(6) Steam sterilization processes (4 points)
(a) How is the air removal carried out in the autoclave shown?
Note: the items to be sterilzed are placed in the grid basket.
(b) Name two disadvantages of the autoclave shown in (a) compared to an autoclave with an external steam generator.
(c) Draw the pressure curve of a steam sterilization process using fractionated vacuum for air elimination and a single vacuum phase for drying.
Steam generation within the chamber – single wall: Batch steam sterilizer
grid basket = Gitterkorb = air can circulated freely but can get unter the basket (enough space between basket & bottom needed for air elimination)
Function:
1) Product placed inside the autoclave
2) water reservoir is direct underneath the plate
3) steam generation with electrical heating (yellow)
4) Elimination of air = steam moves up & displaces the air to the top
- air gets pressed out via air-removal valve out of the chamber
- air must be eliminated otherwise steam cannot reach product; no condensation
picture a) Steam generation within the chamber – single wall: Batch steam sterilizer
disadvantages:
1) slow heat up because steam generation inside the chamber with (cold) product
2) slow cooling because water reservoir inside chamber, takes longer to cool system down (condensate stay inside)
= combination of fractioned vacuum & single vacuum phase
A) Ventilation phase = generated steam replaces the air via air-separator
= Risetime & air elimination:
B) Compensation phase = rest of the air gets eliminated
= Pressure rises: valve get closed & steam accumulates
C) Sterilization time =
D) Cooling & vacuum phase = vacuum helps in drying
(8) Ionizing radiation sterilization (1.5 points)
(a) In which case may ionizing radiation sterilization be used for the terminal sterilization of pharmaceutical products?
(b) Name one disadvantage of gamma irradiation compared to X-ray irradiation.
= use of ionizing radiation possible for: pharmaceutical medical products
dry powder products, non-aqueos liquids & semi-solids if the product is NOT temperature stable! -> e.g. ophthalmic ointments in tubes
if product isn’t heat stable, use ionising radiation with high dose up to 25 kilo Grays
Why is the used of ionising radiation limited? = can change the product characteristics due to the ionisation reaction
= Problem of handling/ disposing radio-active waste -> X-ray has no residues
= gamma-irradiation has lower oenetration capacity than X-ray
(1) Sterility testing (2.5 points)
An infusion solution (large volume parenteral) is to be tested for sterility according to Ph. Eur. 2.6.1. The filling volume per infusion bottle is 500 ml, the batch size is 10.000.
(a) Using the following table from Ph. Eur. 2.6.1, indicate the minimum number of bottles that must be used for sterility testing.
(b) The sterility test is carried out by membrane filtration. Describe the procedure for this test without the subsequent incubation.
a) more than 500 containers (bottles)
2% of 10.000 = 200 -> its too much
whihever is less: minimum 10 bottles for large volume parenterals should be tested
b) Procedure of sterility testing by by membrane filtration
Process: Sample filtration procedure
= should get done under cleanroom, laminar airflow work bench or in isolator
1) 2x empty bottles get connected with an injection needle a hose (Schlauch) through a peristaltic pump with the product bottle
2) Product extraction: with a peristaltic pump the product gets pumped out of the bottle -> product gets transported to the pre-sterilized bottles with a membrane filter
4) product is filtered & empty product bottle gets disconnected
5) a washing solution is placed via hose -> filters get washed to eliminate residuals (esp. of growth inhibiting product)
5) bottles get closed at the bottom
6) Steps for incubation: 2x different nutrient medium get introduced to the bottles to exclude (identify) different bacteria -> incubation for min. 14 days
(3) You have the following data on the heat killing of a microorganism at a temperature of 126.5 °C: a microbial population is reduced by 5 log levels in a time of 240 s. z = 5.5 °C.
(a) What D-value at 121°C results from the data?
(b) After what total time at 121 °C is a SAL of 10^-9 achieved if 100 microorganisms are present at the beginning?
(2.5 points)
a) D-value = decimal reduction value [mins]
= time needed at a certain temperature to kill 90% of germs
= time required to reduce the number of viable organisms to 10 % of the original number
= time that is needed to reduce cfu number by factor 10 or by 1 log 10
Calculation:
Answer: At 121 °C with given data results a D-value of 8 minutes
b) Total time = F-value = time (holding time) at a temperature T in minutes to reduce the population (N0) with the specific D value by the desired number of powers -> often log10 levels (n)
n = from 100 germs to 10^-9 = 11 log levels
F (total time) = 11 * 8 mins = 88 mins
(4) (a) What does the z-value tell us about the heat resistance of a microorganism?
(b) What is the relationship between the F value and the SAL value?
(c) For a steam sterilization process at 134 °C, an F0 value of 50 min is achieved. How is the achieved F0 value of 50 min to be interpreted?
(2 points)
a) Z-value = Temperature change in °C required to alter D-value by factor of 10
high z-value = means large increase in °C is required to achieve 1 log reduction of D-value = means that germs are more heat resistant to changes of temeprature
low z-value = low increase in °C is required to achieve 1 log reduction in the D-value = germs are less heat resistant
e.g. spores = z-value 10°C & vegetative cells = 5,5 °C
b) Relationship between the F value and the SAL value
F-value = thermal death time = holding time [mins] at a temperature T to reduce population N0 with specific D-value by desired number of poweres (log levels) to final value N
SAL = sterility assurance level = probability that germs may be alive
Relationship: with sufficient F-value [holding time in mins] a sterilisation process can ensure that the SAL-value achieves the required standard
high F-value = high holding time = low probability of items remaining non-sterile, low probability that germs stay alive = means a better (lower) SAL-value
low F-Value = low holding time = high probability that sterilisation is not sufficient & germs stay alive = high SAL-value
F0-value [mins] = 1 min means the lethality of a method that works for 1 minute at 121.1 °C (250 °F) and a z-value of 10 °C (18 °F) or an equivalent temperature-time combination
= makes different steam sterilization comparable -> important for pharmacy
50min (F0-value) at 134°C = process achieves the same microbial lethality as the sterilisation at 121,1°C for 975 mins
(12)
(a) According to GMP Annex 1, every company that manufactures sterile medicinal products requires a CCS. What does the abbreviation CCS stand for?
(b) One of the rules of aseptic working is not to interrupt the laminar air flow. GMP Annex 1 refers to this as the first air principle. Briefly explain why this rule must be followed whenever possible?
(c) Which aseptic working rule should class B personnel follow regarding their gloves?
CCS = contamination control strategy (for Aseptic production of sterile drugs)
= a planned set of controls for microorganisms, endotoxin/pyrogen and particles, derived from current product and process understanding to assure process performance & product quality.
= is an important principle for aseptic working
Laminar airflow shouldn't be interrupted because:
1) Contamination control & Sterility maintenance: unidirectional flow of air sweeps away particles from working area -> when interrupted particles/ contaminants can settle on surface (risk of contamination) -> product quality & safety
2) Important if protection of personal required (hazardous subtances) -> interruption can create a risk for personnel
3) Laminar airflow is usually connected with controlled envirenmental conditions (°C/ humidity…) -> interruption can cause disturbances in conditions
(2) (a) What is described by the SAL value?
(b) For a sterilization process, the probability of sterility after the process is P = 0.99999. How many non-sterile containers would have to be expected for a batch size of one million containers?
a) SAL = Sterility Assurance Level
= probability, that germs may be alive
= is expressed as the probability of a non-sterile item after sterilisation
= describes the probability of a unit being non-sterile after undergoing sterilization process
b) P = 0.99999. How many non-sterile containers would have to be expected for a batch size of one million containers?
SAL of 10^−6
= means a probability of not more than 1 viable germ in 1 ×10^6 sterilised items of final product
= of 1 million product containers 1 container is contaminated after sterilization
SAL = 1-0,99999 = 1*10^-5
(1*10^-5) * 10^6 = 10
=> 10 non-sterile containers would be expected for a batch size of 1 Mio. containers (with P = 0,99999)
(14) Barrier systems (2.5 points)
(a) Why is a safety cabinet considered less safe as an open RABS?
(b) Which RABS type is most comparable to an isolator?
(c) When is overpressure used within isolators?
(d) How can it be tested whether an inert gas atmosphere is maintained in an isolator?
at a safety cabinet operator is closer & in direct contact to working area, whereas in RABS interventions are made via gloves (barrier closed system) -> less safety of product & personnel in safety cabinet
RABS enables higher level of sterility (due to enclosed barrier system, no room air & air moves from A to B), whereas safety cabinet is not enclosed (room air gets inside & filtered from B to A)
• Open RABS= vents in the barrier allow air to move from Grade A zone to Grade B area
= closed (active) RABS = closed barrier between operator & critical zone, similar to isolator; glove ports are used
= for aseptic purposes, protection of product -> infeed/ fillig & part of loading area
1) via Inertization test = test to verify that inert atmosphere is mantainted inside isolator is given by monitoring the concentration of oxygen (with oxygen sensor)
2) Gas Sampling = sampling of inert gas from different parts of isolator
3) monitoring of pressure inside isolator = const. pressure means isolator is sealed & inert gas environment is maintained
(16) Preparation of equipment and primary packaging material (2.5 points)
(a) Which medium is used for the final rinse in the washing process of primary packaging materials?
(b) What is the key advantage of using single-use systems in aseptic manufacturing from a patient protection perspective?
(c) What is shown in the figure below?
(d) Why do syringes and cartridges receive an anti-friction coating on the inside?
= Water for injection
= significant reduction of the risk of cross-contamination
= single-use is connected with purity/ sterility & therefore with safety for patient & quality of product
= easy to exchange if necessary (disposable)
= a sterile connector (hose connection with sterile coupling)
= to minimize break loose & gliding forces (minimize friction)
-> without siliconisation on the indeside the stopper doesn’t move at all due to high friction or it moves in an uncontrolled way (no correct dosage of e.g. insulin possible)
6) Steam sterilization processes
(d) Which steam sterilization process is suitable for the sterilization of bottled water for injections besides the hot water spray process?
(e) The figure shows an autoclave tape. What is it used for?
= steam/ air mixture process
2) Chemical indicators (for routine) / biological indicators (for validation)
-> chemical indicator tape = gets placed on product = shows colour change if desired conditions via sterilisation were reached -> indicator to controll success of sterilisation
(13) A stainless steel format part is needed in an isolator for aseptic filling of a medicinal product.
(1) The format part is loaded into a double-door washing machine in class D and then removed in class C.
(2) There, it is packed in a Tyvek bag and loaded in a double-door autoclave.
(3) The sterilized bag with the format part is removed from the autoclave in the filling room class C.
(4) The bag is placed on the work surface of an isolator and the interior of the closed isolator is biodecontaminated with H2O2 vapour.
(5) The bag is opened and the format part is then installed aseptically in class A.
How would you evaluate the process of preparing and transferring the format part: Are all steps (1) to (5) in order with regard to the requirements of Annex 1 and a possible risk to the medicinal product in Class A? Is there potential for optimization? If yes, which one? (2 points)
1) wahing machine from class D to C = is right
2) Packed in a Tyvek-bad = not ok = the used of 2 Tyvek gas is needed to guarantee sterility
3) not ok = Handling & filling of aseptically prepared products in class A with class B environment
4) is right = create class A conditions
5) is right
(7) Dry heat sterilization (1.5 points)
(a) What is meant by compensation time in (dry) heat sterilization processes?
(b) The D value for heat-resistant endotoxins is around 50 °C. To achieve the required 3 log reduction of endotoxins the heat treatment is performed at 280 °C for 3 min. How long would the process duration be at 330 °C to achieve the same effect?
Sterilization with dry heat: = At least 2 h at 160 °C -> Other time/temperature combinations possible -> increase of 10°C = doubles reaction process
= SAL: ≤ 10-6
Compensation time = additonal time required to ensure that the entire load (incl. difficult to reach areas) reaches the desired sterilization temperature.
= time that is needed to reach 160-180°C from the reference measuring point inside sterilizator & all points of product (even coldest spot)
= important to guarantee everywhere 121 °C
= 0,3 min
(17) Fill and Finish (4.5 points + 0.5 bonus points for part h)
(a) Why is weigh dosing the preferred method for aseptic filling for very high-priced products?
(b) What dosing system is shown in the picture?
= enables high specification for dosing exactly to the defined amount
= to use exact amount of an high-prices product (economic aspect)
= (sterile) Rotary piston pump
(18) Why must the monitoring results always be taken into account when releasing an aseptically filled product batch, even though a sterility test is carried out as part of the final product quality control? (1 point)
= because sterility cannot be guaranteed by testing
Monitoring / process control = Recording of process parameters temperature & time -> chemical & biological indicators
= to guarantee aseptic processing for the whole time of process
-> need to be proved that particle concentration is below limits with germ-free environment during & in the end of process
-> guarantees safety of process through recording of parameters & seeing if deviations may have negative impact of product quality (only good sterility testing not enough)
(19) Media fill (3 points)
(a) Media fill is also known as APS. What does APS stand for?
(b) How often must a media fill be performed as part of the introduction of a new product?
(c) An SOP for the aseptic formulation and filling of a liquid product specifies that two excipients and WFI should be added to the active ingredient while stirring gently. How do you take these specifications into account for a media fill?
(d) How long does incubation take after a media fill and how are the incubated containers subsequently analyzed?
APS = aseptic process simulation
for new product = initial validation = 3x successful APS
& periodic revalidation 2x/year
= instead of excepients is the nutrient medium is added
= all process should not have negative impact on microbial growth -> by adding WFI the nutrient medium might be diluted -> better to use pure medium
= media fill hast to be performed as a defined aseptic process according to manufacturing SOP & media fill plan
= incubation for 14 days
Evaluation = via visual inspection & turbidity
(23) The following illustration shows a CIP/SIP-capable sterile sampling valve. The product chamber is on the left of the picture. How can it be ensured that the sealing point is also reliably cleaned and sterilized? (1 point)
d) How can it be ensured that the sealing point is also reliably cleaned and sterilised?
= due to CIP/SIP connection
= valve can get opened for cleaning and sterilisation at sealing point (orange front)
(f) How is the plastic tube produced in the BFS process?
(g) What is the minimum cleanroom class for filling terminally sterilized products?
(h) Bonus question: What is the name of the illustrated method for transferring tubs to class A, which requires double outer packaging of the tubs? Hint: the method is abbreviated NTT.
= aseptic filling of terminally sterilised products is preferred to be done in class A & or at least in class C
= Verarbeitung mit Doppelbeuteln, dem sogentannten No-Touch-Transfer (NTT)
= Dabei werden die Primärpackmittel steril angeliefert und über einen definierten Prozess, welcher kontinuierlich Keim- und Partikelüberwacht ist, in den aseptischen Bereich (Grade A) überführt.
= method designed to maintain the sterility of containers & components when transferring them into a critical cleanroom environment.
(20) Material and surface properties (1.5 points)
(a) An Ra value of 0.8 μm is specified for a surface. What does this mean?
(b) Name a method by which an Ra value of equal to or less than 0.8 μm can be achieved for a stainless steel surface.
(c) A material that comes into contact with a sterile product should be inert to the product. What else must the material be inert to? Specify one aspect.
For stainless steel surfaces a Ra-value of ≤ 0.8 μm achieved by mechanical polishing or machining is recommended or via cold-rolling-process (Kaltwalzen)
= Inert (inaktiv) to the detergents and disinfectants
(10) Sterile filtration (2 points + 0,5 points bonus question)
(a) What type of filtration (in terms of separation limits) does sterile filtration belong to? Tick the correct box.
(b) Bonus question: Specify one common membrane material for sterile filters for the filtration of aqueous products.
(c) What is the unit of the bubble point?
= is always the last option for sterilisation of pharmaceuticals
• Sterilization filtration (microfiltration) = separation of germs by filtration (sieving effect= Siebeffect)
• Membrane filter with an average pore size of < 0.22 μm (or smaller)
Bubble Point [bar] = pressure at which a continuous discharge of air bubbles can be detected (3 to 5 bar) = depends on the filter properties
- big leak = lower pressure
- the higher pressure = the smaller the pore size
10^-5 bar = SI-unit is Pascal (Pa)
(10) Sterile filtration (2 points)
(d) What is the acceptance criterion for the bacterial challenge test that is performed as one of the validation activities for a sterile filtration process?
(e) Sterile filters must be analyzed with regard to extractables, among other things. What are extractables?
= 100% retention of germs, no single reference germ should pass the filter!
(21) The pipe transition shown (flow direction from left to right) has an unfavourable hygienic design. Draw a transition with an ideal hygienic design. (1 point)
(21) The pipe transition shown (flow direction from left to right) has an unfavourable hygienic design. Draw a transition with an ideal hygienic design.
