Exam Questions

a) under aseptic conditions ISO 5 (Class A) in isolator

= sterility testing isolator class A with surrounding area class D

b) Positive growth promotion test (with test microorganisms for each batch)

= prove that special test organisms cannot 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

c) acceptance criterion for sterility testing

= No visible growth after 14 days 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

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

Calculation:

SAL = 1-0,99999 = 1*10^-5

10^6/ 10^5 = 10

=> 10 non-sterile containers woulb be expected for a batch size of 1 mio. containers (with P = 0,99999)

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: 240s = 4min

D-value = mins / log levels = 4mins / 5 = 0,8 mins

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 * 0,8 mins = 8,8 mins

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 temeprature 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 tims 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 aliba = high 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?

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°C for 50 mins

a) Why must sterility testing be carried out in an aseptic process environment?

= to ensure the safety of aseptic process & reliability of the results

= to prevent the introduction of contaminants during the process

= because sterility must be verified for each betach to get released

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) -> antibiotoc 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 => 1ml per container must be tested

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

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 (several drops)

(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?

D = 0,5

25/0,5 = 50

= by 50 log levels reduction

(b) Name one advantage and one disadvantage of electron irradiation compared to gamma irradiation.

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

• Advantages:

  - High penetration capability for sterilization in final packaging

  - suitable for products with high density % large volume (e.g. whole pallets)

  - Rather low dose rate

• Disadvantages:

  - 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

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

=> 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

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

What data are needed for validation with biological indicators?

1) bioburden N0

2) type of microorganisms -> resistence: D & z-value

-> survival & kill-time

Why is sterile compresses air for counterpressure used and not steam?

 = because to generate steam higher °C is needed which raises the pressure: p = p1 + p2 -> container may burst

Describe all further steps or processes required until sterilization is complete. Also address the issue of air removal.

1) water heats up -> generation of steam (with gas burner)

2) steam rises up, reaching the lid -> steam moves down to sterilisation items

3) product is cold -> steam condensates

4) air removal = steam presses the air downwards & out of the system (via open valve)

5) atmopsheric pressure = air is eliminated & temperature can rise to 121 °C -> valve closes when all air is out

a) Reference germs = test organisms

= are required for sterility testing to ensure the effectiveness of the test results

= for positive control: to verify that the sterilization process is effective in killing or inhibiting the growth of microbes

= to proove that test organisms cannot grow in the nutrient medium or that the product doesn’t inhibit their growth

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

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 cannot 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

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

a) (Dual) peristaltic pump

= single-use silicone tube -> tube get sandwiched between rollers (Dialyse-pumpe)

= benefits: single use/ high precision/ quick product change possible

(b) Explain the principle of time-pressure dosing.

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

(c) Why does Annex 1 specify that crimping must be separated from filling and stoppering?

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

(d) Which test must be carried out on glass ampoules after fusion as a 100% check in addition to the visual inspection?

-> closure by fusion, e.g. glass or plastic ampoules should be subject to 100% integrity testing.

(e) What is the main advantage of using nested ready-to-fill syringes in tubs compared to using bulk syringes?

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

(g) What is used to blow up the containers during the blow fill seal process?

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

(a) What is the difference between a passive and active RABS?

• Active RABS = integral HEPA-filtered air supply (HEPA inside)

• Passive RABS = air supply by ceiling mounted HEPA-filters (outside)

Open, active RABS

Open, passive RABS

(b) Which cleanroom class must be used as the background environment for a RABS?

• 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

(c) What are the characteristics of an isolator called "open aseptic"?

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)

(d) How do you perform the leak test of an isolator glove (in addition to the visual inspection)?

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

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

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 grotwoth (zero germs) -> 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)

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

= sufficient SAL is not achieved because not more than 1 viable micro-organism in 10^-6 sterilised items of final product should be found

-> of 1 million product containers 1 container is contaminated

=> SAL of 2*10^-7 means that of 10 million product containers 2 containers are contaminated

= is achieved because SAL calculated lower than 10^-6

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 quality/ cleaning/ roughness for desinfection

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?

(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.

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°

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)

-> check change in concentration through sterile filtration

-> 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%

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

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.

