Pharmaceutical microbiology Flashcards
Tell me the mfg process for ATMP?
- Apheresis: Cell Isolation from patient (autologous) or donner (Allogenic)
- Cell isolation
- Genetic modification (Transduction) with virus vector
- Cell expansion
- Harvest and Formulation
- Cryopreservationan preservation and storage
What are the CCPs in ATMP manufactuig?
- Apheresis - patient/donor identity, transport time/condition, documentation of chain identity
- Transduction - transduction condition (Multiplicity of infection, time, temp)
- Vector Identity, potency, sterility
- Absence of replication-competent virus - Cell Expansion - Culture condition (Temperature, pH, oxgen, co2)
- EM (Grade A/B)
- Monitoring of Growth kinetics and contamination - Formulation and Cryopreservation
* Why critical: Affects product stability and post-thaw viability.
Controls:
* Dimethyl Sulfoxide (preventing Ice Crystal formation) concentration
* Freezing rate and container integrity
* Final product sampling for QC (viability, identity, potency) - Labelling and Traceability
* Why critical: Risk of mismatch or misidentification.
Controls:
* Use of unique identifiers (e.g., barcodes)
* Reconciliation against source and recipient data
* Verification at each transfer step - Storage and Transport
* Why critical: Product viability is highly sensitive to temperature excursions.
Controls:
* Use of validated cryo-storage and dry shippers
* Continuous temperature monitoring
* Transport chain of custody documentation
What are the key QC tests performed for a cell-based Advanced Therapy Medicinal Product (ATMP)?
- Appearance / Visual Inspection
* Purpose: To ensure no particulate matter or visible contamination.
* Check: Colour, clarity, presence of aggregates. - Cell Count and Viability
* Purpose: Confirm therapeutic cell dose and ensure viable cells are delivered.
* Methods: Trypan blue exclusion or flow cytometry.
* Acceptance: Typically ≥70% viability. - Identity
* Purpose: Confirm that the correct cell type is present (e.g., T-cells).
* Method: Flow cytometry using specific surface markers (e.g., CD3 for T-cells).
* Also confirms: Patient/donor match (traceability). - Potency
* Purpose: Demonstrate biological activity of the product.
* Methods:
* Cytotoxicity assay (e.g., killing of target cancer cells)
* Cytokine release (e.g., IFN-γ, IL-2)
* Expression of CAR (e.g., via flow cytometry or qPCR) - Sterility
* Purpose: Ensure product is free from bacterial or fungal contamination.
* Method: Compendial method (Ph. Eur. 2.6.1) or validated rapid sterility test.
* Note: May not be available at release due to short shelf-life—parametric release may be applied. - Endotoxin (Bacterial Pyrogens)
* Purpose: Prevent harmful inflammatory responses in patients.
* Method: LAL test (Limulus Amebocyte Lysate).
* Acceptance: Below threshold based on dose volume (e.g., <5 EU/kg/hour). - Mycoplasma Testing
* Purpose: Detect common contaminants in cell cultures.
* Methods: Culture-based or PCR-based validated methods. - Replication-Competent Virus (RCV) Testing (for viral vector-based ATMPs like CAR-T)
* Purpose: Confirm absence of infectious virus from vector production.
* Timing: Typically performed on the vector and sometimes on the final product. - Adventitious Virus Testing
* Purpose: Detect unexpected viral contaminants.
* Usually performed: On vector seed banks or cell banks, not every batch. - pH / Osmolality / DMSO Concentration
* Purpose: Ensure the product is within physiological limits and formulation is accurate.
Difference between large and small molecule tests?
You’re validating an autoclave for both porous and fluid loads. Can you draw the load types, and explain the purpose of the pulses in the cycle?
Sure. In autoclaving, we typically validate two types of loads:
- Porous loads – such as instruments, filter assemblies, stainless steel parts, rubber bungs, wraps, textiles
- Fluid loads – like water for injection (WFI), growth media, or product in final containers
Draw up pulses.
What are the pulses in the cycle for?
The pulses refer to vacuum and pressure phases in the pre-conditioning stage of a pre-vacuum autoclave cycle.
- Their purpose is to remove air from the chamber and load — especially from porous materials and lumens where air can be trapped.