(11) Validation of sterilization processes (2.5 points)
(a) How do you call the release of terminally sterilized products without batch-related sterility testing, based solely on the measurement of physical process parameters?
(b) What must be considered in the PQ of a steam sterilization process with regard to loading - in addition to worst-case considerations?
(c) Which third key specification of a bioindicator besides the z-value and D-value must be known for the validation of a steam sterilization process?
(d) What should not be included in the URS of a steam sterilizer? Mark with a cross
= test load must contain a product that is routinely handled (loading patterns must be defined)
& the load must represent product families with the greatest difficulty for the sterilisation (worst-case consideration)
= bioburden N0
(d) What should not be included in the URS of a steam sterilizer? Mark with a cross.
URS = user requirements specifications & functional specification document
= a planning document that specifies what a system needs to do => part of DQ (Design Qualification)
NOT included: = WFI-monitoring because use of steam
Sterility testing
(b) The culture medium plays a central role in sterility testing. Which two microbiological tests must be performed on each batch of culture media?
(c) An infusion solution (large volume parenteral) is to be tested for sterility according to Ph. Eur. 2.6.1. The filling volume per container is 250 ml. Using the following table from Ph. Eur. 2.6.1, indicate the minimum volume per container that must be used for sterility testing
b) microbiological tests of culture media (each batch):
1) Sterility testing = must be verified for each batch
-> incubation for 14 days & no visible microbial growth is allowed
2) Positive growth promotion test (with test microorganisms)
= proove that special test organisms can grow in the nutrient medium
-> Test principle: - very small number (100 CFU) of colonies is taken & placed in the nutrient medium (TBS or TM)
- different microorganisms (aerobic/ anaerobic/ fungi/ spores) get incubated to detect if they grow
In addition: biological test = with bioindicators (not culture medium)
c) Table Ph. Eur. 2.6.1
minimum volume per container - infusion solution (large volume parenteral) 250 ml
=> greater than 100ml = 10% of contents of the container (but not less than 20)
10% of 250ml = 25ml per container are used for sterility testing
Questions
What data are needed for validation with biological indicators?
Why is sterile compressed air for counterpressure used and not steam?
1) bioburden N0
2) type of microorganisms -> resistance: D & z-value -> survival & kill-time
= because to generate steam higher °C is needed which raises the pressure: p = p1 + p2 -> container may burst
(a) Within which systems and under which cleanroom class must the sterility test be performed?
(b) The culture medium is of central importance in sterility testing. What test besides checking the sterility of the culture medium must be carried out for each batch of culture medium?
(c) What is the acceptance criterion for sterility testing?
(d) What is the maximum number of containers of a parenteral that must be examined for sterility testing (see table)?
a) under aseptic conditions ISO 5 (Class A) in isolator
= sterility testing isolator class A with surrounding area class D/C
b) Positive growth promotion test (with test microorganisms for each batch)
= prove that special test organisms can grow in the nutrient medium
c) acceptance criterion for sterility testing
= No visible growth (turbidity) after 14 days in the test sample
= a product is sterile if no microbes are detected in the sample after incubation
d) maximum number of containers of a parenteral
= 20 containers
Sterility testing (3 points)
(a) Why are reference germs (Keime) required for sterility testing according to Ph. Eur. 2.6.1?
Name the two purposes for which they are used.
(b) How is the evaluation of the sterility test performed?
(c) A batch of an aseptically filled liquid product (small volume parenteral) consists of 800 disposable syringes. How many syringes must be tested for sterility according to Ph. Eur. 2.6.1 (see table)?
a) Reference germs = test organisms
= are required for sterility testing to ensure the effectiveness of the test results
1) for positive control: to verify that the sterilization process is effective in killing or inhibiting the growth of microbes. By inoculating the test media with known reference germs, it can be demonstrated that the media can support microbial growth. This helps to ensure that a negative test result (no growth) is not due to a failure of the test conditions or media but is a true indication of sterility
2) to proove that the product doesn’t inhibit the growth of test organisms
& to show that the test method can reliably (sensitive & low cfu) detect microorganisms if they are present in the sample.
b) performance evaluation of the sterility test
= visual controll of turbidity after incubation of 2 weeks
c) Table 2.6.1
- more tha 500 containers
- 2% or 20 containers (10 containers for lang volume paranterals) whichever is less => 2% of 800 = 16 is less than 20
(c) How are glass ampoules sealed after the product has been filled?
(d) Which test must be carried out on all glass ampoules as a 100% check after sealing, regardless of whether the product is classified as a parenteral product or not?
(e) You should plan a crimping process as part of a filling and closure line. Specify two aspects in addition to the supply of sterile caps that must be taken into account for the crimping process.
= closure by fusion with flame or laser -> top of the ampoules froms by gravity
d) Which test must be carried out on all glass ampoules as a 100% check after sealing, regardless of whether the product is classified as a parenteral product or not?
= 100% integrity testing -> the integrity of the container closure system = include several tests (CCIT = container closure integrity testing)
• Validation of container closure integrity is important = prove with tests that sterility of product is maintained during storage
Crimping of the cap should be performed as soon as possible after stopper insertion
• while crimping vials a lot of metal particles get generated = non-viable particle
-> therefore, important that crimping is separated from filling area (physical separation)
-> measures to prevent particle contamination & good air extraction are required
(5) Why is "packaging material penetration" an important characteristic that a sterilization medium must fulfil? (1 point)
5) Why is "packaging material penetration" an important characteristic that a sterilization medium must fulfil?
= packaging material must be suitable for sterilization medium
to ensure the efficacy & reliability of the sterilization process
to ensure that every part of packed product get reached by sterilization medium (e.g. steam/ radiation)
for qualified sterilization so that germs cannot survive
(2) Plot a typical heat killing curve of microorganisms (normal, non-logarithmic scaling of the axes) with a decimal reduction time of 1.5 min. (1.5 points)
Typical heat killing curve of microorganisms (normal, non-logarithmic scaling of the axes) with a decimal reduction time of 1.5 min. (1.5 points)
(9) Ionizing radiation sterilization (2 points)
(a) A dose of 25 kGy is reached during irradiation. By how many log levels is a population of the yeast S. cerevisiae (D value: 0.5 kGy) reduced?
(b) Name one advantage and one disadvantage of electron irradiation compared to gamma irradiation.
(c) Why is UV-C radiation not suitable for the irradiation of e.g. medicinal products in sealed tubes?
D = 0,5
25/0,5 = 50
= by 50 log levels reduction
eletron irradiation
Advantages:
- No residues
- More gentle than gamma irradiation
- Continuous process, short irradiation time
Disadvantages:
- Limited penetration, but sterilization of products in final packaging possible
- High dose rate
gamma irradiation
- High penetration capability for sterilization in final packaging
- suitable for products with high density % large volume (e.g. whole pallets)
- Rather low dose rate
- Problem of radio active waste
- Not suitable for all materials (cross-linking / degradation reactions of polymers)
c) Why is UV-C radiation not suitable for the irradiation of e.g. medicinal products in sealed tubes?
= because absorbed on surface
= low penetration depth -> no reach of product
(4) Complete the appropriate terms in the fields with a grey background. (3 points)
The F-Value has the unit … minutes
The higher the F value, the … more extensive (better) is the killing of germs.
The higher the z value, the … more resistant are the germs
The lower the F value, the higher is the SAL value achieved (high probability of germs staying alive)
With increasing temperature the D-value … decreases -> high °C results faster killing & a less D-value
The F0 value is always connected with a temperature of … 121.1°C (for 1 min)
Higher z-value: Microorganisms are more resistant to temperature changes
Lower z-value: Microorganisms are more sensitive to temperature changes
Higher F-value: More extensive killing of germs.
Lower F-value: Less extensive killing of germs.
High F-value: More extensive killing of germs, resulting in a lower SAL value (higher assurance of sterility).
Low F-value: Less extensive killing of germs, resulting in a higher SAL value (lower assurance of sterility).
Increasing Temperature: The D-value decreases because higher temperatures increase the rate of microbial inactivation, leading to a shorter time required to achieve the same level of reduction in the microbial population.
(a) Why can sterility testing alone not guarantee the sterility of a batch?
(b) What is examined in the product-specific suitability test?
(c) A liquid vaccine is to be tested for sterility according to Ph. Eur. 2.6.1. The filling volume per container is 1.2 ml. What is the minimum volume per container that must be tested for sterility?
a) Why can sterility testing alone not guarantee the sterility of a batch?
sterility means = absence of viable microorganisms
= because its not possible to test every container without damaging the product/ batch
= in reality only small sample (max. 20 sample size) get taken for testing -> there is always a statistical probability that the test might miss a contaminated unit if the contamination level is low
=> exponential killing (e^-x) of microorgamisms never reaches zero = never 100 % sterility possible -> D-value never zero
-> sterility has to be assured by the application of a suitably validated production process (geeignetes validiertes Produktionsverfahren)
b) product-specific suitability test
= to prove that the product doesn’t inhibit the growth of the 6 test microorganisms
=> product shouldn’t kill organisms that might me brought in a container during process (e.g. aseptic filling) to detect a contamination
2 Techniques of sterility testing = membrane filtration & direct inoculation
c) minimum volume per container - liquid vaccine 1.2ml
=> liquids 1-40ml = half the contents of each container but not less than 1mL -> half the contents 1.2/2 = 0.6ml
=> 1 ml per container must be testes
(8) Dry heat sterilization (1.5 points)
Hot air sterilization tunnels are used to transfer washed glass containers to the aseptic filling area.
(a) Name two approaches to prevent particles / microorganisms from entering the aseptic area from the glass washer area in addition to the high temperature.
(b) The EMA specifies a minimum temperature of 160 °C for hot-air sterilization, but the pharmacopoeia requires at least 220 °C for hot-air sterilization tunnels. What is the objective (Ziel) of the higher temperature in the pharmacopoeia?
a) Name two approaches to prevent particles / microorganisms from entering the aseptic area from the glass washer area in addition to the high temperature
1) pressure cascade
2) Forced air circulation with HEPA filtered hot air (laminar airflow (LAF) = to remove particles & to control pressure conditions
b) What is the objective (Ziel) of the higher temperature in the pharmacopoeia?
= to ensure a more effective sterilization process -> Increased Sterilization Efficacy, high sterility assurance
= to enable depyrogenation which starts above 220 °C (for glass) to reduce heat resistant endotoxins
= to fulfill all Pharmacopoeial Standards for sterilization -> product & patient safety
(10) Ethylene oxide sterilization (2 points + 0.5 bonus points)
Cartons of individually wrapped disposable syringes (stacked on a pallet) are to be sterilized using ethylene oxide.
Describe the complete sequence of the procedure.
Describe the complete sequence of sterilization using ethylene oxide.
= generally, only acceptable if no other method of sterilisation is possible” -> use in food sector prohibited
= in pharma limits of dosage are defined due to toxic residues
-> is very explosive when in contact to O2
Process sequence (negative pressure process)
= runs under vacuum because of 2 purposes:
I) test of integrity of sterilization chamber
II) to take out air because it forms explosive mixture with ETO
(5) (a) A requirement for pure steam is the “correlation with dry saturated steam”. Why does pure steam have to be dry and saturated?
(b) Why is there also a limit value for bacterial endotoxins in pure steam?
(1.5 points)
a) Why does pure steam have to be dry and saturated?
• Pure steam = pharmazeutischer Reinstdampf
= steam with special purity, for direct exposure to group II sterilization goods
-> condensed pure steam has quality of water for injection (highest water quality)
saturated steam (100% dry) = contains 100% of the latent heat available at that pressure
Saturated water = has no latent heat & 0% dryness, contains low heat.
= has to be dry & satured for effective sterilization:
1) Optimal heat transfer = dry satureated steam has highes amount of energy and can transfer this energy more effectively (weat has lower energy transfer is & takes too long)
2) can condensate immediately -> because saturated team has boiling point of water ensuring a concitent temperature (better effectiveness)
3) with dry saturated steam germs are exposed to the max. thermal energy leading to more reliable sterilization (as with via wet steam, droplets)
= pure steam can get in direct contact with pharmaceutical products
(e. g. primary packaging, solutions for production of aseptic medicinal products, parts in contact with medicals)
-> limit value to ensure that product is not cntaminated -> to ensure patient safety, product integrity, quality & positive (desired) clinical effect of the drug
(11) Sterile filtration (1.5 points)
(a) What is the typical pore size of filters for sterile filtration?
(b) It is to be examined whether product is lost during sterile filtration, i.e. whether active substance is adsorbed in the filter. What is the procedure for this adsorption test?
a) What is the typical pore size of filters for sterile filtration?
< 0.22 micron (or smaller)
b) It is to be examined whether product is lost during sterile filtration, i.e. whether active substance is adsorbed in the filter. What is the procedure for this adsorption test?
Adsorption test = for sterile filterable products
= checking the amount of product loss; muss be 100% (no loss)
Procedure:
1) check change in concentration through sterile filtration
2) measure concentration of API before & after filtration
-> calculation of recovery = must be 100% no change in concentration
recovery = Wiederfindung: amount of product after & before sterilization = should be 100%
(12) Validation of sterilization processes (2.5 points)
(a) What is checked during the OQ of a steam sterilizer?
(b) What is the general name of test strips with bacterial spores used to validate sterilization procedures?
(c) What should not be included in the URS of a steam sterilizer? Mark with a cross.
a) What is checked during the OQ of a steam sterilizer?
OQ = Operational Qualification = Checking basic functions of equipment within the working area including chamber integrity
-> checking if:
1) sterilization possible
2) are required °C possible to achieve
3) chamber integrity -> possible with vacuum
b) What is the general name of test strips with bacterial spores used to validate sterilization procedures?
= biological indicator (microbial tests)
= filter paper strips
c) What should not be included in the URS of a steam sterilizer? Mark with a cross.
DQ = Design Qualification = Determination of requirements for equipment
(16) Barrier systems (3 points)
(a) What is the difference between a passive and active RABS?
(b) Which cleanroom class must be used as the background environment for a RABS?
(c) What are the characteristics of an isolator called "open aseptic"?
(d) How do you perform the leak test of an isolator glove (in addition to the visual inspection)?
• Active RABS = integral HEPA-filtered air supply (HEPA inside)
• Passive RABS = air supply by sealing mounted HEPA-filters (outside)
Open, active RABS = HEPA filter is inside, independent air supply unit
Open, passive RABS = air flows aout of the system, not for toxic products
• Surrounding room classification should be ISO 7 minimum in operation -> surrounding area of RABS = class B
Isolator surrounding = for open isolator class C & for closed isolator class D
Product protection isolators = “Aseptic isolator”
II) Open isolator systems = allows continuous material flow, hermetic seal for decontamination, with unidirectional air flow
= designed for continuous or semi-continuous ingress and/or egress of materials (Ein-/ Austritt) during operations through one or more openings -> mouse holes
= Openings must be engineered (e.g. using continuous overpressure) that no external air/ contaminant get into the isolator
-> has tiny openings (“mouse holes”) with inside overpressure to ensure that no particles get inside
-> min. grade C surrounding (because of holes)
2) Leak testing:
2.1) Pressure decay test = gloves get blowed up with pressure
2.2) airflow test at constant pressure = air gets inside of gloves & measurement of volume that is needed to keep pressure constant => if tight, no additional air needed
(14) In preparation for aseptic filling of a medicinal product, the formulated product is usually first subjected to germ-reducing filtration and then finally to sterile filtration in class A.
(a) Why does the formulation of the product have to be carried out at least in clean room class C, when subsequently two filtration steps are carried out to achieve sterility?
(b) What must be checked on each filter before and after use?
= to ensure a low & controlled bioburden before getting to the filtration
-> according to EMA-guidline = Frequent bioburden limit: ≤ 10 CFU / 100 ml -> to fulfill this clean room conditions necessary
= the lower the bioburden, the slower cake formation during filtration (during dead-end filtration)
= Filter integrity testing– GMP Guidline
= The integrity of the filter must be checked before & after use
-> after usage = easy to perform -> filter can get taken out for a quality control in lab or in production area
-> before usage = is critical because it must be assured that filter doesn’t get contaminated -> 2 possibilities to keep filter sterile:
1) sterilization in autoclave after filter testing -> validation important
2) filter gets installed in production area & gets sterilized in place -> filter testing can be done in place
(13)
(a) Which prerequisite must be fulfilled for an aqueous medicinal product to be prepared aseptically at all?
(b) The condition from (a) is given and a drug is to be prepared aseptically. However, it cannot be sterile-filtered.
What is the aseptic production process in this case?
a) Which prerequisite must be fulfilled for an aqueous medicinal product to be prepared aseptically at all?
