Data Review and Trend Analysis: “Data would be trended over time. Any alert or action limit excursions would trigger OOS/OOT investigations as per SOP. Regular trend reviews would be performed by QA and microbiology teams.”

Answer 101

o I asked clarifying questions, expressed concerns, and concluded that I would reject the batch. I explained the reasoning (potential contamination risk and GMP non-compliance). They seemed satisfied with my structured and assertive response. Scenario Setup: * During Grade B pre-fill operations, particulate matter (referred to as “debris”) is found. * The sterile product is aseptically filled in an isolator (Grade A) situated within a Grade B background. * Pre-fill compounding occurred in Grade C. * The fill-finish operator used RABS or isolator. ________________________________________ Clarifying Questions Asked by Candidate: 1. What is the nature of the debris? (e.g., visible fibre, plastic, glass) 2. Where exactly was it found — floor, equipment, air return grille? 3. What was the status of the batch — in process, filled, released? 4. Was any intervention performed? If so, was it recorded and reviewed? 5. Are there any EM (environmental monitoring) excursions in Grade B/C? 6. What are the results for the Grade A isolator? o Answer: Isolator EM results are “fine”. 7. Were there any operator gowning issues or EM failures? o Answer: Yes, finger dab results were out of limit. 8. Any recent cleaning? o Answer: The cleaning regime was missed for 3 days in Grade B/C areas. 9. Any recent changes in disinfectants or SOPs? o Answer: Yes, disinfectant was changed, but no validation performed. ________________________________________ Additional Information Provided by Assessors: * Operators not trained on the new disinfectant or revised cleaning SOP. * Investigation reveals multiple PQS failures: o Lack of cleaning validation for the new disinfectant. o Training gaps. o Poor documentation and cleaning traceability. * Despite acceptable isolator EM, multiple EM excursions in Grade B and C were reported. * Operator who failed finger dab test had conducted critical interventions. ________________________________________ Candidate’s Assessment & Response: Immediate Concerns: “Grade B debris, coupled with missed cleaning, failed finger dabs, and poor PQS oversight, raises significant concerns about aseptic assurance and potential cross-contamination risk.” ________________________________________ Structured Investigation Plan: 1. Containment: * Quarantine any affected batches and halt operations. * Secure EM and intervention records. 2. EM and APS Review: * Review EM trends from Grade A to D for: o Viable (settle/contact/finger dabs). o Non-viable (particle counts). * Cross-check interventions against APS (media fill) performance. * Review video recordings of interventions. 3. Personnel & Training Assessment: * Verify training records for all involved staff. * Retrain or disqualify if needed. * Review gowning qualification, retraining, and requalification. 4. Cleaning Validation & Disinfectant Qualification: * Identify root cause — no cleaning validation for the new disinfectant. * Immediate CAPA: validate disinfectants (e.g., using EN13697 or USP <1072>), document microbial efficacy. ________________________________________ Final QP Decision: “Given the combination of: * Particles in Grade B * Finger dab failure * Missed cleaning * No disinfectant validation * Operator intervention in isolator I would not certify the batch at this time. The sterility assurance cannot be verified. However, if a full investigation demonstrates no link between the Grade B excursion and Grade A operations, and risk to product is acceptably low, certification may be reconsidered — subject to QA oversight, a detailed risk assessment, and review of APS data and video evidence.” ________________________________________ Model Answer Summary: “Finding debris in a Grade B area during sterile manufacture is a red flag. In this case, the missed cleaning, operator failure, and unvalidated disinfectant raise systemic PQS concerns. While the isolator EM results are acceptable, I would need to: 1. Assess risk to product sterility via EM trends, operator intervention logs, and video footage. 2. Initiate CAPA on PQS failures (training, cleaning, disinfectant qualification). 3. Conduct a robust investigation aligned with Annex 1, Sections 9 and 10. Unless confident that product sterility assurance is intact, I would withhold certification.”

Pharmaceutical microbiology Flashcards

(126 cards)