- Air inhibits steam contact, and even a small air pocket can prevent sterilisation.
- Pulsing alternates vacuum and steam injections to create pressure changes that ‘suck out’ air from the deepest parts of the load.
This is why porous loads need pre-vacuum cycles with pulsing.
Fluid loads, on the other hand, typically use gravity or slow exhaust cycles, with air removal via steam displacement.
What’s the difference between porous and fluid loads in validation?
- Porous Loads
Use pre-vacuum cycles with multiple pulses
Validated using Bowie-Dick test for air removal
Challenged using biological indicators (BIs) in wrapped instruments
Risk = air pockets - Fluid Loads
Often use gravity or slow-exhaust cycles
Validated using FP sensors to confirm time/temp
Challenged using BIs and temp probes inside liquid containers
Risk = delayed heating/cooling (cold spots)
What’s the risk of air pockets?”
“Air pockets prevent direct contact between saturated steam and the item’s surface, which is necessary for efficient heat transfer and microbial kill.
A single air pocket can result in an unsterilised zone. That’s why:
- Air removal is critical in porous load sterilisation
- We verify this with Bowie-Dick tests and thermometric mapping
- Load configurations must minimise crevices and ensure proper drainage”
For an Aseptic filling line, it was given a schematic diagram of a Grade C cleanroom with Isolator, showing location of environmental monitoring in both the Grade C room and the Grade A isolator. The product being filled was a biologic (mab) not subjected to terminal sterilisation.
You are a QP at this site (MIA) reviewing the batch docs for certification. 6 cfu had been recovered from the finger dab (Gram +ve Cocci/skin commensals) contact plate for 1 of the operators, all other Ems were within limits. How do you proceed?
spent a great length asking questions about the Product, and Process (PRICE-PRISM and drew out Ishikawa analysis to identify the issue). Turned out that root cause was faulty VHP transfer sanitisation system. I was challenged/questioned for almost all the questions I asked in this scenario i.e., why did I want to know that, what was the relevance to my decision making?
Micro OOS for a dispensing booth in non-sterile manufacture, what do you do? Micro OOS due to increased capacity, impact on batches; same scenario but in sterile manufacture what would you do;
Micro OOS due to increased capacity, impact on batches
Same scenario but in sterile manufacture what would you do?
Grade D micro limits as per Annex 1?
A
Air sample CFU /m3 - No growth
Settle plates CFU /4 hours - No growth
Contact plates CFU / plate - No growth
Glove print, Including 5 fingers on
both hands CFU / glove - No growth
B
Air sample CFU /m3 - 10
Settle plates CFU /4 hours - 5
Contact plates CFU / plate - 5
Glove print, Including 5 fingers on
both hands CFU / glove - 5
C
Air sample CFU /m3 - 100
Settle plates CFU /4 hours - 50
Contact plates CFU / plate - 25
Glove print, Including 5 fingers on
both hands CFU / glove - not required
D
Air sample CFU /m3 - 200
Settle plates CFU /4 hours - 100
Contact plates CFU / plate - 50
Glove print, Including 5 fingers on
both hands CFU / glove - not required
Grade of rooms for different types of activities i.e. dispensing in sterile vs non sterile?
What do you understand about media fills?
You are doing your routine media fill and have 4 vials with growth – what would you do?
If the microorganism was confirmed to be gram positive can you suggest any potential sources of contamination? Do you know the latin names ?
If it was confirmed as a gram negative can you suggest any potential sources of contamination?
Turns out it was a skin organism caused by a new operator – he hadn’t been present in any routine manufacture so no impact to lots already manufactured – what would you need to do before returning to routine manufacture?
You are a QP at the sight and came to know that the personal finger dabs failed which was taken before the aseptic processing. What are the limits for finger dabs in Tablets and creams manufacturing?
You are a QP at a sterile manufacturing site and you received a call from QC informing you of a Micro OOS for a unit going turbid. This was still incubation and at day 5. This was part of Media Fill. What are your concerns? 8 batches manufactured since last Media Fill. 4 onsite and 4 on the market. What do you do? You have been told that the Micro is Micrococcus.
What different types of sterilisation methods are there?
How would you validate a steam autoclave?