1) sterile material & equipment
2) class A conditions (environment)
3) sterile transport
4) Isolator
5) sterile product
b) The condition from (a) is given and a drug is to be prepared aseptically. However, it cannot be sterile-filtered.
= product is not heat stable at all & cannot be filtered then: use pre-sterilised individual or mixed components & aseptic processing
= all product components need to be sterilized separately beforehand & bring/ fill everything in aseptic processing together (Aseptic formulation: class A)
Important: Terminal sterilization is considered as much more saver as the aseptic processing because final product is completely closed in the final packaging. It cannot get contaminated because of the validated container closure integrity
(17) Transfer systems (2 points)
(a) Describe the principle of a transfer using a decontamination airlock into an isolator during operation.
(b) What transfer system is shown in the picture?
Decontamination airlock (Luftschleuse)/ transfer hatch
= has 2 decontamination airlocks (small chambers) on each side of the isolator that are connected to working area
-> material can get inside & out of the isolator
-> double door system
RTP = Rapid Transfer Port
= for transfer of materials, stoppers, caps into aseptic area while processing
(18) Preparation of equipment and primary packaging material
(2 points + 0.5 bonus points for part d)
(a) Why is primary packaging material considered more critical then secondary packaging material?
(b) Name one way in which two silicone tubes can be connected aseptically.
(c) Why are cartridges siliconized on the inside?
(d) At which point in the process does siliconization take place?
= its the first layer of packaging that is in direct contact with pharmaceutical product (e.g. vials/ ampoules/ syringes/ stoppers/ caps)
-> shouldn’t cause recontamination or have negative impact on product -> must be sterile (depyrogenation) / inert/ abrasive proof etc.
-> Sterilization tunnel (continuous sterilization) = used for sterilization & depyrogenation of primary packaging materials (glass containers) -> not for plastic because high °C!
without siliconization stopper doesn’t move at all due to high friction or stopper suddenly moves & stops -> syringe doesn’t work well, no correct dosage (insulin) -> dangerous for patient
break loose force = force that is needed to start movement of a stoper
gliding force = force that is needed to keep stopper moving
1) siliconisation of packaging (syringe/ cartridge) usually before sterilisation (after washing & drying) -> often applied backed on siliconization which has formation of silicon layer during (hot air) sterilization
2) siliconisation of syringe & cartridge stoppers = optionally after sterilization -> stoppers must move to achieve equal distribution of silicone
7)
a) Why is there a limit for non-condensable gases for pharmaceutical pure steam?
b) What is the key advantage of using saturated steam compared to superheated steam?
a) pure steam = has special purity & high quality so it can get in contact with pharmaceutical
-> if there is a lot of air or non-condensable gases inside sterilization system the steam cannot reach every area of product
=> results: bad sterilisation quality
- no condensation possible
- bad energy transfer -> needed °C cannot be reached for desired duration
b) Advantage of saturated steam = condensates quickly -> shorter process time
(super-heates steam = needs to cool down to condensate -> longer)
(15) Name four other approaches, different in terms of protection mechanism, as an addition to the use of HEPA filters, how the open product in cleanroom class A can be protected from contamination?
How can the open product in cleanroom class A can be protected from contamination?
1) physical protection
- Walls/ Barriers
- Shell concept: surrounding class B
2) aerodynamic protection
- protection via laminar air flow
- pressure cascade between zones
3) Personnel (human factor) = try to avoid human interaction through using automatic system
4) Use materials (incl. API & excipients) with very low bioburden
5) Surfaces in production surrounding = compatible hygenic design -> quality/ cleaning/ roughness for desinfection
(3)
You have the following data on the heat killing of a micro-organism at a temperature of 121 °C: a germ population is reduced by 6 log levels in a period of 180 s.
(a) What D value at 121 °C results from the data?
(b) After what total time is a SAL of 10^-12 achieved if 10000 germs are present at the beginning?
a) What D value at 121 °C results from the data?
b) After what total time is a SAL of 10^-12 achieved if 10000 germs are present at the beginning?
(9) Describe the steps that are needed to biodecontaminate a cleanroom with hydrogen peroxide vapour (VHP process)? (2 points)
9) Describe the steps that are needed to biodecontaminate a cleanroom with hydrogen peroxide vapour (VHP process)?
(19) Fill and finish - Dosing systems
(a) What dosing system is shown in the picture?
(b) Explain the principle of time-pressure dosing.
(c) Why does Annex 1 specify that crimping must be separated from filling and stoppering?
(d) Which test must be carried out on glass ampoules after fusion as a 100% check in addition to the visual inspection?
(e) What is the main advantage of using nested ready-to-fill syringes in tubs compared to using bulk syringes?
(f) How do you call the process for manufacturing infusion or parenteral nutrition bags?
(g) What is used to blow up the containers during the blow fill seal process?
a) (Dual) peristaltic pump
= single-use silicone tube -> tube get sandwiched between rollers (Dialyse-pumpe)
= benefits: single use/ high precision/ quick product change possible
Time-pressure system = gravity & viscosity dependent system
- benefit: ultra-high output/ CIP-SIP compartible
- not for: viscous products & products that are sensitive to silicone tubes
Principle:
- pressure inside vessel must stay constant -> precise monitoring of pressure
- pinched valves are opened & then after defined dosed product valves get closed
- temperature very important (high impact on viscosity) -> influences flow through
Crimping = aluminium cap gets placed on the stopper
• while crimping vials a lot of metal particles get generated -> non-viable particle => therefore, important that crimping is separated from filling area (physical separation)
=> to prevent particle contamination & good air extraction are required
-> closure by fusion, e.g. glass or plastic ampoules should be subject to 100% integrity testing.
bulk syringes = Washing, drying, siliconisation, sterilization necessary before filling
nested ready-to-fill syringes = just opening & filling & closure (ready to use) -> no washing & sterilization etc.
(f) How do you call the process for manufacturing infusion or parenteral nutrition bags? = form- fill- seal production
Blow/ fill/ seal system = purpose-built machine in which, in one continuous operation, containers are formed from a thermoplastics granulate, filled & sealed, all in one production line
Blow mould closes, bottom is welded -> blowing with sterilized compressed air
(23) Hygienic design of plant components (4.5 points)
(a) Draw in a design improvement for the pictured container bottom.
(b) Name two criteria for the design of pipework that are not related to the properties of the material / surfaces.
(c) Sketch / draw the compression of a seal with an axial stop.
(d) In the picture you can see two possible sealings of the longitunial movement of a valve rod. Which one is better with regard to hygienic design / contamination risk? Please briefly justify your choice.
(e) Why is a simple mechanical seal not suitable for sealing agitator shafts?
a) Draw in a design improvement for the pictured container bottom.
• Self-drainable -> have special slope (are not straight)
• Fixed connections prefered to detachable ones
c) Sketch / draw the compression of a seal with an axial stop
d) Which one is better with regard to hygienic design / contamination risk? Please briefly justify your choice.
Sealing of longitudinal movements
a) dynamic sealing
b) better: hermetic sealing = because hermetically sealed & no germs can enter
= is NOT 100% sealed = not 100% safe: contamination get through sealing point
-> cannot be used for aseptic purposes & hazardous media -> used for non-sterile/ non-hazardous product/ pumps -> microbial contamination can get through sliding point
(21) Media fill (2 points)
(a) During a media fill, the aseptic process defined in the SOP is tested for sterility. What must be considered in addition to the routine process when planning a media fill?
(b) In a media fill, 600 containers are filled with nutrient medium. How many of them are allowed to show germ growth after incubation?
(c) Which special aspects have to be considered for a media fill if a freeze-drying process is part of the procedure to be validated?
a) What must be considered in addition to the routine process when planning a media fill?
Media fill = should imitate as closely as possible the routine aseptic process & include all critical manufacturing steps -> microbial monitoring
= should consider various interventions known to occur during normal production & worst-case situations -> risk analysis
b) In a media fill, 600 containers are filled with nutrient medium. How many of them are allowed to show germ growth after incubation?
= no growth (0 germs/ growth) -> otherwise something is not right in production
c) Which special aspects have to be considered for a media fill if a freeze-drying process is part of the procedure to be validated?
Lyophilizates -> partial drying process
- FDA recommends that unsealed containers be exposed to partial evacuation of the chamber that simulates the process.
- Vials should not be frozen, and all precautions should be taken so that microbial growth get supported (for evaluation)
(2)
For a steam sterilization process, the resulting probability of sterility is P = 0.9999998.
(a) What SAL value is achieved here?
(b) Is the SAL value achieved sufficient for a pharmaceutical steam sterilization process or not? Please justify.
a) SAL + Psterile = 1 = 100%
SAL = probability, that germs may be alive
Psterile = probability of germs that are death
1 - P = 0,00000002 SAL = 2*10^-7
b) Is the SAL value achieved sufficient for a pharmaceutical steam sterilization process or not? Please justify.
-> in pharmacy = aim to achieve SAL level of equal to or less 1*10^-6
= is achieved & even axceeded because SALof 2*10^-7 means that 2 containers of 10 Mio are unsterile (1 unsterile container of 5 Mio)
-> SAL of 1 * 10^-6 means 1 container of 1 Mio unsterile
(15) Transfer systems (2 points)
(a) What is meant by mouseholes in barrier systems and for what purpose are they used?
(b) When using RTP systems, how is it ensured that no contaminated air enters the barrier system (see illustration)? Specify two aspects.
b) When using RTP systems, how is it ensured that no contaminated air enters the barrier system (see illustration)? Specify two aspects
2) connection must be air-tight to avoid decontamination
3) opening of lid only possible when fully connected
4) higher pressure inside transfer pressure for product protection
Sterility testing (2 points)
(a) Why must sterility testing be carried out in an aseptic process environment?
(b) Why can the direct inoculation method not be used for sterility testing of a sterile antibiotic?
(c) A liquid vaccine is to be tested for sterility according to Ph. Eur. 2.6.1. The filling volume per container is 2.4 ml. What is the minimum volume per container that must be tested for sterility?
a) Why must sterility testing be carried out in an aseptic process environment?
= to avoid recontamination during testing, leading to wrong results
(which may wrongly identify that processing was not sterile enough while contamination was brought inside after processing)
b) Why can the direct inoculation method not be used for sterility testing of a sterile antibiotic?
sterile antibiotic = inhibits the growth of germs (function of antibiotic)
-> if product inhibits the growth of microbes = direct inoculation cannot be used because its not possible to do Positive method suitability test ( = to prove that the product doesn’t inhibit the growth) -> antibiotic does inhibit growth!
-> other sterility test must be applied
c) 2.4 mL => 1-40mL -> half the contents of ech container but not less tha 1 mL
-> half the content 2.4/ 2 = 1,2 => min. 1ml per container must be tested
(2) Sterility testing - SAL
(a) How are the SAL value and the probability of sterility related?
(b) For a batch size of 1,500,000 containers, how many non-sterile containers would be expected if the SAL value present would be 10^-5?
c) What is the acceptance criterion for the SAL value of a pharmaceutical steam sterilization process?
b) 10^-5 = 1 container of 100.000 is non-sterile
1,500,000 containers * 10^-5 = 15 containers are non-sterile
c) acceptance criterion for the SAL value
= min. SAL of 10^-6 for pharmaceuticals
-> in reality pharma companies chieve SAL of at least 10^-12
(3) Sterility testing - SAL
a) For a sterilization process, the SAL value achieved is 1.8 * 10^-11.
What was the initial bacterial count N0 before sterilization if this SAL value was reached after a reduction by 13 log steps?
b) For a sterilization process, the SAL value is 3 * 10^-2. What is the resulting probability of sterility in percent?
a) SAL 1.8 * 10^-11
Redution: 13 log steps -> 11.8 * 10^(-11+13)
initial baterial count N0 = SAL + log steps = 1.8 * 10^2 = 180
b) Probability in percent:
SAL + Pst = 1
Pst = 1 - SAL = 1 - (3 * 10^-2) = 0.97 = 97%
(a) What does the SAL value describe?
(b) What is the probability of sterility achieved if the bioburden before sterilization is 20 cfu/ml in a 5 ml volume and sterilisation results in a 7 log reduction?
a) What does the SAL value describe?
SAL = sterility assurance level = probability, that germs may be alive
SAL of a sterilation process = degree of assurance with which the process makes a population of items sterile
= is expressed as the probability of a non-sterile item in that population
b) What is the probability of sterility achieved if the bioburden before sterilization is 20 cfu/ml in a 5 ml volume and sterilisation results in a 7 log reduction?
(4) Sterility testing - z value
The following diagram shows the killing curve of a microorganism (z = 5 °C) at a temperature of 121 °C.
(a) Which D value at 121 °C results from the diagram?
(b) What is the D value at a temperature of 131 °C?
calculate: D-value = mins/ log-levels = 8mins/ 4 log levels = 2 mins
or read in diagram
b) What is the D value at a temperature of 131 °C?
(5) What role does the bioburden of the sterilization items play in the design of a sterilization process? (1 point)
What role does the bioburden of the sterilization items play in the design of a sterilization process?
the lower the bioburden that easier is it to guarantee sterility
reduction of bioburden includes washing/ cleaning/ desinfection & sterilization in the end
The level of bioburden affects the required lethality of the sterilization process. Higher bioburden = requires more strict sterilization parameters (e.g., higher temperatures, longer exposure times) to ensure good microbial killing
Knowledge of bioburden helps to select the appropriate sterilization method (e.g., steam, dry heat, radiation) and optimized parameters such as temperature, pressure, and duration to achieve the desired Sterility Assurance Level (SAL).
(4) z-value & D-value
(a) A low bioburden before sterilization is not only important to achieve the shortest possible sterilization time and the highest possible sterility assurance. What other reason is there?
(b) How are the D-value and the z-value related?
Prevention of undesirable by-Products -> metabolic-products of germs may begatively influence the quality of product
low bioburden is advantageous for sterile filtration
-> filter capacity = with high bioburden gets quickly blocked by germs (cake formation)
-> depending on amount of bioburden the filter must be huge enough
relationship in formula:
z-value = Temperature change in °C, which is required to alter the D value by a factor of 10 -> temperature dependency of D-value
(5) Sterility testing - z value
(a) The following z value (temperature coefficient) is given in connection with a steam sterilization process: z = 6 °C. What is the D value at 121 °C if the D value at 109 °C is exactly 30 s?
(c) For a steam sterilization process at 134 °C, an F0 value of 18 min is achieved. How long would you have to sterilize at 121.1 °C to achieve the same lethality?
Formula:
a) The following z value (temperature coefficient) is given in connection with a steam sterilization process: z = 6 °C. What is the D value at 121 °C if the D value at 109 °C is exactly 30 s?
c) Formula cannot be used because no z value given!
(6) Sterility testing
(a) Explain the effect of air pockets within the sterilization load during steam sterilization.
(b) How is the pressure in an autoclave related to the amount of non-condensable gases in the autoclave chamber?
(c) How is venting / air elimination performed in a steam sterilization process that uses the gravity or flow principle?
Steam sterilization (= autoclaving) relies on the presence of saturated steam to transfer heat efficiently to all parts of the load.
-> when saturated steam is coming into an autoclave = it reaches a cold product surface & gets condensate because the steam cools quickly down & transfers all its saved energy to the product -> so product heats up
-> condensation = very fast & powerful energy transfer process
Effects of air pockets:
1) incomplete sterilization: - Air pockets act as barriers to the penetration of steam -> due to air pocket area may not reach the required temperature for effective sterilization -> creation of cold spots -> higher risk of surviving germs -> air prevents steam for reaching the product -> bad sterilization
2) longer sterilization time: - Steam provides higher efficient heat transfer than air -> areas with air pockets require longer exposure times to reach the required temperature -> extention of overall cycle time
b) How is the pressure in an autoclave related to the amount of noncondensable gases in the autoclave chamber?
limit < 40ml/kg non-condensable gases (NCGs)
3) Pressure increase: In a closed system the pressure rises up (because of high energy & steam needs more space) so the vaporization curve reaching 212°C and it becomes a saturated steam
= if pressure rises to high, then there is a lot if air left causing overpressure (p1+p2= Ptotal)
Interference with Steam Penetration: NCGs can form pockets or layers that disturb the distribution of steam, leading to uneven heating & insufficient sterilization.
Lower Effective Sterilization Temperature: The presence of NCGs can lower the effective sterilization temperature. For example, if the chamber pressure is read as 121.1 °C (1 atm of steam pressure), but part of this pressure is due to NCGs, the actual steam temperature will be lower than 121.1 °C. -> reduced SAL & uneffective strilization
(a) A mean z-value of 5.5 °C is usually assumed for vegetative
microorganisms. What does this z value of 5.5 °C indicate?