What is the aseptic production process in this case?

= product is not heat stable at all & cannot be filtered then: use pre-sterilised individual or mixed components & aseptic processing

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.

• Self-drainable -> have special slope (are not straight)

• Fixed connections prefered to detachable ones

Avoidance of

• dead legs (no flow of media) Ú rule: L < 3 x D

• sharp-edged profile extensions & reductions (no corners)

• large-scale flow around / flow shadow (creates dead leg)

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: hyrmetic sealing = because hermetically sealed & no germs can enter

(e) Why is a simple mechanical seal not suitable for sealing agitator shafts?

Single mechanical seal

1) has a rotating stirrer shaft & rotating ring

2) in the housing is a fixed static ring -> while rotating these 2 parts sliding on each other & spring presses sliding ring against static ring

principle: 2 rings slide on each other (friction & heat creation)

= 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

a) SAL + Psterile = 1 = 100%

SAL = probability, that germs may be alive

Psterile = probability of germs that are death

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

a) 13 -1 = 12 12/8 (log-levels) = 1,5 D-value

b) D-value = 1.5 -> from 10^8 to 10^-6 = 14 log levels

1.5 * 14 (log levels) = 21 minuten

c) above 13 mins -> F0 at 121.1°C

a) What does this z value of 5.5 °C indicate?

z-value of 5,5 °C = temperature to reduce cfu of factor of 10 log levels

b) How are the F0 value and the SAL value related?

F0-value = special F-value for 121 °C conditions

-> the higher F-value = the lower SAL-value

The F-Value has the unit … minutes

The higher the F value, the … slower is the killing of germs.

The highert the z value, the … more resistant are the germs

The lower the F value, the faster is the SAL value achieved

With increasing temperature the D-value … decreases

The F0 value is always connected with a temperature of … 121.1°C (for 1 min)

The larger the F value, the lower is the SAL value achieved

-> high °C results faster killing & a less D-value

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%

a) SAL 1.8 * 10^-11

Redution: 13 log steps

initial baterial count N0 = SAL + log steps = 1.8 * 10^2

b) Probability in percent:

SAL + Pst = 1

Pst = 1 - SAL = 1 - (3 * 10^2) = 0.97 = 97%

a)

c) Formula cannot be used because no z value given!

How do the z-values for dry heat differ from those for steam sterilisation? Give a reason for the difference as well.

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

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

• to minimise the bioburden several precautions are necessary e.g.:

  - use of components with low degree of microbial contamination

  - microbial monitoring

  - row materials with low bioburden

  - max. bioburden must be defined (via validation)

• reduction of bioburden includes washing/ cleaning/ desinfection & sterilization in the end

a) What is meant by compensation time in (dry) heat sterilization processes?

Compensation time = time that is needed to reach 121°C from the

 reference measuring point inside chamber & all points of product (even coldest spot)

= important to guarantee everywhere 121 °C

= should be as short as possible for good heat transfer

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 sruface of syringe

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 cracks)

(a) Describe the venting process of a steam sterilization process using the fractionated vacuum method.

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

(b) How is container bursting prevented in the hot water spray process?

= through creation of counterpressure by introducing sterile compressed air into container

a) For which sterilization goods is the steam-air mixture process used?

1) steam/air mixture process

(b) Why does the steam-air mixture process use a steam-air mixture and not pure saturated steam?

 = 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

• steam-air mixture allows better control over temperature. Pure saturated steam can have a high temperature & cause damage on produts. By introducing air into the steam, the overall temperature can be moderated to prevent overheating

• addition of air to the steam helps to improve heat penetration and distribution throughout the load. Air is a less efficient conductor of heat compared to steam, and the presence of air pockets can aid in achieving more uniform heat distribution within the sterilization chamber

(a) Why are the vacuum-nitrogen cycles required?

ETO = Extremely flammable, forms explosive mixtures with air (O2)

-> 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)

(b) What is the purpose of the dwell period?

dwell-period = Sterilization time = exposure time of product under constant pressure & temperature

(c) Name one advantage of ethylene oxide sterilization over hydrogen peroxide sterilization.