Talk about equipment qualification, validation protocol, load cycles, mapping, Bis, F0 calculations, drew cycles, routine checks and water quality
Why is non condensable gas important?
Manufacturing a non-sterile ointment and micro is 100 CFU/g. It is gram-negative. What is the source and what do you do?
An operator tells you that they have identified an OOS in your purified water system, what do you do?
*Microorganisms is staphylococcus aureus
What would you do if you confirmed that your water system is contaminated and you need it ASAP to manufacture urgent batches?
What chemicals will you use to sanitise it?
Please tell us Purified water system and acceptance criteria as per pharmacopeia ?
You are manufacturing a cream and during testing your have a micro failure for a required absence of microorganism. Explain what you would do.
- The microorganism is pseudomonas aeruginosa . Thoughts?
You’ve found the same microorganism when sampling your purified water system but the labs have put it down to sampling errors. Thoughts?
How would you validate a purified water system?
What are the requirements of WFI compared with Purified Water? That are the CPPs for each step in the process.
Can youTalk through an aseptic drug manufacturing process?
That are the CPPs for each step in the process?
Purified Water failure – Above action Limit
8Phase 1a/1b investigation. - What agar would you use for water testing?
What organisms would you use for growth promotions?
8Why are you interested in the ID of the organism? Ps. aeruginosa – no other organisms.
What checks can you do on your water system to confirm that it is functioning appropriately?
Uncovered that all other plates from that day were also contaminated – revealed that it was a bank holiday, so the samples were left at room temp over the long weekend. Is everything okay now? Requested checks on the plates for subsequent days testing – no growth so happy with water – but not with the lab!
You have a call that there is an OOS result for a user point on your PW system 200cfu/100ml sample – in house limit exceeded.
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 .
How would you set up environmental monitoring? They have shown a table and asked me how would I set up viable and non-viable monitoring on the table?
What is endotoxin, and where is it found?
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).
Your injectable product filled in Grade A is OOS for endotoxin. What are your next steps?
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.
How do you validate a depyrogenation tunnel?
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.
What are the differences between terminal sterilisation and aseptic processing?
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.
Media Fill Failure (Sterile Scenario)
Q8. You have a single contaminated unit from a media fill. What do you do?
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.
What is the purpose of preservative efficacy testing (PET) in sterile pharmaceutical products?
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.
Which microorganisms are used in PET according to Ph. Eur. 5.1.3?
Ph. Eur. lists the following standard test organisms: ESCAPZ:
Escherichia coli (for oral preparations);
Staphylococcus aureus; Candida albicans; Aspergillus brasiliensis; Pseudomonas aeruginosa; Zygosaccharomyces rouxii for sugar-rich oral preparations.
Describe the preparation of inoculum for PET.
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.
What are the acceptance criteria for PET according to Ph. Eur. Table 5.1.3-1?
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.
What does NR and NI stand for in PET evaluation?
NR: No Recovery — No viable organisms detected.
NI: No Increase — No increase in viable count compared to the previous reading.
Would you require PET for a single-dose sterile product?
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.
In a multi-dose sterile product, what would you do if PET results only met criterion B?
In a multi-dose sterile product, what would you do if PET results only met criterion B?
What could cause PET failure in a sterile multi-dose formulation?
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
What is the principle of the sterility test?
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.
What methods are described in Ph. Eur. for sterility testing?
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.
When would you choose membrane filtration over direct inoculation?
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.
What are the controls used in sterility testing?
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.
How do you validate the sterility test for a new product?
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.
What are the implications if the sterility test fails?
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.).
Is sterility testing sufficient to confirm product sterility?
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.
What is the incubation duration and conditions for sterility testing?
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.
What microorganisms are used for a sterility test?
SBC CAP
Staohylococcus aureus (aerobe)
Bacillus subtilis (aerobe)
Clostridium sporogenes (anaerobe)
Candida albicans (yeast)
Aspergilus brasiliensis (mold)
Pseudomonas aeruginosa (aerobe)
Explain the significance of Sterility Assurance Level (SAL) in sterilisation.
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.
What is moist heat sterilisation?
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.
Validation of Moist Heat Sterilisation (general)
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.
What product type would be suitable for Polous load. And how would you validate?
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.