(b) How are the F0 value and the SAL value related?
a) What does this z value of 5.5 °C indicate?
z-value of 5,5 °C = Temperature change of 5,5 °C, which is required to alter the D value by a factor of 10 -> 1 log reduction
= measures the heat resistance of microorganisms
= low z-value means that germs are sensitive to °C (e.g. vegetative cells are not heat resistant)
b) How are the F0 value and the SAL value related?
F0-value = special F-value for 121 °C conditions
the higher F-value (long holding time) = the lower SAL-value (low probablity that germs stay alive, sufficient sterilisation)
the higher the F-value (short holding time) = the higher the SAL-value (high probablity that germs stay alive, no sufficent sterilisation)
(7)
(a) For which pharmaceutical products can the hot water spray process be used?
(b) How is the counterpressure built up in the hot water spray process to prevent the product containers from bursting?
(c) In which steam sterilization processes is the Bowie-Dick test used?
a) For which pharmaceutical products can the hot water spray process be used?
Hot water spray = Counter pressure sterilization processes
= for aqueous liquids (infusion) in closed containers
= e.g. for bottles, vials made of glass/ plastics & single-multi bags
= via controlled application of compressed air in the chamber -> to balance the internal pressure inside the containers with the external pressure in the sterilization chamber
As the temperature inside the chamber rises, the pressure also increases. To prevent the containers from expanding or bursting due to the increased internal pressure from the heating, compressed air is introduced into the chamber. => The introduction of compressed air increases the external pressure in the chamber, creating a counterpressure that balances the internal pressure of the containers.
= for saturated steam sterilisation with vacuum processes
= proof if sufficient air removal achieved & steam penetration into product sufficient
(20) Specify two aspects that must be taken into account with regard to personnel during visual inspection. (1 point)
1) visual inspection is tiring for eyes -> looking 5 secons & making regular pauses
2) Personnel should follow hygiene practices, to prevent contamination of the products during visual inspection
3) Adequate and good lighting conditions are important for effective visual inspection
4) personnel friendly working condition (temperature/ humidity) are important -> creation of particles through sweating/ low concentration
(7) Sterility testing
(a) Describe the venting process of a steam sterilization process using the fractionated vacuum method.
(b) How is container bursting prevented in the hot water spray process?
II) fractioned pre-vacuum process
= for special products with very difficult air elimination
- replay change of vacuum phase & steam introduction
- steam gets introduced to reach desired °C
- fractionated cooling & drying: vacuum & air
= through creation of counterpressure by introducing sterile compressed air into container
(22)
(a) Name two further requirements in addition to easy cleanability & smooth surface for materials in contact with the product with regard to their hygienic design.
(b) What is designed in a hygienically unfavourable way here (see picture)? Name one point of criticism.
Requirements for materials - hygenic design
1) Smooth, easy to clean
2) Corrosion resistant -> stainless steel
3) Abrasion-proof, mechanically stable
4) Stable to aging & not change over time -> rubber may break in peaces
5) Inert (inaktiv) to the product & detergents and disinfectants
b) What is designed in a hygienically unfavourable way here (see picture)? Name one point of criticism.
- Sharp corners (≤ 90°) must be avoided
- surfaces hall always slope to one side with a minimum angle of 3°
- not self-draining
(11) Dry heat sterilization
In a pharmaceutical plant, a hot air sterilization tunnel is used to sterilize glass cartridges.
(a) In addition to sterilization, what two purposes are met by treatment in the hot air sterilization tunnel?
(b) Give a common operating temperature for the sterilization zone.
(c) Which zone of the hot air sterilization tunnel has the highest ambient pressure?
a) in hot air tunnel:
1) Depyrogenation (while sterilization phase) = to reduce heat resistant endotoxins
2) seliconization = silicon layer is spread / made up to reduce friction (for glass or in syringes)
b) Give a common operating temperature for the sterilization zone.
= 280 - 350°C
c) pressure zones in hot air tunnel:
1) washing zone = +2 Pa (refer to class C pressure)
2) heating zone = +4 Pa (sterilization)
3) cooling zone = +6 Pa (class A conditions) = to make shure that no air is getting inside sterilization tunnel
(8) Dry heat sterilization (3 points)
(b) In a pharmaceutical plant, a hot air sterilization tunnel is used to sterilize glass cartridges.
In addition to sterilization and depyrogenation, what third purpose is met by the treatment in the hot air sterilization tunnel?
(c) The z value for heat resistant endotoxins is about 46 °C. What does this mean when you compare the required process times at 280 °C and 326 °C?
(d) Why is it important to cool glass containers slowly after hot air treatment?
a) What is meant by compensation time in (dry) heat sterilization processes?
b) In addition to sterilization and depyrogenation, what third purpose is met by the treatment in the hot air sterilization tunnel?
= siliconization : silicon layer (anti-friction coating) is made up to minimize break loose & gliding forces
-> for cartridges & syringes = have stopper inside, it has to move while injection
-> silicone oil or emulsion is sprayed to the inner surface of syringe
t = 326-280°C/ 46°C = 1
=> 1* z-value (reduction of 10log)
= Process tima at 326°C will be reduced by factor of 10 compared to 280°C
d) Why is it important to cool glass containers slowly after hot air treatment?
= to prevent thermal stress and potential breakage
= to avoide class breakage (due to possible micro-cracks)
= to guarantee product safety (no contamination through glass crackes)
(4) Sterility testing
The following data on heat-killing of a microorganism at a temperature of 125 °C are available to you:
(a) What D value at 125 °C results from the data?
(b) After what total time at 125 °C is a SAL of ≤ 10^-6 achieved?
(c) Is the F0 value reached at the time point 13 min
- below 13 min,
- exactly 13 min or
- above 13 min?
Please justify your choice.
(3 points)
a) D-value = mins/ log levels
13 -1 = 12mins -> 12/8 (log-levels) = 1,5 D-value
b) D-value = 1.5 -> n = log-levels: from 10^8 to 10^-6 = 14 log levels
1.5 * 14 (log levels) = 21 minutes
c)
The F0- value is reached below 13 minutes at 125 °C.
The sterilization process at 125 °C achieves the required F0 value much earlier than 13 minutes.
(9) Ionizing radiation sterilization (1.5 points)
(a) Which type of electromagnetic radiation used for sterilization has the highest frequency?
(b) Name one disadvantage of electron irradiation over gamma irradiation.
24) What is the advantage of electron radiation over gamma radiation?
(c) In which unit do you specify the dose for radiation sterilization?
= gamma irradiation ( gamma rays)
Electromagnetic spectrum
b) Name one disadvantage of electron irradiation over gamma irradiation.
= no radioactive waste & high dosage rate
(b) According to which mode of action does UV radiation, which is not one of the ionizing types of radiation, kill microorganisms?
(c) Which of the ionizing radiation types does not belong to the electromagnetic radiation?
= if it is NOT possible to sterilize pharmaceuticals by heat or steam (not heat stable product), then ionizing radiation sterilization may be applied
= if product cannot be sterilised by dry heat at 160°C for 120 min or by any other alternative temperature achieving SAL < 10^-6, the use ionizing radiation
-> for: pharmaceutical medical products; dry powder products, non-aqueos liquids & semi-solids if the product is NOT temperature stable!
e.g. ophthalmic ointments in tubes
• Damages DNA (maximum at 265 nm) -> kiling of germs due to disruption of DNA replication & transcription
= DNA is absorbing UV-energy causing apoptosis of cells/ mutations
= alpha & beta rays
(9) Dry heat tunnel (2 points)
(a) Why are hot air sterilization tunnels usually operated at temperatures of 300 - 350 °C, when according to regulations 160 °C would be sufficient for dry heat sterilization? Give two reasons.
(b) Which zone of a hot air sterilization tunnel has the lowest ambient pressure?
(c) Why is it important that glassware cools slowly after hot air sterilization?
a) Why are hot air sterilization tunnels usually operated at temperatures of 300 - 350 °C?
1) to eliminate pyrogens (depyrogenation) -> above 220 °C
2) because its a (faster) process (sterilization cycle) resulting better killing & higher sterility assurance
b) Which zone of a hot air sterilization tunnel has the lowest ambient pressure?
= the drying/ preheating (infeed) zone (class c) = +2Pa
-> highest pressure in cooling zone (after sterilization to ensure sterility)
c) Why is it important that glassware cools slowly after hot air sterilization?
= to avoide class breakage (due to possible micro-cracks) & to guarantee product quality (no cracks, no residues in product, risk of contamination)
For a sterilization process, the SAL value is 5 * 10^-7.
(a) What was the initial bacterial count N0 before sterilization if this SAL value was achieved after a reduction of 8 log steps?
(b) What is the probability of sterility in percent associated with the SAL value?
a) What was the initial bacterial count N0 before sterilization if this SAL valuewas achieved after a reduction of 8 log steps?
5* 10^-7+8 = 5*10^1 = 50
b) What is the probability of sterility in percent associated with the SAL value?
(12) Ionizing radiation sterilization (1.5 points)
(a) Which two irradiation methods are described in Annex 12 of the GMP guideline and are thus applicable for terminal radiation sterilization?
(b) Irradiation is also used for sterile transfer, among other things. Give a specific example of use.
a) Which two irradiation methods are thus applicable for terminal radiation sterilization (Annex 12)?
1) electron irradiation
2) gamma irradiation
b) Irradiation for sterile transfer
= E-beam tunnel -> transfer from class C to class A
(10) Ionizing radiation sterilization (1.5 points)
(a) What is the common mechanism of action of all types of ionizing radiation sterilization?
(b) For pallet sterilization of consumables, a radiation sterilization process with the highest penetration capacity should be selected. Material compatibility is given. Which type of radiation can be considered besides X-ray?
(c) Name an area of application for decontamination with UV radiation.
= induction or damage to the genetic material (DNA or RNA) of microorganisms (indirect or indirect way)
- Ionizing radiation has enough energy to ionize atoms/ molecules , leading to the formation of highly reactive ions / free radicals. These can then interact with and damage the genetic material of microorganisms, making them unable to reproduce & causing their destruction.
Gamma irradiation
= High penetration capability sterilization of products in final packaging
= suitable for products with high density & large volume (e.g. whole pallets)
= in water treatment -> used to disinfect and decontaminate water
1) Sterility testing (3 points)
(a) How is sterility defined according to Ph. Eur.?
(b) What are the two sterility test types?
(c) An investigational medicinal product should be tested for sterility according to Ph. Eur. 2.6.1. The investigational medicinal product is eye drops. The batch size is 180. Using the following table from Ph. Eur. 2.6.1, state the minimum number of containers to be tested.
a) How is sterility defined according to Ph. Eur.?
= absence of viable micro-organisms
=> defined by a sterility assurance level (SAL) equal to or less than 10^−6
b) Two sterility test types
1) Membrane filtration
2) Direct inoculation
c) Minimum number of containers - eye drops 180 batch
=> more than 100 but not more than 500 = min. 10 containers
Because eye drops are usually in single-dose containers
(10) Gamma irradiation
Gamma irradiation and gassing with ethylene oxide are often used for the sterilisation of entire pallets with consumables.
(a) What is the mechanism of action of gamma irradiation?
(b) Give a reason why gamma irradiation is preferred over electron irradiation for this application.
(c) Name one advantage and one disadvantage of gamma irradiation compared to sterilization with ethylene oxide.
(d) Give a reason why ethylene oxide is used for that application but not hydrogen peroxide.(2.5 points)
= use of high-energy gamma rays to disrupt the DNA or RNA of microorganisms, making them incapable of reproduction
mechanism of action:
1) Generation of Ionizing Radiation: = cobalt-60 creates during radioactive decay high-energy gamma rays
2) Penetration of Materials = Gamma rays have high energy and short wavelengths, allowing them to penetrate deeply into materials (sterilization)
3) Ionization of Atoms & Molecules = As gamma rays pass through a material, causing ionization of atoms -> results formation of highly reactive ions & free radicals -> killing germs
electron irradiation = Limited penetration, but sterilization of products in final packaging possible -> may be not sufficient to penetrate whole pallet (bad sterilization)
Gamma irradiation = High penetration capability sterilization of products in final packaging -> higher assurance level (to penetrate whole pallet)
Gamma Irradiation
High penetration capability sterilization of products in final packaging & no residuces
Problem of radio active waste
Advantages - ETO
1) Broad spectrum of activity
2) Alkylation of nucleic acids, proteins (requires H2O)
3) Excellent penetration capacity
Disadvantages - ETO
1) Carcinogenic
2) Extremely flammable, forms explosive mixtures with air
3) Leaves toxic residues -> not allowed in food sector
(d) Give a reason why ethylene oxide is used for that application but not hydrogen peroxide = sterilisation of entire pallets with consumables.
because: ETO has excellent penetration capacity & broad spetrum of activitity -> better for entire pallets with undefined consumables
hydrogen peroxide
= Very strong oxidizing agent
= Highly corrosive
= Penetration capacity limited
(8) z-values
How do the z-values for dry heat differ from those for steam sterilisation?
Give a reason for the difference as well. (1 point)
dry heat = 160° for 125 min
steam sterilization = 121.1°C for 1 min
Effectiveness = dry heat is less effective than steam sterilization at the same temperature because no condensation!
because:
1) microorganisms/ spores are more heat resistant under dry conditions (= D-values are approx. 10 times higher at the same temperature) => z-value 10°C
2) dry air has a lower heat capacity & poorer heat transfer and a higher risk of cold islands
= z-values about twice as high compared to steam sterilization because higher D-value necessary -> less effective/ no condensation
- FH value (instead of F0) - for dry heat sterilization
(10) In the diagram the sequence of an ethylene oxide sterilization is shown.
(a) Why are the vacuum-nitrogen cycles required?
(b) What is the purpose of the dwell period?
(c) Name one advantage of ethylene oxide sterilization over hydrogen peroxide sterilization.
ETO = highly effective sterilant but extremely flammable, forms explosive mixtures with air (O2) & toxic/ hazardous to human
before sterilization:
-> therefore O2 needs to be brought out of chamber by vacuum & for gas sterilization is nitrogen used
-> through pulsation vacuumed input of nitrogen air gets better removed (better displacement)
after sterilization: ETO = is toxic and may be potentially hazardous to humans. After the sterilization process is completed, it is crucial to remove residual ethylene oxide gas from the sterilized items to ensure they are safe for use. -> with vacuum ETO residues get sucked out & nitrogen (inert gas) gets introduced in chamber displacing ETO
dwell-period = Sterilization time = exposure time of product under constant pressure / temperature & desirend conditions with sterilization agent (e.g. ETO)
- enables efficient penetration/ contact/ uniform distribution of sterlization agent over product
Advantages - ETO = Excellent penetration capacity (highly effective)
(10) Gas sterilization (2 points)
(a) What steps are needed to biodecontaminate an isolator with hydrogen peroxide?
(b) Why should condensation of H2O2 be prevented if possible?
VHP = vaporized hydrogen peroxide = surface sterilization process / used for biodecontamination -> decrease in 10log levels
1) because drops on surface of product increase corrosion
2) duration process takes much longer if H2O2 gets to vapor
(11) Sterile filtration
What is the so-called bubble point of a sterile filter and how is it determined? (1.5 points)
What is the so-called bubble point of a sterile filter and how is it determined?
1) Bubble point test (manual application)
= Membrane gets wetted with liquid product & pressure (air) gets applied from one side of the filter to press out the solvents out through the filter
-> Solvent is pressed out of filter with increasing pressure
Bubble Point = pressure at which a continuous discharge of air bubbles can be detected (3 to 5 bar) => depends on the filter properties
(12) Validation of sterilization processes (2 points)
(b) What is checked during the OQ of a steam sterilizer?
(c) What is the acceptance criterion for the Bacterial Challenge Test (BCT) that must be performed as part of the validation of a sterile filtration process?
b) What is checked during the OQ of a steam sterilizer?
OQ = Operational Qualification
= Checking basic functions of equipment within the working area including chamber integrity (pressure & vacuum test)
(c) What is the acceptance criterion for the Bacterial Challenge Test (BCT)?
Checking sterility after filtration = 100 % retention of germs (acceptance criterion) -> no single cell of reference germ should pass the filter!
(a) During the DQ you have to select a steam sterilization process for the sterilization of 2 m long silicone tubes used for aseptic filling. Which process do you choose? Give a justification for your choice.
(b) What is the name of the document in which the user demands on a sterilization system are recorded as part of the DQ?
(c) What is checked during thermoelectric testing with an empty sterilization chamber as part of the PQ of a steam sterilization process?