Advantages - ETO

1) Broad spectrum of activity

2) Excellent penetration capacity

Hydrogen peroxide (H2O2) - Wasserstoffperoxid  

• Very strong oxidizing agent

• Broad antimicrobial spectrum

• Penetration capacity limited

• Highly corrosive

(a) Explain the effect of air pockets within the sterilization load during steam sterilization.

-> 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

-> air prevent steam for reaching the product -> bad sterilization

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

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

-> the lower the pressure, the lower amount of non-condensable gases

= if pressure rises to high, then there is a lot if air left causing overpressure

(c) How is venting / air elimination performed in a steam sterilization process that uses the gravity or flow principle?

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

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

(a) Which type of electromagnetic radiation used for sterilization has the highest frequency?

= gamma irradiation

b) Name one disadvantage of electron irradiation over gamma irradiation.

I) Electron irradiation - Characteristics

• Limited penetration, sterilization in final pakaging possible

• High dose rate

• No residues

• More gentle than gamma irradiation

II) Gamma irradiation - type of ionizing radiation: Annex 12 GMP

Characteristics

• High penetration capability sterilization of products in final packaging

• suitable for products with high density and large volume (e.g. whole pallets)

• Rather low dose rate

• No residues -> Problem of radio active waste

• Not suitable for all materials (cross-linking / degradation reactions of polymers)

(c) In which unit do you specify the dose for radiation sterilization?

Dose - ionizing radiation: Gy = Gray

• Target absorbed dose: ≥ 25 kGy (very high!) for def. SAL

• Unit: 1 Gray = 1 Gy = 1 Joule / kg = 1 Nm/ kg = 1 m^2/ s^2

What is the advantage of electron radiation over gamma radiation?

= no radioactive waste & high dosage rate

(a) What is the common mechanism of action of all types of ionizing radiation sterilization?

= induction or damage to the genetic material (DNA or RNA) of microorganisms.

- 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.

(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?

Gamma irradiation = High penetration capability sterilization of products in final packaging

= suitable for products with high density & large volume (e.g. whole pallets)

(c) Name an area of application for decontamination with UV radiation.

= in healthcare (hospitals) for disinfection of surfaces and air to reduce the risk of healthcare-associated infections

(a) In which case may ionizing radiation sterilization be used for the terminal sterilization of pharmaceutical products?

= if it is NOT possible to sterilize pharmaceuticals by heat or steam, then ionizing radiation sterilization may applied

-> 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

(b) According to which mode of action does UV radiation, which is not one of the ionizing types of radiation, kill microorganisms?

I) UV irradiation - type of NOT- ionizing radiation

Characteristics

• Disinfection of smooth surfaces, water and air

• Strong absorption, therefore low penetration depth

• Damages DNA (maximum at 265 nm) -> kiling of germs

• No residues

(c) Which of the ionizing radiation types does not belong to the electromagnetic radiation?

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) saturated steam = condensates quickly -> shorter process time

(super-heates steam = needs to cool down to condensate -> longer)

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

(a) When may sterile filtration be used to sterilize a liquid medicinal product?

= 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

(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.

Bacterial Challenge Test, BCT – biological test for filtration

1) Preparation: performance of Viability test (with reference microorganism Brevundimonas diminuta)

= checking if product has bactericidal effect -> germ killing 1log level in 45mins

-> if product is bactericidal = then it needs to get filtered first & then with adding germ suspension

-> if product isn’t bactericidal = germs get added generating a solution with > 107cfu/cm² filter area

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!

(a) What is the mechanism of action of gamma irradiation?

= use of high-energy gamma rays to disrupt the DNA or RNA of microorganisms, making them incapable of reproduction

- Generation of Ionizing Radiation: = cobalt-60 creates during radioactive decay high-energy gamma rays

- Penetration of Materials = Gamma rays have high energy and short wavelengths, allowing them to penetrate deeply into materials (sterilization)

- 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

(b) Give a reason why gamma irradiation is preferred over electron irradiation for this application.

electron irradiation = Limited penetration, but sterilization of products in final packaging possible

Gamma irradiation = High penetration capability sterilization of products in final packaging -> higher assurance level

(c) Name one advantage and one disadvantage of gamma irradiation compared to sterilization with ethylene oxide.