What product type would be suitable for Fluid load And how would you validate?
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.
Scinario: You are a QP in a semisolid manufacturing site making antiseptic cream. Your QC manager says he has an atypical test result on TAMC test of a batch and one OOS result in another batch TAMC. Are you concerned? (About 15 minutes).
Follow up: ae. What is atypical result?
Follow up: af. What checks will you do in Phase 1b?
Follow up: ag. You never seen this organism in the baseline flora. It is a bacillus. Where would you get these organisms? For your OOS you have ID ed and found Pesuodomonas. Why are you concerned?
Follow up: ah. Talk me through the controls of the water system? What are the acceptance limits? Why TOC?
Follow up: ai. What data will you like to see? Why CUSUM charts? Any other things you will like to do with the data? What is normalisation?
Follow up: aj. So, you have 4 batches affected. What you are going to do with this?
Follow up: ak. So, you have 4 batches affected. What you are going to do with this?
Follow up: al. How will you sanitise the water system?
Follow-up : am. OK now let’s concentrate on the atypical bacillus. Your investigation said its coming from a door work in your microbiology lab. So, when are you releasing the batch?
Follow up: an. You have explored everything and is still the door.
Follow up: ao. Your sales guys are pushing they need this batch out to meet the end of month target.
What is Parametric Release?
• 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.
What is a Pyrogen?
A substance that causes fever. Example: Endotoxin from Gram-negative bacteria.
What is an Endotoxin?
Lipopolysaccharide component of Gram-negative bacterial cell wall, pyrogenic.
What is an Endospore?
Dormant, resistant form of bacteria (e.g., Bacillus) for survival in harsh conditions.
Commensal vs Pathogen vs Opportunistic Pathogen
Commensal (normal flora), Pathogen (disease-causing), Opportunistic Pathogen (causes disease in immunocompromised).
Gram Positive Bacteria
Bacillus, Staphylococcus aureus.
Gram Negative Bacteria
E. coli, Pseudomonas aeruginosa.
Mould Examples
Aspergillus niger (environment), Penicillium (food spoilage).
Source of Contamination
Animal-origin: Prions, viruses; Vegetable-origin: Yeasts, moulds; Minerals: Spore-formers; Sugars: Yeasts, moulds.
Disinfectant Qualification
Surface efficacy testing, rotating disinfectants to prevent resistance.
Micro prep1: Where can microorganisms exist in your facility?
Micro prep2: What about in your raw materials? What controls can you put in place?
Micro prep3: Different methods to count bacteria?
Micro prep4: Describe good sampling practice of purified water points? How frequent should you sample?
Micro prep5:What is the difference between disinfection and sanitization?
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.
micro prep6: Scenario: You are a QP, CEO wants a new sterile product manufactured, asked how I would
perform EM qualification of the Suite. Wanted everything for this including Laminar flow
controls, viable/nonviable limits for Grade A&B rooms, positions of the plates and air
sampling within the Grade A (I said close to point of fil which led to discussion around risk of product hitting the plates). Spent a little time on utilities and WFI testing (QC and Micro).
Micro Prep7. What considerations would you have for setting up an environmental monitoring
programme in your cream manufacture suite? What are you looking for? Objectives? How do you determine sampling sites?
‐ high traffic areas, areas with poor airflow, sinks
Micro prep8: What are the Annex 1, microbiological requirements within a clean room, use of agar plates,
grades of area
Micro prep9: scenario: Your micro department informs you that they have discovered a trend of Micrococcus luteus
and Bacillus subtilis in the tablet compression room of your grade D facility. What are your concerns?
Follow-Up: What type of micro are these?
Follow-up: Are they pathogenic?
Follow-up: Where is guidance found on clean rooms?
Follow-up: Where is guidance found on micro for tablets?
Follow-up: Was originally above alert level, but now above action level. What action would you take?
Micro prep8: Scenario: You have a cream product that has failed TYMC (yeasts and moulds). You have retested and it passes, you therefore have one passing result and one failing result.
Follow-up: What would you investigate?
Follow-up: What is the preservative efficacy test?
Follow-up: What organisms would you use?
Follow-up: How long would it last?
Follow-up: You now have a second batch, and the same thing happens – what would you do
now?