DQ = Design Qualification = Determination of requirements for the equipment ->(URS = user requirements specifications & functional specification document)
Justification:
with SIP is effectively sterilization of long tubing systems possible
tubes can remain in their installed positions within the system during sterilization, maintaining aseptic integrity
SIP systems are designed to ensure uniform distribution of steam, which is critical for sterilizing long silicone tubes uniformly. This prevents any cold spots or areas where steam might not reach effectively.
= uniformity of temeprature & pressure
(14) Sterile filtration (2 points)
(a) How is the bubble point of a sterile filter related to the pore diameter?
(b) In preparation for aseptic filling of a drug product, the drug is usually first subjected to germ-reducing filtration and then finally to sterile filtration in class A. Give two reasons why an additional germ-reducing filtration is carried out.
a) bobble point & pore diameter
bubble point = pressure at which a continuous discharge of air bubbles can be detected
=> the smaller the pore size = the higher the pressure (3-5 bar)
= the higher the bubble point
b) Give two reasons why an additional germ-reducing filtration is carried out.
bioburden reduction prefilters & sterilising grade filters
1) to prevent built up of the filter cake
2) if 1-st filter is having a leak the 2-nd filter enables filtration = increase safety of sterility
( 3) separation in 2 steps possible: one in class C area & one in final step)
(12) Sterile filtration (2.5 points)
(b) In preparation for the aseptic filling of a liquid medicinal product, the drug is usually subjected to two filtration steps. The second step is sterile filtration. What is the purpose of the first filtration step?
(c) Sterile filters must be examined for leachables. What are leachables?
bioburden reduction prefilters and/or sterilising grade filters may be used at multiple points during manufacturing process to ensure a low & controlled bioburden before getting to the final sterilising filter
Purposes:
Leachables (if required) = only required if toxic extractables detected
= Substances leached from the filter material in contact with the product under standard process conditions
= substances that can migrate or leach from materials used in the manufacturing process into the final product
-> for sterile filters, leachables refer to any chemicals or substances that may be released from the filter material and contaminate the filtered product.
(11) Sterile filtration (2 points)
(a) When may sterile filtration be used to sterilize a liquid medicinal product?
(b) As part of the validation of a sterile filtration process the bacterial challengetest (BCT) must be performed. How does it work?
Note: the viability test doesnot need to be addressed.
= if product is not heat stable at all, use sterile filtration
= if steam sterilization is not possible then sterile filtration with microbial retentive filter is the last option
=> must be combined with aseptic processing
with presterilized containers -> only final sterile filtration & filling is done in class A
Note: the viability test does not need to be addressed.
Bacterial Challenge Test = BCT – biological test for filtration
1) Preparation: performance of Viability test
2) Provide filtration process = under worst case conditions (with product, filter elements, contact time, pressure, temperature)-> check the filter retention capacity = validation of retention of germs on membrane filters
3) Checking sterility after filtration = 100 % retention of germs (acceptance criterion) -> no single cell of reference germ should pass the filter!
(15) Validation of sterilization processes (1.5 points)
(a) List four different non-economic requirements that should be included in a user requirement specification (URS) for an autoclave.
(b) What must be considered in the PQ of a steam sterilization process with regard to loading - in addition to the loading schemes of routine production?
(a) List four different non-economic requirements that should be included in a user requirement specification (URS) for an autoclave
1) Location / Environment / Dimensioning
· Place of installation/ Environment / zone requirements
· Constructional conditions, e.g. available area, floor load
2) Process
· Procedure / operating mode (e.g. FRVV)
· Special requirements for the process (e.g. accelerated cooling)
· Design / Type, (cylindrical/rectangular; horizontal/vertical)
· Working range (pressure / temperature)/ Programms
3) Monitoring / Measurement technology
· Sensors/ Accuracy/ Additional features
4) Materials and surfaces
· Materials
· Surface properties
· Hygienic Design
5) PQ = Performance Qualification = Verification of sterilization success based on relevant loads -> actual process validation
PQ of a steam sterilization process = involves demonstrating that the sterilizer operates effectively and consistently within the defined parameters to achieve the desired level of sterility.
1) load & packaging system should contain product & be identical to the packaging used for routine production
2) Worst-Case Loading & Conditions should be included (max/ min/ cold spots)
3) loading configuration/ amount/ distribution inside (spacing) must be defined
(b) What should be considered when validating a steam sterilisation process with regard to loading?
(c) What is the general name of test strips with bacterial spores used to validate sterilization procedures?
b) What should be considered when validating a steam sterilisation process with regard to loading?
Standard and Worst-Case Loads: Validate using both routine (standard) and worst-case load configurations. Worst-case scenarios may include maximum and minimum load capacities, as well as challenging load items that are difficult to sterilize.
Uniformity & Spacing : Ensure that the load is uniformly distributed within the sterilizer chamber to allow for proper steam penetration and heat distribution. Maintain enough spacing between items and ensure proper orientation to facilitate steam access to all surfaces and areas of the load.
PQ = Performance Qualification = Verification of sterilization success based on relevant loads -> actual process validation
test load of the sterilizer contains products which are routinely handled -> loading patterns must be defined
load should represent product families which have the greatest difficulty for the sterilisation -> include worst case conditions
the loading configuration & amount of load must be defined Ò size and/or the mass of the sterilizer load
Diverse Materials: Include a variety of materials (e.g., metal, glass, plastic, textiles) that are commonly processed to ensure that the sterilization process is effective for all types.
Container Sizes and Types: Validate using different container sizes and types (e.g., vials, bottles, pouches, trays) to ensure steam penetration and effectiveness across all configurations.
Sensor Placement: Strategically place temperature and pressure sensors (thermocouples) throughout the load and chamber, especially in hard-to-sterilize areas, to ensure uniform conditions.
Environmental Mapping: Conduct temperature and pressure mapping studies to identify potential cold spots or areas where steam penetration might be insufficient.
Biological Indicators (BIs): Place BIs in the most challenging locations within the load to provide direct evidence of effective sterilization. BIs should contain highly resistant microorganisms (e.g., Geobacillus stearothermophilus) to validate the process.
Chemical Indicators (CIs): Use CIs as visual markers to confirm that sterilization parameters (e.g., temperature, steam penetration) have been met throughout the load.
Cycle Parameters: Validate all critical cycle parameters, including temperature, pressure, and exposure time, to ensure they consistently achieve the required conditions.
Steam Quality: Verify that the steam used meets the required quality specifications, including purity and dryness fraction, to ensure effective sterilization.
Multiple Runs: Conduct multiple validation runs to demonstrate that the sterilization process is reproducible and consistently achieves the desired outcomes under varying load conditions.
Statistical Analysis: Analyze the data statistically to ensure consistency and reliability across all validation runs.
Compliance Standards: Ensure the validation process meets relevant regulatory standards and guidelines, such as those provided by the FDA, EMA, ISO, and other pertinent bodies.
Documentation: Maintain thorough and accurate documentation of all validation activities, including protocols, test results, and analyses, to provide evidence of compliance and process control.
Dense and Light Loads: Validate both densely packed and lightly packed loads to ensure steam can penetrate effectively regardless of load density.
Mixed Loads: Include mixed loads with different item types and materials to validate the versatility of the sterilization process.
c) What is the general name of test strips with bacterial spores used to validate sterilization procedures?
The general name for test strips with bacterial spores used to validate sterilization procedures is "biological indicators" or "biological indicator strips”
biological indicator (for validation) = filter paper strips with spores/ spore suspensions with colour indicator
These strips contain a known number of highly resistant bacterial spores, such as Bacillus subtilis or Geobacillus stearothermophilus. Biological indicators are employed to assess the efficacy of sterilization processes, for steam (autoclaving), ethylene oxide gas, hydrogen peroxide gas etc.
(9) F0 value
For a steam sterilization process at 125°C, an F0 value of 30 min is achieved. How is the achieved F0 value of 30 min to be interpreted?
(1 point)
9)
It means that the steam sterilization process for 30min at 125°C reaches an equal lethality as the process for 74 mins at 121.1°C.
(4) F0 & D value
(a) How are the F-value and the D-value related?
(b) For a given z-value of 10 °C, how does the D-value change if steam sterilisation is carried out at 120 °C (increased pressure, e.g. pressure cooker) instead of 100 °C (atmospheric pressure)?
(c) For a steam sterilisation process, the F0 value is 30 min. How can this value be interpreted?
F-value [mins] = holding time at a temperature T in minutes to reduce the existing population (N0) with the specific D value by the desired number of powers often/ log10 levels (n) to a final value (N)
D-value = duration of sterilization to kill 90% of germs (of N0)
F-value is directly proportional to D-value.
low F-value = short sterilization (holding time) => means low D-value = short duration of sterilisation to kill 90% of germs = steep curve
high F-value = long sterilization (holding time) => means huge D-value = long duration of sterilisation to kill 90% of germs = flat curve
The lower the F value, the slower is the SAL value achieved (low assurance of sterility)
With increasing temperature the D-value [mins]… decreases -> high °C results faster killing & a less D-value
= D-value decreases for factor 10^-2
F0 = 1 min means the lethality of a method that works for 1 minute at 121.1 °C (250 °F) and a z-value of 10 °C (18 °F) or an equivalent temperature-time combination
F0 value of 30min = means the same lethality of a method that works for 1 minute at 121.1°C
(3) F0 & D value
a) You shall define the minimum duration of a pharmaceutical steam sterilization procedure. The D value of the microorganisms present in the material to be sterilized at 121 °C is given as D = 1.25 min. What is the minimum time you need to sterilize at 121 °C if the bioburden before sterilization is N0 = 10^6?
(b) What is the F value at 121 °C for the data in (a)?
F0 & D-value
a) minimum SAL 10^-6 = 12 log levels
F-value = holding time (process time) to reduce N0 (with specific D-vlaue) by desired log-levels to final value N
In preparation for aseptic filling of a drug product, the product formulation is usually first filtered in a germ-reducing step and then finally sterile filtered in class A.
(a) What would be the consequence if the product formulation was not carried out in at least class C, as required, but in class D? Provide an explanation.
(a) What would be the consequence if the product formulation was not carried out in at least class C, as required, but in class D?
Provide an explanation.
1) higher risk of product contamination of particles out of air or materials (“dirty” environment)
2) Impact on product quality -> may have unwanted particles -> danger for patient
3) Risk of bad sterility -> not sterile enough, to high bioburden, bad filtration -> no release of product (additional costs)
b) What must be checked on each filter before and after use?
-> before usage = is critical because it must be assured that filter doesn’t get contaminated
2 possibilities to keep filter sterile before usage:
1) sterilization in autoclave after filter testing Ò validation important
2) filter gets installed in production area & gets sterilized in place Ò filter testing can be done in place
(a) How can you draw conclusions about the integrity of a sterile filter from the measured value of the bubble point?
(b) How is possible product loss during sterile filtration investigated?
- the higher the bubble point / pressure = the smaller the pore size
- The smaller the average pore size of a filter, the higher the pressure. If there is a leak in the filter the pressure (bubble point) stays low
with Adsorption test = for sterile filterable products
= measure concentration of API before & after filtration
-> calculation of recovery = bust be 100% no change in concentration
(8) Dry heat sterilization (2.5 points)
(a) What can be criticized about the loading of this hot air sterilization oven?
(b) For depyrogenation, hot air sterilization tunnels must be operated at over 220 °C according to the pharmacopoeia. The z value for depyrogenation is around 50 °C. What does this mean when you compare the required process time at 320 °C and 220 °C?
load should be putted in right position in the autolave (straight)
product shouldn't stand on top of each other (air cannot get inside)
air distribution = Air must be able to circulate freely
-> metal sterilization cassettes with holes = product should be placed correctly on the trays (Tablett)
z-value =Temperature change in °C, which is required to alter the D value by a factor of 10 -> 1 log reduction
= process time is longer at 220°C
= process time at 320°C is reduced by factor 100 compared to the time at 220°C
(15min = 900sek -> 900sek/9sek = 100)
a) For a pharmaceutical steam sterilization process, a SAL value of 1.4 * 10^-6 is achieved. Evaluate whether this process meets the specifications of the EMA.
Note: An evaluation includes a justification. (1.5 points)
(b) What is the probability of sterility after a single germ has been subjected to a process that reduces it by one log level?
a) SAL value of 1.4 * 10^-6 is NOT achieved. = 1.4 container of 1 mio non-sterile
EMA - specifiation: 1* 10^-6 = max 1 container of 1 mio non sterile
= the process doesn’t meet the specification of EMA because 1.4 is higher than 1
b) cfu/ml = 1
1 - log (10) = 0
Probability of sterility = 100%
Plot a typical heat killing curve of microorganisms with a decimal reduction time of 1.5 min. Start with a bioburden of 10^6 and end the curve after 9 min. Note: Do not forget to label the axes and use logarithmic scale for the y-axis.
(13) Chemical sterilization (1 point)
(a) What is the advantage of gas sterilization with hydrogen peroxide, which operates below the saturation concentration of H2O2 and thus without micro-condensation?
(b) Peracetic acid can also be used for gas sterilization. Give one reason why hydrogen peroxide is preferred over peracetic acid.
(a) advantage of gas sterilization with hydrogen peroxide, which operates below the saturation concentration of H2O2 and thus without micro-condensation:
1) Reduced Moisture Exposure: Operating below the saturation concentration means there is less moisture present during the sterilization process. Reduced moisture exposure is beneficial for materials sensitive to humidity e.g. electronics, medical devices
2) Faster Processing: faster sterilization because no condensation
Hydrogen peroxide vapor sterilization systems, e.g. used in healthcare facilities and pharmaceutical manufacturing, often operate below the saturation concentration to achieve effective sterilization while lowering the risks associated with excessed moisture.
b) Give one reason why hydrogen peroxide is preferred over peracetic acid.
Peracetic acid = bad smell
= is strong irritant (Reiz), may get in high dose toxic or have a negative impact on product
hydrogen peroxide = doesnt leave any persistent chemical residues, less toxic, environmental friendly
(6) The autoclave shown is to be used for sterilization at 121 °C. As a starting situation, assume that water of suitable quality is present in the vessel, the items to be sterilized are positioned, the lid (Deckel) is tightly closed and the valve is open. Describe all further steps or processes required until sterilization is complete. Also address the issue of air removal. (3 points)
Describe all further steps or processes required until sterilization is complete. Also address the issue of air removal.
2) water heats up -> generation of steam (with gas burner)
3) stean flows inside the vessel
4) steam rises up, reaching the lid -> steam moves down to sterilisation items
-> product is cold -> steam condensates
-> air removal = steam presses the air downwards & out of the system (via air separator, open valve)
5) atmopsheric pressure = air is eliminated & temperature can rise to 121 °C -> valve closes when all air is out -> pressure inside rises to achiieve p= 2 bar
6) Sterilisation gets done for desired time ( at least 15 mins) at 121°C
7) Cooling dwn of system -> until temperature below 80°C
(5) F0 & z-values
(a) By a holding time of 30 seconds at an unknown sterilization temperature, an F0 value of 50 min is achieved. The z-value is 5.2 °C. What is the sterilization temperature of the process?
(b) How is the achieved F0 value of 50 min to be interpreted?
a) = 131,5
b) How is the achieved F0 value of 50 min to be interpreted?
the F0 -value of 50 minutes = SAME lethality of germs that is achieved for 50 minutes at 121.1 °C
7) Sterility testing
(a) For which sterilization goods is the steam-air mixture process used?
(b) Why does the steam-air mixture process use a steam-air mixture and not pure saturated steam?
a) For which sterilization goods is the steam-air mixture process used?
1) steam/air mixture process
= because to generate steam higher °C is needed, which raises the pressure: p = p1 + p2 -> container may burst
-> higher °C to create steam may have negativ impact on product
Reasons for using a steam-air mixture:
I) Temperature Control:
Reduced Temperature: Pure saturated steam provides uniform heating at fixed temperature corresponding to its pressure (e.g., 121.1°C at 1 atm). But for sterilization processes with lower or more precised temperatures stea,-air mixtures are necessary.
Mixture Adjustments: By mixing steam with air, the overall temperature can be adjusted below the saturation temperature of pure steam. This enables more controlled heating environment for delicate items or processes that cannot withstand higher temperatures.
II) Uniform Heating
The addition of air can help in distributing heat more uniformly
Avoiding Condensation Issues: Pure steam can condense on cooler surfaces, leading to uneven heating and potential cold spots. A steam-air mixture can reduce the rate of condensation, leading to a more uniform temperature profile within the chamber.
steam-air mixture allows better control over temperature. Pure saturated steam can have a high temperature & cause damage on products. By introducing air into the steam, the overall temperature can be moderated to prevent overheating
(22) Static and dynamic seals (2.5 points)
(a) The illustration shows a static seal. When you take a look at the very left side of the seal, what two positive aspects with regard to hygienically designed static seals are fulfilled here?