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

(a) advantage of gas sterilization with hydrogen peroxide, which operates below the saturation concentration of H2O2 and thus without micro-condensation:

= faster sterilization because no condensation

b) Give one reason why hydrogen peroxide is preferred over peracetic acid.

Peracetic acid = bad smell

= leaves residues (organic produts) that may get i high dooese toxic or have a negative impact on produt

hydrogen peroxide = doesnt leave any persistent chemical residues

= environment friendly

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

- big leak = lower pressure

- the higher pressure = the smaller the pore size

(a) How can you draw conclusions about the integrity of a sterile filter from the measured value of the bubble point?

- big leak = lower pressure

- 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

(b) How is possible product loss during sterile filtration investigated?

with Adsorption test = for sterile filterable products

= checking the amount of product loss; muss be 100% (no loss)

= measure concentration of API before & after filtration

-> calculation of recovery = bust be 100% no change in concentration

recovery = Wiederfindung: amount of product after & before sterilization = should be 100%

(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?

= 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)

(b) What must be checked on each filter before and after use?

= 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

(a) What can be criticized about the loading of this hot air sterilization oven?

• load should be putted in right position in the autolave (straight)

• air distribution = Air must be able to circulate freely

  -> metal sterilization cassettes with holes = product should be placed correctly

(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?

= process needs longer at 220°C

D value increse when °C insrease

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-multig bags

(b) How is the counterpressure built up in the hot water spray process to prevent the product containers from bursting?

 = compressed air gets introduced in the chamber to build up the counterpressure

(c) In which steam sterilization processes is the Bowie-Dick test used?

= for saturated steam sterilisation with vacuum processes

= proof if sufficient air removal achieved & steam penetration into product sufficient

(a) Why is primary packaging material considered more critical then secondary packaging material?

= 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.

(b) Name one way in which two silicone tubes can be connected aseptically.

Disposal systems: Aseptic connection

Aseptic connection via

1) Sterile connectors/ couplings = presterilized system with membraned (gets taken out) for product transfer

2) Silicone hose connectors = used in class A environment of 2 sterile tubes

3) 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 (+200°C) -> class A air supply

(c) Why are cartridges siliconized on the inside?

= siliconisation with PDMS = polydimethylsiloxane

• very important for Syringes & cartridges

= get covered with anti-friction coating (silicone) to minimize break loose & gliding forces

• 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

(d) At which point in the process does siliconization take place?

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

(a) What steps are needed to biodecontaminate an isolator with hydrogen peroxide?

VHP = vaporized hydrogen peroxide = surface sterilization process / used for biodecontamination -> decrease in 10log levels

General procedure/phases of VHP-process

= in isolators for decontamination/ for transfer airlocks

1) Dehumidification = same starting humidity is important

 = reduce of humidity with dehumidifier because H2O2 vapours & creates moisture

2) Conditioning = H2O2 vaporises & gets in sterilisation chamber = concentration of H2O2 & humidity rises

3) Decontamination = desired H2O2 concentration is reached & kept on the same level through adding more H2O2

-> H2O2 concentration must be below 100% saturation, to avoid condensation on the surfaces

4) Aeration (Belüftung) = vapor (gaseous H2O2) can get easily out of the system (as liquid) enabling aeration with fresh air from outside = shorter process time

(b) Why should condensation of H2O2 be prevented if possible?

1) because drops on surface of product increase corrosion

2) duration process takes much longer if H2O2 gets to vapor

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 was the initial bacterial count N0 before sterilization if this SAL valuewas achieved after a reduction of 8 log steps?

b) What is the probability of sterility in percent associated with the SAL value?

(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?

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:

- 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

(c) Sterile filters must be examined for leachables. What are leachables?

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.

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)

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 can survive

a) Why are hot air sterilization tunnels usually operated at temperatures of 300 - 350 °C?

1) to eliminate pyrogens (depyrogenation)

2) because its a (faster) process resulting better killing & higher sterility assurance

b) Which zone of a hot air sterilization tunnel has the lowest ambient pressure?