(b) What is the role of the axial stop in (a)?
(c) What are the two general possibilities to seal the rotational movement of a stirrer shaft in aseptic processing?
Improvements a) & b) = how hygienic O-ring sealing should look like
= O-ring sits in the middle of 2 metal parts
a) sealing with from A
left side of the seal:
- front-flush sealing is available with maximum contact pressure at sealing point
- direct sealing of product area from the rest
other advanteages
- little expansion space & axial stop is defined
- special centring so metal parts are exactly aligned
= the axial stop deefines the compression of O-ring otherweise too much squeezing can damage O-ring (no full sealing then)
1) magnetic coupling
2) double mechanical seal
(14)
In preparation for aseptic filling of a drug product, the formulation is usually first subjected to germ-reducing filtration and then finally to sterile filtration in class A.
(a) What is the minimum cleanroom class in which the preparation must take place?
(b) Give two reasons why additional germ-reducing filtration is carried out.
= Class C
b) Give two reasons why additional germ-reducing filtration is carried out.
bioburden reduction prefilters and sterilising grade filters
1) to prevent the build up of filter cake, first filter takes the main germs load out of product
2) if the first filter is having a lack the second filter enables the filtration (safety of process)
3) increases the process safety to ensure a sterile product
4) separation of the 2 steps is possible, so one is done in class C area & the other in the final filtration
(16) Give two reasons why, in critical cleanroom classes, cleanroom personnel should make only slow, controlled movements? (1 point)
Why, in critical cleanroom classes, cleanroom personnel should make only slow, controlled movements?
at high clean room classes are high requirement for aseptic working
slow controlled movements to avoid:
1) interruption of unidirectional flow or creation of air tubulances
2) contamination of products or spread out additional particles
A silicone tubing shall be used for the aseptic filling of a drug. The preparation of the silicone tubing consists of the following steps:
[1] In class D it is placed in a double-door washing machine, washed therein, and then removed to class C.
[2] In class C the hoses are double-wrapped in tyvek bags and loaded into a pass-through autoclave.
[3] The removal of the sterilized hoses takes place in class B.
[4] The hoses are then transferred to a previously decontaminated RABS. During transfer, the door of the RABS is briefly opened and the outer tyvek bag is removed.
[5] Finally, with the doors closed, glove ports are used to open the inner tyvek bag and install the tubing.
How do you evaluate the process?
Are all steps (1 to 5) in compliance with the requirements of Annex 1 and a minimal contamination risk for the drug processed in class A?
Is there potential for optimization? If yes, which?
= the process doesn’t fulfill the criteria of aseptic preparation
Are all steps (1 to 5) in compliance with the requirements of annex 1 and a minimal contamination risk for the drug processed in class A?
= No are not, high contamination risk due to wrong unpacking in wrong zone
[1] ist right
[2] & [3] = tubs get unpacked/ opened (removal of second packaging tyvek bag) in class C -> surface sterilization from C to B is correct
[4] door of RABS shouldn't get opened -> high contamination risk (class A inside) & unpackaging of second packaging layer has to be in class C
[5] doors shouldn't open (transporttation via decontamiantion airlock or RTP better) - inner tyvek bag better to open in class B area & transfer sterile tub to class A for final filling/ processing
Main steps:
I) Class C/D: 1) tubs get mechanically unpacked/ opened -> HEPA filtered air
2) tubs get introduced into E-beam tunnel -> through airlock doors
3) tubs get transferred to irradiation zone -> surface sterilisation
II) Class B: 4) tubs get transported trough class B zone to class A
III) Class A: 5) filling zone -> presterilized syringes/ vials get filled
(15)
State two principles of aseptic work in addition to slow, controlled movements, and briefly indicate in each case what the consequences of not following the principle would be for the production environment or process.
State two principles of aseptic work. What the consequences of not following the principle would be for the production environment or process.
Principles of aseptic working
1) Do not work above the product = risk of product contamination & risk for inhalation of (toxic/ bad) substances for personnel
2) Desinfect sterile gloves regularly or change them = to avoid spread out of particles, parts of skin (sweat) getting to product or particels from product getting through the gloves (protection of personnel)
(17) Leak test isolator glove
What is the leak test procedure for an isolator glove (in addition to the visual inspection)? (1 point)
What is the leak test procedure for an isolator glove?
2.2) airflow test at constant pressure = air gets inside of gloves & measurement of volume that is needed to keep pressure constant -> if tight, no additional air needed
(19) Validation of sterilization processes
Preparation of equipment and primary packaging material (2 points)
(a) What is measured to monitor CIP processes?
(b) Sterile couplings are used for single-use systems. What is their purpose?
(c) In addition to the glass body, which parts of a finished syringe are also siliconized?
= is a Validated automatic cleaning without disassembly
-> Mobile or fixed systems with connection to SIP
Monitoring = measurement of Conductivity (possibly also pH / TOC = total org. carbon) in the effluent (Abwasser)
Conductivity = property of water to conduct electrical current. Is influenced by the concentration of ions in the water. => used as an indicator of overall water quality.
High conductivity = high concentration of dissolved ions in water
= may suggest the presence of contaminants or changes in the composition of water.
Sterile connectors/ couplings = presterilized system with membraned (gets taken out) for product transfer
= for Hose connection with sterile couplings (under class A air) = disposable system for product transfer in class A to maintain sterility
- tiny hole in the wall between class C & class A (overpressure)
- tube can be fixed in this hole & get connected with sterile coupling
-> reduced risk of recontamination
= syringe stopper & needle (if present)
(18) Transfer
(a) Which two transfer technologies are best suited for transferring crimping pliers (Crimp-Zange) into an isolator during operation?
(b) E-beam tunnels are one of the technologies used to transfer presterilized prefilled syringes in tubs to the filling area (class A).
How is the ingress of germs from the loading area (insertion of the still film-wrapped tubs) into the filling area (Class A) prevented?
(a) Which two transfer technologies are best suited for transferring crimping pliers into an isolator during operation?
1) RTP = Rapid Transfer Port = double door transfer systems
= dog removable storage/ containers bag -> not directly connected to isolator, connection via alpha port
= for transport of stoppers, caps & Liquids
2) Decontamination airlock
(b) E-beam tunnels: How is the ingress of germs from the loading area (insertion of the still film-wrapped tubs) into the filling area (Class A) prevented?
= through pressure cascade & physical barrier (walls)
(16) Barrier systems
(a) From what can it be determined that the RABS shown is an open, active RABS?
(b) Which GMP clean room class is required as the background environment if aseptic production takes place in this RABS?
c) What is the difference between RABS & Isolator?
open = air circulation in & outside chamber (from A to B class)
active = HEPA Filter inside
-> independent air supply unit
= class B environment (for RABS)
(19) Barrier systems (2 points)
(a) Which RABS type is shown in the figure?
(b) Give two differences between a product protection isolator and a personal protection isolator (for handling a non-sterile toxic product).
a) open active RABS
b) differences between a product protection isolator and a personal protection isolator (for handling a non-sterile toxic product).
product protection isolator
1) protection of product -> operation at overpressure +
2) working with gloves
3) Fully sterilizable (H2O2 decontamination)
personal protection isolator
1) protection of personnel
- Negative pressure = operation for non-aseptic products
+ Overpressure = for aseptic/ toxic products
2) working with special manipulators (NO gloves!)
3) Spray cleaning (CIP) = manual spray cleaning from outside by personnel
4) exhaust air protected = air gets filtered bevor leaving isolator
(15) Barrier systems (3 points)
(a) What characterizes an open, passive RABS?
(b) What characterizes an open isolator?
(c) When is underpressure used within isolators?
(d) How often / at what frequency should the leak test of an isolator glove be carried out?
- Negative pressure = operation for toxic/ hazardous products
= MUST be done at each batch change before every biodecontamination -> prove if isolator & gloves are tight/ closed
You want to transfer forceps into an isolator in which a product is currently being aseptically filled.
A beta container is to be used for the transfer.
Describe the process for transferring the forceps. (2 points)
A beta container is to be used for the transfer. Describe the process for transferring the forceps
1) Rapid Transfer Port = RTP
- double door transfer system
- consists of an Alpha-port which is installed in the wall of isolator or RABS
- for transfer a Beta-container/ bag gets connected to Alpha-port
-> beta-container fits exactly to alpha-port
1) Beta container = stainless steel with pipe connector & hydrophobic sterile filter = forceps are put in the beta container
2) container (with forceps inside) gets autoclaved with vacuum
3) Connection of Isolator + container -> connected lids get opened from inside for material transfer = must be air-tight to avoid decontamination
-> lids must be airtight connected ->opening of lid only possible when fully connected-> by rotation 60°
4) forceps can be transfered into isolator & there used
-> higher pressure inside transfer pressure for product protection
(16) Barrier systems (2 points)
(a) Which RABS type is shown in the figure below and what is the main difference to an isolator?
(b) How often / at what frequency should the leak test of an isolator glove be carried out?
open = air circulation between zones (A to B)
active = HEPA filter Unit inside
= open, active RABS
Main difference to Isolator = surrounding environment ISO 7 which is equivalent to class B -> Isolator has an surrounding environment of C/D
(20) Barrier systems (2.5 points)
(a) RABS and isolators are operated by glove/sleeve systems. What checks/tests must be performed on the gloves before usage?
(b) Describe the principle of a transfer into an isolator during operation using a decontamination airlock.
(c) Indicate one way in which a continuous transfer of material can be made out of an isolator?
a) What checks/tests must be performed on the gloves before usage?
1) Visual inspection of the gloves -> if leak detected no further test needed
b) principle of a transfer into an isolator during operation using a decontamination airlock.
c) Indicate one way in which a continuous transfer of material can be made out of an isolator?
(17) Barrier systems (4 points)
(a) What is the meaning of the abbreviation RABS?
(b) What characterizes an active, closed RABS?
= Restricted Access Barrier System
= always for aseptic purposes = + over-pressure inside (protection of product)
(17) Preparation of equipment and primary packaging material (2 points)
(a) Which medium is used in a CIP process for the final rinse of a format part that is later used in an aseptic process?
(b) Name one way in which two silicone tubes can be aseptically connected.
(c) What process takes place for cartridges after washing before treatment in the hot air sterilization tunnel?
(d) What is the final process step of an automated stopper treatment?
= WFI = Water for injection
= aseptic connection via:
Sterile welding of thermos plastic tubes = 2 tubes get brought together & closed in a special machine (welder) -> inside machine: tubes get cut & welded to each other -> class A air supply
= siliconization
Capping/ Crimping = after placing a stopper on a vial, capping must be perform to fully seal the vial
= ONLY after this step container is considered ready & sealed -> can leave class A
(18) Preparation of equipment and primary packaging material (2.5 points)
(a) In CIP processes, it is important that no spray shadows occur. What is meant by this?
(b) Stoppers that will later be used in syringes in an aseptic process are to be prepared. Outline the process steps of automatic stopper treatment (excluding transfer and storage processes).
(c) What process takes place for syringes after final rinsing and blow-out with compressed air before treatment in the hot air sterilization tunnel?
"spray shadows" = areas or surfaces within a processing system that are not effectively reached or cleaned by the spray nozzles during the CIP cycle.
Automatic stopper treatment
1) Washing & cleaning = to remove any contaminants, particles, or residues.
2) Rinsing = After cleaning, the stoppers undergo rinsing to eliminate any remaining cleaning agents or impurities
3) Drying = to remove moisture
4) Sterilization = autoclaving, gamma irradiation
5) Siliconization = application of a thin layer of silicone oil to the stopper surfaces. This is done to facilitate the movement of the stoppers within the vial or syrange and reduce frction & enebale correct dosing
6) Inspection and Quality Control = to ensure that quality: visual inspection for defects, dimensional checks, and testing for sterility.
7) insert stopper in syringe (with pipe or vacuum)
(21) Preparation of equipment and primary packaging material (2.5 points)
(b) Which test is performed in the pharmaceutical sector for the validation of CIP processes?
(c) When in the course of the process does siliconization of glass syringes take place?
(d) Name one disadvantage that oily siliconization has compared to baked-on siliconization.
(e) For which pharmaceutical products is the final drying of the stoppers after automatic stopper treatment of particular importance?
Validation = Riboflavin test (at least 2 times): flush is fluorescence (green) substance & good water soluble
1) contamination of the inner surfaces with a riboflavin solution
2) drying
3) CIP verification of successful cleaning with UV lamp
-> no green fluorescence should be visible! otherwise dirt left
CIP = Cleaning in Place
= before sterilization (glass syringes) -> baked-on siliconization preferred & to reduce friction while transport/ sterilization
d) Name one disadvantage that oily siliconization has compared to baked-on siliconization.
oily siliconization = thick layer -> may cause mechanicall problems e.g. in syringe due to limited space
-> backed-on silionization = due to treatment by heat (during sterilization) more robust silocon layer results, lower risk of silicone migration
e) For which pharmaceutical products is the final drying of the stoppers after automatic stopper treatment of particular importance?
Lyophilized (Freeze-Dried) Products = are highly sensitive to moisture (API)
= Dual-chamber technology - syringes
• Chamber 1 = lyophilized drug
• Chamber 2 = diluent for reconstitution
-> Used because: dilution of drug is easier, safer & higher dosage precision
(18) Preparation of equipment and primary packaging material (1.5 points)
(a) What is the riboflavin test used for?
(b) Which medium is used for the final rinse in the washing process of primary packaging materials?
(c) Why are syringes and cartridges siliconized?
a) What is the riboflavin test used for?
= for cleaning validation -> validation of CIP
b) Which medium is used for the final rinse in the washing process of primary packaging materials?
= water for injection (WFI) due to contact with product
c) Why are syringes and cartridges siliconized?
= to reduce friction & to achieve easy/ precised application (easier stoppes movement)
(18) Aseptic filling (2.5 points)
(a) Name two dosing systems besides the peristaltic pump that are used for aseptic filling.
(b) What is the purpose of a stopper sorting bowl? Name two aspects.
(c) What needs to be considered when placing a lyophilization stopper following aseptic filling?
1) Rotary piston pump = Drehkolbenpumpe: for temperature sensitive product
= used for: viscous products & for high precision filling (low volumes)
-> not for: metal sensitive products & quick product changes
- cylinder (no valve) & a piston that moves up/ down
- groove of upper part can rotate
- product get inside, groove rotates (180°C) & get filled to final container
2) Rolling diaphragm pump = multi-use or single use pumps
- intake port open & product gets inside, piston moves inside
- discharge port opens & product gets out, piston moves out
- diaphragm inside housing covers completely the piston,
no connection to outside (closed system)
(b) What is the purpose of a stopper sorting bowl? Name two aspects
Feeding of stoppers (Zufuhr von Stopfen) = Singularization (Vereinzelung) & correct stopper alignment with (vibratory) sorting bowl
- inside barrier system get transported to a vibratory sorting bowl (is rotation & vibrating)
- only stoppers that are located in right direct/ position are get transferred for capping
Lyophilization = performed in free dryer under class A conditions
-> because: - vials have special lyophilization stopper (with opening), so the solvent/ vapor can get out
- opening in stopper = risk of contamination -> class A surrounding & CIP/SIP capable (steam sterilization/ H2O2)
(20) Aseptic filling (2 points)
(a) What is controlled fully automatically during aseptic filling? Name two aspects.
(b) Why are cartridges always filled to the top?
(c) What is the name of the illustrated dosing system?
1) environmental monitoring (particle & microbial) -> Air circulation/ distribution & pressure cascades -> temperature
2) Weight check (while dosing; Filling weight) = check if enough volume inside (not too less/ not too much)
Filling Process according of cartridges to bulk syringes, but
= Filling to the edge (100 %) to exclude air inclusions /bubbles
c) Rotary piston pump = Drehkolbenpumpe: for temperature sensitive product
(22) Aseptic filling (2.5 points)
(a) Which routes can be used to transfer the product to be filled to the filling area (class A)? Name two possibilities.
(b) Name one advantage that peristaltic pumps offer over rotary piston pumps in aseptic filling.
(c) Explain the general principle of time-pressure dosing.
a) Which routes can be used to transfer the product to be filled to the filling area (class A)? Name two possibilities.
single use silicone tube = higher assurance of sterility, to washing necessary -> quick product change possible
Dosing system: Time-pressure system
(19) Aseptic filling
An infusion bottle containing 1 liter of 0.9 % saline solution is to be produced using the blow-fill-seal process.
Specify all production steps starting from extrusion to the finished, sterile product. (2.5 points + up to 1 bonus point)
blow-fill-seal process:
Cyclic process (BFS)
1) Extrusion: Melting & homogenization of plastic granules in an extruder -> at 170°C get automatically sterilized
2) Pressing the liquid plastic through a ring nozzle -> a hose (schlauch) is formed
3) Extrusion & cutting hose below the nozzle -> most critical
4) Blow mould closes, bottom is welded -> blowing with sterilized compressed air
4.1) Or Inflating (aufblasen) to a container with sterile compressed air (optional vacuum in the blow mould)
6) Filling with the product via the mandrel (Dorn)
7) Seal: Lifting of the mandrel, closure of the head mold & formation of the head closure (with vacuum)
8) Opening of the blow mould
9) Visual inspection for integrity necessary -> if plastic is to cold while forming because it creates holes -> bubble test
10) Cap welding -> on cap welding station
(21) Blow fill seal (1.5 points)
(a) Which step of the blow-fill-seal process is the most critical step for the integrity of the container? In addition, provide a brief justification.
(b) Name one advantage of the blow-fill-seal process over traditional filling in glass containers besides the exclusion of glass breakage
Critical:
• sealing step = not good sealed container has a high risk of contamination
The sealing step is crucial because it is the point at which the container is permanently closed. Any defect or contamination during sealing can compromise the sterility of product, leading to potential contamination
Proper sealing ensures that the container is leak-proof, which is essential for maintaining the integrity of the product throughout its shelf life.
The seal must be strong and durable to withstand handling, transport, and storage conditions. A weak or defective seal can lead to container failure, which can have negative impacts on the quality of product inside.
= is used for aseptic production
Advantage: BFS gives very high process & product safety (no transport routes, sterilization of vessels)
(24) Form fill seal / blow fill seal (1.5 points)
(a) What primary packaging materials are created in form-fill-seal processes?
(b) What is used in the BFS process as a supplement in the "Blow" step when very small containers are filled or a more complex container geometry is realized?
= sealed pouches (versiegelte Beutel) & infusion bags
(b) What is used in the BFS process as a supplement (Ergänzung) in the "Blow" step when very small containers are filled or a more complex container geometry is realized?
= Blow: Inflating to a container with sterile compressed air (with vacuum in the blow mould)
-> to form small or complex structure containers always vacuum is needed!
(22) A parenteral product was aseptically filled into glass vials and sealed with a stopper and crimp cap. The product is then visually inspected.
What is checked during this inspection process? Name two points.
What is checked during this visual inspection process? Name two points.
Inspection of:
1) visible particles in product
2) container integrity
3) correct crimping
4) other defects/ deviatiations
(23) Media fill (3 points)
(a) In which documents is the execution of a media fill specified?
(b) How often / with what frequency must a media fill be repeated after successful initial validation?
(c) An SOP for aseptic formulation and filling of a product requires that the product is freeze-dried and nitrogen is applied for inertisation before closing the stopper after freeze-drying. How do you take these process steps into account for a media fill?
= manufacturing SOPs & media fill plan (Plan for the media fill)
Frequency - EU GMP Annex 1
-> APS should be performed as initial validation = including 3 successful APS that cover all working shifts when significant modification in process occurs = again initial validation is required
-> periodic re-validation = APS should be repeated twice a year (2x/ year) per shift & process
-> Every employee who is routinely involved in aseptic processes must participate in 1x successful APS min. 1x/ year with an appropriate job profile.
c) An SOP for aseptic formulation and filling of a product requires that the product is freeze-dried and nitrogen is applied for inertisation before closing the stopper after freeze-drying. How do you take these process steps into account for a media fill?
Execution of Media Fill: = done by following the manufacturing SOPs & media fill plan
instead of nitrogen (inert gas) must be replaced by replaced by sterile air toguarantee perfect growth condition for usually aerobic (need O2) germs
no freezing steps = only simulation of time without lowering the °C because it may affect the growth of germs
(20) Media fill (2.5 points)
(a) What is the purpose of a media fill?
(b) A media fill plan includes extended microbiological environmental monitoring during a media fill compared to routine production. How do you evaluate this?
(c) How is a media fill evaluated after incubation?
a) What is the purpose of a media fill?
= to verify that process has high capability in assuring product sterility
= to guarantee sterile processing via process simulation considering critical steps & various interventions (worst-case situations -> risk analysis)
b) A media fill plan includes extended microbiological environmental monitoring during a media fill compared to routine production.
How do you evaluate this?
Evaluation: Visual inspection for growth, germ identification if necessary -> Objective: zerogrowth
The number of units processed (filled) for APS should be sufficient to effectively simulate all activities that are representative of the aseptic manufacturing process.
Typically, a minimum of 5000 to 10000 units are filled.
For small batches (e.g. those under 5000 units), the number of containers for APS should at least equal the size of the production batch.”
c) How is a media fill evaluated after incubation?
= visual inspection & microscope
= no turbidity
(21) Media Fill (2.5 points)
(a) What should be considered in a media fill with regard to personnel besides going for the maximum number of persons involved in the routine process? Name one additional point.
(b) An SOP for aseptic formulation and filling of a liquid product states that two excipients and WFI are added to the active ingredient for formulation, followed by stirring at 300 rpm for 30 min ± 10 min. How do you take these specifications into account for a media fill?
(c) Which number is missing in the following extract from Annex 1?
“Process simulation tests should be performed as initial validation with ____ consecutive satisfactory simulation tests”.
a) What should be considered in a media fill with regard to personnel besides going for the maximum number of persons involved in the routine process? Name one additional point.
APS = Aceptic process simulation
b) An SOP for aseptic formulation and filling of a liquid product states that two excipients and WFI are added to the active ingredient for formulation, followed by stirring at 300 rpm for 30 min ± 10 min. How do you take these specifications into account for a media fill?
1) Create a plan for the media fill
2) all excipients should get replaced with nutrient medium & WFI as well!
3) stirring of medium should be part of process simulation -> simulation of time without affecting the growth of germs is allowed
c) Which number is missing in the following extract from Annex 1?
“Process simulation tests should be performed as initial validation with _3___ consecutive satisfactory simulation tests”.
(20) Media fill (3 points)
(a) What should be considered during a media fill with regard to personnel? Name two points.
(b) An SOP for aseptic formulation and filling of a liquid product specifies stirring for three hours as part of the aseptic batch formulation and storing the product at -20°C after filling into syringes. How do you take these specifications into account for a media fill?
• max. Number of personnel & their activities -> must do their normal work
• Shift changes, breaks, and gown changes (Kleiderwechsel) (when applicable)
-> Every employee who is routinely involved in aseptic processes must participate in 1x successful APS min. 1x/ year with an appropriate job profile
b) An SOP for aseptic formulation and filling of a liquid product specifies stirring for three hours as part of the aseptic batch formulation and storing the product at -20°C after filling into syringes. How do you take these specifications into account for a media fill?
= all aseptic operations of process must be included
= EVERYTHING is replaced by nutrient medium (all excipients)!
1) The stirring or recirculation should be part of the simulation: means that 3 hours of stirring under sterile conditions must be done (includes real process duration)
1)
2) Media should not be frozen at any stage during simulation = syringes should not be frozen, and precautions should be taken that ensure that the medium remains in an aerobic state to avoid potentially inhibiting the growth of microorganisms.
2)
Crystallisation of the medium should be prevented because it may reduce the likelihood of recovery of organisms.
(26) Media fill (3 points)
(a) List two worst-case situations that could be considered in a media fill of an aseptic filling.
(b) An SOP for aseptic formulation and filling of a liquid product specifies that for formulation two excipients are added to the active ingredient while stirring. The product is then sterile filtered and aseptically filled. After filling, the product is stored at -80°C. How do you take these process steps into account for a media fill?
Stopping of a machine -> extended process duration -> vials stay open (high contamination risk)
maximum number of resonable interventions that my occure in a process
-> Each intervention has a risk of contamination, process shooul stay sterile despite frequent disruptions.
1) all excipients & API should get replaced with nutrient medium
2) stirring process should get performed as real as possible (incl. duration)
3) Media fill (process simulation) includes the steps of sterile filtration -> medium should be capable of being filtered through the same filter as used in production.
4) aseptical filling must also be simulated according to reald process
5) storage at -80°C not allowed: Media should not be frozen at any stage during simulation = product should not be frozen, and precautions should be taken that ensure that the medium remains in an aerobic state to avoid potentially inhibiting the growth of microorganisms -> only simulation of storage time possible
(24) Hygenic design
(a) How must surfaces be designed so that they can be certified as hygienic? Name two different aspects in addition to a suitable Ra value.
(b) Which stainless steel surface finishing process is used if an optimum surface finish is to be achieved?
1) smooth, inpervious, unbroken, no scratches
2) chemical (corrosion) resistant & inert, stable to aging
3) not attracted for particles -> not 90°C surfaces, no recesses (Winkel)
= Electro-polishing
(25) Name two positive and additionally one negative aspect regarding the compression of the seal shown.
Name two positive and additionally one negative aspect regarding the compression of the seal shown.
negative = huge gap, sealing far awy from product area -> not good O-ring sealing ( no front flush sealing)
positive:
1) enough expansion space for O-ring with axial stop (= to avoid total squeezing)
2) O-ring sits in a groove, its captive within the 2 Parts
(26) Hygenic design
(a) Why is there the rule L < 3 * D in piping systems or what does this rule say?
(b) What is the most important criterion for the design of vessels / tanks that is definitely fulfilled for the reactor shown below?
a) 3-D rule: L < 3 x D
L = length of the dead leg from the pipe wall
D = diameter of the pipe (connection piece)
In pipes for aseptic processing ( e.g. biorechtor pipes) dead ends & dead legs should be avoided because there is no flow of media possible & it may assemble there. Pipes must be designed to be self-drainable.
For a good hygenic design (if connection piece cannot be avoided) its length has to be lower than the 3x times the diamater of a pipe end
b) What is the most important criterion for the design of vessels / tanks that is definitely fulfilled for the reactor shown below?
smooth surface & no edges (must be smooth) -> sel drainable
(27) What is the difference between a double mechanical seal and a single mechanical seal? (1 point)
What is the difference between a double mechanical seal and a single mechanical seal? (1 point)
= primary difference between a double mechanical seal and a single mechanical seal lies in the number of seal faces and the barrier fluid used for sealing
(22) Design of a piping systems (2 points)
a) This arragement should be avoided because:
its not self-drainable -> no slope, 90° possition not hygenic
creation of dead ends -> medium may accumulate in the edges
permament welding connections preferred to detachable ones
b) Why is there a guidline value of L<3xD for the length of connecton pieces/ dead spaces in pipping systems?
(23) Hygienic design of plant components (3 points)
(a) What is the name of the static connection shown in the figure?
(b) The figure below shows a hygienic static connection with front-flush sealing. One of the requirements for hygienic static seals is that there should be the maximum contact pressure at the sealing point. Is this requirement fulfilled here? Please give a brief justification for your answer.
= Flange connection
= yes the requirement is fulfilled: ideal hygienic O-ring sealing -> O-ring sits in the middle of 2 metal parts
= no product can get through the sealing
(23) Hygienic design of plant components
(c) In the figure below a sterile sampling valve with bellows coating is shown. Explain one aspect that may be critical to successful cleaning of all valve internal surfaces?
(d) How can rotational movements of stirrer shafts be hermetically sealed?
c) In the figure below a sterile sampling valve with bellows coating is shown. Explain one aspect that may be critical to successful cleaning of all valve internal surfaces?
- narrow space between bellows & the corners may be critical for cleaning
- area of conical sealing is also critical due to less space, dirt can badly wash out
- all sealing points in bioreactor: MUST be opened so that the point can be cleaned
-> not suitable design for real use in bioreactors
(28) Hygienic design of plant components (3 points)
(a) The following figure shows a tank with an unfavorable hygienic design. Indicate what needs to be improved for the two marked points.
sharp edges -> should be rounded
connection pipe should be short or avoides (liquid remains left)
endegs of connection pipe should also be rounded
avoide horizontal installations (pipe on bottom)
c) Positive aspects of the design:
- Part 1: metal parts are exactly aligned & axial stop is defined
- Part 2: little expansion space is defined
- sealing sits in a groove, its captive within the 2 components
front-flush sealing is available = direct sealing of product area from the rest
enough expansion space & good fixation of the seal with axial stop
maximum contact pressure & rounded contact in space
(23) Hygienic design of plant components (4 points)
(a) Which two aspects should be improved in this reducing piece? Note: the arrow indicates the direction of flow.
(b) Draw the compression of a front-flush seal with axial stop and label everything.
a) Two aspects should be improved in this reducing piece?
sharp edges should be rounded ( no innter/ outer corners)
no angle (alpha); should be a straight or rounded area for liquid to flow
b) front-flush seal with axial stop
(c) The housing of which valve type is shown here?
(d) In the figure below you can see a valve for sterile sampling. The product chamber is on the left of the picture. How can it be ensured that the sealing point is also reliably cleaned and sterilised?
c) the housing of diaphragm valve
(a) The illustration shows a hygienically designed static seal. For the positions marked 1 and 2, please indicate which positive aspects regarding hygienic design have been realised here. Please note: For position 2 it is already specified that it is a front-flush seal. This is to be supplemented by another aspect.
b) sealing with form B (different shape)
- 1: good fixation of the seal with axial stop
- 2: front-flush sealing is available = direct sealing of product area from the rest (O-ring shaped)
- enough expansion space (up & down)
- seal sits inbetween Part 1 & 2 in good fixed position
(b) How can a longitudinal movement of a valve rod be hermetically sealed. Name one possibility.
(c) Mark the location within the sampling valve that would be most difficult to clean in a CIP procedure (besides the sealing point) and justify your choice
(d) What is an alternative to the magnetic coupling for hygienic sealing of rotational movements that require high forces?
= valve rod with a membrane or diaphragm
= sampling valve with diaphragm membrane
all sealing points (shoulder seal & O-ring seal) must be pned for cleaning
shoulder seal = difficult to reach for cleaning media
= double mechanical sealing
(6) A suitable sterilization process is to be selected for the sterilization of a new aqueous pharmaceutical product.
The product is sterile-filterable.
Other sterilization processes have not yet been tested for their suitability. Describe how, following the guidelines of the EMA, the selection of the process has to be done. (2 points)
Describe how, following the guidelines of the EMA, the selection of the process has to be done.
EMA: choice of sterilization method should be justified
-> according to decision tree = sterile filterable aquous product is NOT heat stable => normal steam sterilization not possible
1) product not heat stable = can be sterilised with F0-value > 8mins
-> lower °C & longer time to protect product
2) if product is not heat stable at all = use sterile filtration
3) product is sterile filterable (class A conditions) = apply combination of aseptic processing & filling
(10) Autoclave
(a) Which principle is used for air removal in the autoclave shown?
(b) For what kind of sterilization items are counterpressure autoclaves used and how is the counterpressure generated?
= air removal by grvity/ flow principle via air separator (in the bottom of system)
-> air is 1,5 times heavier than steam = it moves down & get pressed out of system
-> valve of air separator is open so air gets out
-> temeprature inside autoclave rises & get detected via temperature measurement -> at 100°C no cold air flow out & valve closes
b) For what kind of sterilization items are counterpressure autoclaves used and how is the counterpressure generated?
counterpressure autoclaves are used for = aqueoues liquids (e.g. infusions) in closed containers -> air elimination not possible so counterpressure necessary
-> e.g. for bottles/ ampoules/ contect lenses…
generation of counterpressure = additional pressure gets introduced in the autoclave chamber with sterile compressed air
(17) Terminally sterilised products
Sterile drugs can be manufactured either by terminal sterilization or aseptic processing. In this context, complete the missing information in each of the following excerpts from Annex 1 of the GMP guide. (1.5 points)
Terminally sterilised products:
28. Preparation of components and most products should be done in at least a grade D environment […].
29. Filling of products for terminal sterilisation should be carried out in at least a grade C environment.
Aseptic preparation:
32. Preparation of solutions which are to be sterile filtered during the process should be done in a grade C environment
33. Handling and filling of aseptically prepared products should be done in a grade A environment […] with class B environment
(18)
Annex 1 of the GMP guideline contains the following remark on the purpose of cleanroom class B: "For aseptic preparation and filling, this is the background environment for the grade A zone."
When is a Class B cleanroom not required?
List two possibilities.
1) for terminally sterilised products = classes A/C/D required
2) for aseptic preparations = classes A/C/D required
(25) Annex 1 of the GMP guide specifies: „124. Filled containers of parenteral products should be inspected individually for extraneous contamination or other defects.“
What is the procedure for testing if it is done manually?
What is the procedure for testing of parenterals if it is done manually?
= visual inspection by pesonnel
1) filling weight
2) particles
(23) Glass vials
(a) A product is filled aseptically. Glass vials serve as the primary packaging material. Rubber stoppers and aluminium caps are used as the closure system. At what point in the process are the containers considered to be completely sealed?