= the drying/ preheating zone (class c) = +2Pa

-> highes 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 cracks)

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

= to enable depyrogenation which starts above 220 °C (for glass) to reduce heat resistant endotoxins

(a) Which D value at 121 °C results from the diagram?

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?

a) = 30 seconds

(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?

• 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

(b) How are the D-value and the z-value related?

= Temperature change in °C, which is required to alter the D value by a factor of 10.

= temperature dependency of D-value

(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?

Parametric release – Ph.Eur.5.1.1

= can be applied when for the terminal sterilization of pharmaceuticals steam sterilization, dry heat sterilization or ionising radiation are used

-> Precondition: sterilization processes must be validated & be reliable = to get permission by authorities

What is parametric release?

= Release of terminally sterilized products without batch-related sterility testing -> for results of sterility testing 14 days needed

= release of product is based solely on the measurement of physical process parameters -> monitoring of parameters

= possible because standard conditions are defined via regulations & are performed in reality with overkilling phase (far above minimal requirements, reaching low SAL) which guarantees sterilisation safety

-> standard conditions (121 °C / 15 min steam sterilization) represent overkill = killing efficiency  sterilization safety

-> But must be approved by the authorities

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) Describe the principle of a transfer using a decontamination airlock into an isolator during operation.

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

(b) What transfer system is shown in the picture?

RTP = Rapid Transfer Port

= for transfer of materials, stoppers, caps into aseptic area while processing

Principle:

- double door transfer system

- consists of Alpha-port which is installed in the wall of isolator/ RABS

- for transfer a Beta-container/ bag gets connected to Alpha-port

 - beta-container fits exactly to alpha-port

Principle

1) Beta container = stainless steel with pipe connector & hydrophobic sterile filter

2) container gets autoclaved with vacuum

3) Connection of Isolator + container -> connected lids get opened from inside for material transfer => lids must be airtight connected -> by rotation 60°

(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)

(a) What characterizes an open, passive RABS?

(b) What characterizes an open 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)

(c) When is underpressure used within isolators?

- Negative pressure = operation for toxic/ hazardous products

(d) How often / at what frequency should the leak test of an isolator glove be carried out?

= MUST be done at each batch change before every biodecontamination -> prove if isolator & gloves are tight/ closed

(a) Which RABS type is shown in the figure below and what is the main difference to an isolator?

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

(b) How often / at what frequency should the leak test of an isolator glove be carried out?

= MUST be done at each batch change before every biodecontamination -> prove if isolator & gloves are tight/ closed

(a) What is measured to monitor CIP processes?

= 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.

(b) Sterile couplings are used for single-use systems. What is their purpose?

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

- tiny hole in the wall between class C & class A (overpressure)

- tube can be fixed in this hole & get connected with sterile coupling

(c) In addition to the glass body, which parts of a finished syringe are also siliconized?

- syringe plunger & needle & stopper

(a) From what can it be determined that the RABS shown is an open, active RABS?

open = air circulation in & outside chamber (from A to B class)

active = HEPA Filter inside

(b) Which GMP clean room class is required as the background environment if aseptic production takes place in this RABS?

= class B environment (for RABS)

What is the leak test procedure for an isolator glove?

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

(a) What is the meaning of the abbreviation RABS?

= Restricted Access Barrier System

(b) What characterizes an active, closed RABS?

(c) When is overpressure used within isolators?

= always for aseptic purposes = + over-pressure inside (protection of product)

(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?

= is a Validated automatic cleaning without disassembly

->Mobile or fixed systems with connection to SIP

CIP – steps = 2 water qualities required

1) Pre-rinse with purified water (pre-rinse)

2) If necessary, wash cycle with (alkaline) detergent

3) If necessary, rinse with purified water

4) Rinsing with water for injection (final rinse)

5) Drying -> otherwise can cause dilution of product

(b) Name one way in which two silicone tubes can be aseptically connected.

= 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

(c) What process takes place for cartridges after washing before treatment in the hot air sterilization tunnel?

= siliconization

(d) What is the final process step of an automated stopper treatment?

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

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 chieve easy/ precised application (easier stoppes movement)

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

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

b) principle of a transfer into an isolator during operation using a decontamination airlock.