(b) Indicate one way the stopper can be placed in syringes without pushing product out of the syringe.
fully sealed means:
- vials = when stopper & cap are crimped
= with vacuum to suck in the stopper inside syringe
(27)
(a) A material in contact with the product should be inert to the product. Explain why this requirement is very important in terms of patient safety?
(b) Which parameter is used to describe the surface finish / roughness of a material?
(c) Why are scratches/crevices to be avoided on a surface in contact with the product?
= Inert (inaktiv) to the product & detergents and disinfectants to avoid interaction of material & product -> properties & quality of product my change/ cause toxic reactions -> can be dangerous for patients
= for secure storage/ long-term stability
Roughness = Ra-value = gets measured perpendicular to grinding direction
-> Reference value: Ra ≤ 0.8 μm for surfaces in contact with the product
=> ideal: Ra ≤ 0.5 μm
-> For stainless steel surfaces a Ra-value of ≤ 0.8 μm achieved by mechanical polishing or machinings is recommended
-> most microbes are around 1 μm so they don’t fit in a roughness profile of ≤ 0.8 μm
= germs can accumulate in the rough profile (scratch)
-> difficult to clean
(29) Sealing of rotational movements (1 point)
(a) What is the main advantage of a magnetic coupling compared to a double mechanical seal?
(b) In addition to removing possible contamination, what purpose does the barrier medium serve in a double mechanical seal?
magnetic coupling = benefit: guarantee hermetic sealing & for hazardous media => 100% sealing
3) Barrier interspace (CIP/SIP-capable) between the two seal faces continuously flushed with barrier fluid -> if there is a contamination the barrier fluid will transport/ wash it out of the sealing
4) Barrier fluid = MUST be compatible with the product, as small quantities can
penetrate through sliding point -> e.g. WFI (water for injection) = serves also as lubricant (Schmiermittel)/ for cooling -> to cool down the seal
(due to sliding friction get heat created which may destroy sealing)
(5)
(a) By which process is most of the thermal energy transferred to the sterilisation load during steam sterilisation?
(b) DIN 58950 specifies for pure steam that the superheat must be below 5°C. Why is this specification important?
= saturated steam
= Ensures Efficient Heat Transfer: super-heated steam needs to cool down to condensate (reduced efficancy of heat)
= better sterilization efficacy: Effective steam sterilization relies on the combination of heat & moisture.
Immediate condensation of saturated steam provides both, creating good denaturation of proteins which destroys cellular structures of germs. Superheated steam may not provide sufficient moisture, reducing its microbial killing efficacy.
(6)
(a) Draw the pressure curve of a steam sterilisation process using fractionated vacuum for air elimination and a single vacuum phase for drying.
(b) How can sufficient air removal be verified / checked? Name one possible way to do this.
= Bowie Dick test
Bowie-Dick test = for vacuum processes = proof of sufficient air removal & steam penetration into product
(7) Glass bottles with WFI are to be terminally heat-sterilised by a hot water spray process. Describe the basic principle of the process, including how to prevent the glass bottles from bursting. (2 points)
Describe the basic principle of the Hot water spray process, including how to prevent the glass bottles from bursting. (2 points)
(9) Hot air sterilization tunnel (1.5 points)
(a) Hot air sterilization tunnels are a transfer system for primary packaging materials. Between which cleanroom classes does the transfer take place?
(b) Why is there the highest pressure in the cooling zone?
a) sterilization tunnel: Between which cleanroom classes does the transfer take place?
= C to A -> because in class C washing of items is taking place
b) Why is there the highest pressure in the cooling zone?
= to avoid entrance of air or particles from outside & to prevent recontamination of sterilized products
(13) A buffer is required for the aseptic formulation of a medicinal product which is not finally sterile-filterable. The buffer is prepared in cleanroom class D and subjected to germ-reducing filtration. For aseptic formulation in cleanroom class A, the sterile active substance is added directly to this buffer. Which two points are incorrect here? (2 points)
A buffer is required for the aseptic formulation of a medicinal product which is not finally sterile-filterable.
The buffer is prepared in cleanroom class D and subjected to germ-reducing filtration. For aseptic formulation in cleanroom class A, the sterile active substance is added directly to this buffer.
Which two points are incorrect here?
1) not sterile filterable product = aseptic processing required
Preparation of buffer has to be done at least in class C (preparations of solutions to be filtered)
2) The buffer should be subjected to sterile filtration (through a 0.2 micron filter) rather than just germ-reducing filtration. This ensures that the buffer is sterile before it is used in the aseptic formulation process.
all product components need to be sterilized separately beforehand
and then bring/ fill everything in aseptic processing together under class A conditions
What to do if filtration is not possible? = all product components need to be sterilized separately beforehand & bring/ fill everything in aseptic processing together (Aseptic formulation: class A)
-> Aseptic processing has higher contamination risk because product gets filled into pre-sterilized open container systems
(14) State one activity/step in the aseptic manufacture of a pharmaceutical product carried out in clean room class B. (1 point)
State one activity/step in the aseptic manufacture of a pharmaceutical product carried out in clean room class B.
= conveying & staging/ aseptic preparation of equipment, materials, components, items for introduction in class A
(a) Why is one of the principles of aseptic working that (as far as possible) no work should be done over the open product?
(b) During the media fill experiment, sterile forceps were made unsterile by touching a potentially contaminated surface, but were still used for handling sterile stoppers. Which principle of aseptic working was violated?
a) Why is one of the principles of aseptic working that (as far as possible) no work should be done over the open product?
to avoide contamination of product —> product protection (particle distribution from human & microbial contamination, sweating)
working over product can disturb the laminar airflow, protect prodcut integrity
for self-protection of personnel -> not to inhale substances
b) During the media fill experiment, sterile forceps were made unsterile by touching a potentially contaminated surface, but were still used for handling sterile stoppers. Which principle of aseptic working was violated (verletzt)?
• Only touch sterile materials with sterile equipment
• Keep sterile equipment under Class A conditions & protect it from contamination; exchange it in case of contamination
(17)
(a) Describe the way to accomplish the transfer of sterilized rubber stoppers, which are present in a beta bag, into an isolator during operation.
(b) The stoppers are used to close vials. For this purpose, the stoppers are singularised and placed on the vials by means of pick & place. What is the principle of pick & place?
a) Describe the way to accomplish (erreichen) the transfer of sterilized rubber stoppers, which are present in a beta bag, into an isolator during operation.
= via RTP
Stopper placement methods:
1) Pick and place = stopper is held by automated system via vacuum (pick) & when vial is underneath stopper gets pressed on it
(19) Fill and finish (4 points)
(a) What is shown in the figure below?
(b) Why is fill by weight preferred for high-priced medicinal products?
(c) Why are two-hose and not single-hose peristaltic pumps usually used?
(d) In which cleanroom class must freeze-drying take place after aseptic filling?
Hose connection with sterile couplings (under class A air) = disposable system
-> tiny hole in the wall between class C & class A (overpressure)
-> tube can be fixed in this hole & get connected with sterile coupling
= exact dosage possible (fine filling) -> program controlled panel, automated filling & weigh measure while filling
= minimal waste -> when desired amount closed valve closes
= automated weight system can be integrated in production line
2) (Dual) peristaltic pump = single-use silicone tube Ò tube get sandwiched between rollers (Dialyse-pumpe)
mostly two-hose (2x Schläuche) (and not single-hose) peristaltic pumps are used due to precised & realibale fluid handling
= provide more consistent & accurate flow control
= reduced pulsation (higher precision)
= delivery of 2 different fluids -> minimal risk of cross-contamination
= in class A conditions
(e) Why does Annex 1 specify that crimping must be separated from filling and stoppering?
(f) How are ampoules closed?
(g) How do you call the process for manufacturing infusion or parenteral nutrition bags?
(h) How is the plastic hose produced in the blow fill seal process?
= by fusion with flame or laser
= form fill seal
(h) How is the plastic hose (schlauch) produced in the blow fill seal process?
20) Which two data / parameters / documents must not be taken into account when releasing an aseptically manufactured product batch? (1 point)
Which two data / parameters / documents must not be taken into account when releasing an aseptically manufactured product batch?
site master file = document from manufacturer with detailed information about production, operations, control; describes quality management system of a manufacturing site
Product quality review = is typically an annual retrospective analysis of the quality of products
(a) Materials in contact with a pharmaceutical product should be smooth (low Ra value), easy to clean and inert. State two further requirements in addition to that.
(b) What is the guideline value for the Ra value of surfaces in contact with products in the pharmaceutical sector?
a) Materials in contact with a pharmaceutical product should be smooth (low Ra value), easy to clean and inert. State two further requirements in addition to that.
Requirements for materials
b) What is the guideline value for the Ra value of surfaces in contact with products in the pharmaceutical sector?
Many sterilization items that are steam sterilized are difficult to vent / remove air from.
(a) Name a sterilization item that is difficult to vent other than textiles.
(b) How can sufficient air removal be verified / checked? Name two possible ways to do this.
= rigid hollow instruments or devices -> e.g. narrow lumens (tubes), cannulated devices (needles)
-> products with easy air elimination = gravitation processes preferred
-> products with difficult air elimination = only vacuum processes
Bowie-Dick test & Test strips
(a) Describe how the air removal is done in the autoclave shown below?
(b) Why does the temperature measurement take place at the bottom of the system (see illustration)?
a) Describe how the air removal is done in the autoclave shown below?
1) Steam gets introduced from outside inside the steam chamber
2) Inside chamber are cold prodcuts & air
4) Elimination of air -> by gravity/ flow principle
- In the beginning product is cold & contains a lot of air
- air must be eliminated otherwise steam cannot reach product, no condensation
- air is 1,5 times heavier than the steam = moves down & get pressed out of the system
- it reaches air separator (Z) with a valve -> at 100°C no cold air flows out & valve closes
5) more steam generation -> pressure inside rises to achieve p = 2 bar & 121,1 °C
6) heating up of product = with condensation Ò gives high heat energy transfer
b) Why does the temperature measurement take place at the bottom of the system (see illustration)?
= because cold air is 1,5 times heavier than the steam and moves down
-> to guerantee that required temperature is reached (means the cold air is out) the thermometer is placed down -> potential coldest spot/ area of autoclave
(11) Describe the steps that are needed to biodecontaminate an isolator with hydrogen peroxide
(VHP process). (2 points)
Describe the steps that are needed to biodecontaminate an isolator with hydrogen peroxide (VHP process).
(8) Dry heat sterilization processes
In dry heat sterilization processes, the aim is to achieve the shortest possible compensation time. Illustrate the compensation time graphically.
Note: Do not forget to label the axes. (2 points)
In heat sterilization processes, the aim is to achieve the shortest possible compensation time. Illustrate the compensation time graphically.
(13) What should be considered when validating a steam sterilization process with regard to loading? (1 point)
What should be considered when validating a steam sterilization process with regard to loading?
1) the test load of the sterilizer must contain products which are routinely handled -> loading patterns must be defined
2) laod should represent product families which have the greatest difficulty for the sterilisation -> include worst case conditions
3) the loading configuration & amount of load must be defined -> size and/or the mass of the load
4) based on relevant loads -> Optimization of steam penetration, venting etc. for different loads (cycle development)
5) the packaging system of load must be identical to the packaging used for routine production
(14) (a) A company wants to produce a new sterile product via aseptic processing, but without any robust data on the sterilizability of the product. You do not agree with this and suggest another procedure to the company. Which one?
(b) What is the minimum cleanroom grade for the preparation of a medicinal product that is later filled aseptically?
(a) A company wants to produce a new sterile product via aseptic processing, but without any robust data on the sterilizability of the product.
You do not agree with this and suggest another procedure to the company.
Which one?
if it is an aquous product = apply combination of sterile filtration & aseptic processing
if it is a dry poweder, non-aquous liquid or semi-solid product = use sterile friltration, pre-sterilized containers & aseptic processing
Only Aseptic processing is the last option if fterile filtration of product is not possible = has higher contamination risk because product gets filled into pre-sterilized open container systems -> combination with sterile filtration better
= at least class C environment
(15) Silicone tubes are used for the aseptic filling of a medicinal product.
[1] These are inserted into a double-door washing machine in a D zone, washed and dried therein and then removed to class C.
[2] There, the tubes are packed in a Tyvek bag, sterilized in an autoclave and removed again in class C. The bag is then placed in a disinfected material transfer hatch in class C and removed from it in class B.
[3] In class B, the transfer takes place into a previously decontaminated RABS. The Tyvek bag is placed in class A via a small opening in the window of the RABS.
[4] Finally, with the doors of the RABS closed, the opening of the Tyvek bag and the installation of the tubing are carried out via glove ports.
How do you evaluate the process? Are all steps (1 to 4) in compliance with the requirements of annex 1 and a minimal contamination risk for the drug processed in class A? Is there potential for optimization? If yes, which?
(3 points + 1 bonus point)
Nr. 1) is right according to Annex 1
Nr. 2) is wrong:
- for class B use pass through autoclave
- make biodecontamination
- the use of 2 Tyvek bags is needed for this process
Nr. 3) is wrong: small opening is not good in RABS
Nr. 4) is right according to Annex 1
(16)
(a) Give two reasons why, in critical cleanroom classes, cleanroom personnel should make only slow, controlled movements?
(b) Name two more basic rules of aseptic working.
a) Give two reasons why, in critical cleanroom classes, cleanroom personnel should make only slow, controlled movements
1) to avoid additional particle generation & distribution
2) to avoid interruption of lamina airflow & creation of air turbulances
3) to reduce risk of product contamination
• Always perform aseptic procedures from the side, do not work above the product
• Do not speak in critical areas if possible
• Disinfect sterile gloves regularly or change them
8)
8) The larger the F value (holding time), the lower is the SAL value achieved
= long holding time means longer sterilization & low probability that germs stay alive (low SAL)
low F-Value = low holding time = high probability that sterilisation is not sufficient & germs stay aliba = high SAL-value
With a D value of 1 min, an F value of 5 min results in 5 log levels of germ reduction. -> F = n*D
(19) Fill and finish (4.5 points)
(a) What is checked during aseptic filling as part of an in-process control? Name one aspect.
(b) Name one advantage that rotary piston pumps can offer over peristaltic pumps in aseptic filling.
(d) What is part of the aseptic filling process for oxidation-sensitive products?
(e) Which stopper placement method is shown here?
In-process control
1) environmental monitoring
2) sterility
3) filling weight check & optical check
= for sensitive & viscous products with low volumes
= pick and place
(f) What is the main advantage of using nested ready-to-fill syringes in tubs over bulk syringes?
(g) What are the two options for forming the plastic container in blow-fill-seal processes?
(h) What must be checked for all finished BFS containers, just as for ampoules, regardless of whether the product contained is administered parenterally?
= no washing/ drying/ sterilization needed
Blow mould closes, bottom is welded Ò blowing with sterilized compressed air
Or inflating to a container with sterile compressed air (optional vacuum in the blow mould)
(21)
(a) The surface quality is usually described by the Ra value. What is the Ra value or does the Ra value express?
(b) A material in contact with a sterile product should be abrasion-proof. Why?
a) The surface quality is usually described by the Ra value. What is the Ra value or does the Ra value express?
= Reference value: Ra ≤ 0.8 μm for surfaces in contact with the product
b) A material in contact with a sterile product should be abrasion-proof. Why?
1) for prevention of Particle Generation & crosscontamination
2) for maintaining Product Integrity: Abrasion can cause physical damage to materials, leading to cracks, scratches
3) to enhance Long-Term Durability: Sterile products are often intended for use over an extended period. Abrasion-proof materials contribute to the long-term durability to maintain the quality of product
(22) Why are metal-to-metal contact surfaces problematic in terms of hygienic design? (1 point)
Direct metal-to-metal joints shall not be used other than welding
= may harbour soil, liquids and could corrode
= create huge gap because metal is not flexible,
small pieces can break through friction & be dangerous for product
Why are hot air sterilization tunnels dseigned in such a way that they become slightly wider in the course of the sterilization zone?
= because of elongation of glass containers that get wider through heating -> containers expand
-> otherwise they may get truck & glass can break
-> glass breaking is a very huge problem because its very difficult to clean it up in the tunnel
Question: In preparation of aseptic filling of a drug product, the drug is usually first subjected to germ-reducing filtration
& then finally to sterile filtration in class A.
Give reasons why an additional germ-reducing filtration is carried out.
- to prevent the build up of filter cake, first filter takes the main germs load out of product
- if the first filter is having a lack the second filter enables the filtration (safety of process)
- increases the process safety to ensure a sterile product
- separation of the 2 steps is possible, so one is done in class C area & the other in the final filtration
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