3.1) Decontamination airlock/ 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

airlock = Luftschleuse

c) Indicate one way in which a continuous transfer of material can be made out of an isolator?

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

Mousehole – transfer system

= for continuous material transfer between different pressure zones within the isolator or out of isolator

-> Pressure tight closure for decontamination

-> fit exactly to the maximum dimension of the transported material (e.g. vials)

• Active mousehole = with suction (additional protection) Ò overpressure cascade inside isolator

(a) In CIP processes, it is important that no spray shadows occur. What is meant by this?

"spray shadows" = areas or surfaces within a processing system that are not effectively reached or cleaned by the spray nozzles during the CIP cycle.

(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).

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)

(c) What process takes place for syringes after final rinsing and blow-out with compressed air before treatment in the hot air sterilization tunnel? = siliconization

(a) What is controlled fully automatically during aseptic filling? Name two aspects.

1) Air circulation/ distribution & pressure cascades -> temperature

2) Filling weight

(b) Why are cartridges always filled to the top?

Filling Process according of cartridges to bulk syringes, but

1) Stopper first

2) Filling to the edge (100 %) to exclude air inclusions /bubbles

3) Closure with stopper and flanged cap or special closure system

c) 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

 Principle:

- 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

(a) Name two dosing systems besides the peristaltic pump that are used for 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

 Principle:

- 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

(c) What needs to be considered when placing a lyophilization stopper following aseptic filling?

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)

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

5) Inflating 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

A beta container is to be used for the transfer. Describe the process for transferring the forceps

1) Rapid Transfer Port = RTP

= for transfer of materials, stoppers, caps into aseptic area while processing

Principle:

- 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

(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.

Critical:

• sealing step = not good sealed container is a high risk of contamination

(b) Name one advantage of the blow-fill-seal process over traditional filling in glass containers besides the exclusion of glass breakage

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

= is used for aseptic production

Advantage: BFS gives very high process & product safety (no transport routes, sterilization of vessels)

(a) What primary packaging materials are created in form-fill-seal processes?

= sealed pouches & infusion bags

(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?

4) Blow mould closes, bottom is welded -> blowing with sterilized compressed air

-> to form small or complex structure containers always vacuum is needed!

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

(a) In which documents is the execution of a media fill specified?

= Validation plan

(b) How often / with what frequency must a media fill be repeated after successful initial validation?

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?

• 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)

a) What is the purpose of a media fill?

Medial fill – EU GMP Annex 1

= filling of a nutrient medium instead of product for aseptic process simulation

= to verify that process has high capability in assuring product sterility

= all aseptic operations of process must be included

-> process simulation test = should imitate as closely as possible the routine aseptic manufacturing process & include all the critical subsequent manufacturing steps -> microbial monitoring

= should consider various interventions known to occur during normal production & 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?

Biological indicator

Evaluation = Visual inspection for growth by personnel, germ identification/turbidity  -> Objective: zero growth

c) How is a media fill evaluated after incubation?

= visual inspection & microscope

= no turbidity

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) How must surfaces be designed so that they can be certified as hygienic? Name two different aspects in addition to a suitable Ra value.

1) smooth, no scratches

2) chemical (corrosion) resistant & inert

3) not attracted for particles -> not 90°C surfaces

(b) Which stainless steel surface finishing process is used if an optimum surface finish is to be achieved?

= mechanical polishung

a) (25) Name two positive and additionally one negative aspect regarding the compression of the seal shown.

• negative = huge gap -> sealing not safe

• positive = enough expansion space for sealing (get pressed) & rounded contact of seal to metall

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 or media 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 time diamater.

b)

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

a) Which routes can be used to transfer the product to be filled to the filling area (class A)? Name two possibilities.

1) from class D/C to B the to A = e.g. RABS/ decontamination airlock

2) from class C to A -> inside sterilization tunnel (via via conveyor belt)

a) two aspects should be improved in this reducing piece?

• sharp edges should be rounded

• no engle (alpha); should be a straight area for liquid to flow

b) front-flush seal with axial stop

c) the housing of diaphragm valve

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

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 substances for personnel

2) Keep sterile equipment under Class A conditions & protect

it from contamination; exchange it in case of contamination = risk of product contamination/ risk of cross-contamination/ if toxic subsatnce risk for personnel

(b) Which test is performed in the pharmaceutical sector for the validation of CIP processes?

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

(c) When in the course of the process does siliconization of glass syringes take place?

= before sterilization -> 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 syrange dur to limited space

-> backed-on silionization = due to treatment by heat (during sterilization) mire 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

= Dual-chamber technology - syringes

• Chamber 1 = lyophilized drug

• Chamber 2 = diluent for reconstitution

-> Used because: dilution of drug is easier,

safer & higher dosage precision

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

(a) Which principle is used for air removal in the autoclave shown?

= 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) counterpressure in autoclaves

• 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

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

When is a Class B cleanroom not required?

1) for terminally sterilised products = classes A/C/D required

2) for aseptic preparations = classes A/C/D required

What is the procedure for testing if it is done manually?

= visual inspection by pesonnel

1) filling weight

2) particles

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

(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?

= Inert (inaktiv) to the product & detergents and disinfectants to avoid interaction of material & product -> proterties & quality of product my change/ cause toxic reactions -> can be dangerous for patients

= for recure storage/ ling term stability

(b) Which parameter is used to describe the surface finish / roughness of a material?

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

(c) Why are scratches/crevices to be avoided on a surface in contact with the product?

= germs can accumulate  in the rough profile (scratch)

-> difficult to clean

(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?

(a) By which process is most of the thermal energy transferred to the sterilisation load during steam sterilisation?

= saturated steam

(b) DIN 58950 specifies for pure steam that the superheat must be below 5°C. Why is this specification important?

(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

Hot water spray

a) sterilization tunnel: Between which cleanroom classes does the transfer take place?

= C to A -> because in claass C washing of items is taking place

b) Why is there the highest pressure in the cooling zone?

= to avoid entrance of air inside or particles & to prevent recontamination of sterilized products

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?

State one activity/step in the aseptic manufacture of a pharmaceutical product carried out in clean room class B.

Double-door / pass-through autoclaves

Usage: - for manufacturing sterile pharmaceuticals -> aseptic production

 - as transfer system

- always has 2 doors: loading from the dirty side & unload from clean side

- installed between class C (loading) & class B (unloading sterile products)

-> high assurance that no contamination in class B (only 1 door opens at a time)

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

• for self-protection of personnel -> not to inhale substances

b) Which principle of aseptic working was violated?

• Only touch sterile materials with sterile equipment

• Keep sterile equipment under Class A conditions & protect it from contamination; exchange it in case of contamination

Which two data / parameters / documents must not be taken into account when releasing an aseptically manufactured product batch?

• site master file

• fulfillment of product specofocations

For batch release must be taken into account:

1) Manufacturing documentation

2) In-process & final product controls = Sterility of product: all quality controls must be approved!

3) Controlling all Monitoring data (environment, personnel, WFI, etc.)

4) check deviations (Abweichungen): e.g. interventions, shutdown, equipment malfunction, CAPA

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

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 the guideline value for the Ra value of surfaces in contact with products in the pharmaceutical sector?

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

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)?

Describe the steps that are needed to biodecontaminate an isolator with hydrogen peroxide (VHP process).

VHP = vaporized hydrogen peroxide

= surface sterilization process -> decrease in 10log levels

= used for biodecontamination

General procedure/phases of VHP-process

= in isolators for decontamination/ for transfer airlocks

1) Dehumidification = same starting humidity is important

 = reduce of humidity with dehumidified because H2O2 vapours & creates moisture

2) Conditioning = H2O2 vaporises & gets in sterilisation chamber

= concentration of H2O2 & humidity rises

3) Decontamination = desired H2O2 concentration is reached & kept on the same level through adding more H2O2

-> H2O2 concentration must be below 100% saturation, to avoid condensation on the surfaces

4) Aeration (Belüftung) = vapor (gaseous H2O2) can get easily out of the system (as liquid) enabling aeration with fresh air from outside

-> shorter process time

Why is it good to avoid condensation of H2O2?

1) because drops on surface of product increase corrosion

2) duration process takes much longer if H2O2 gets to vapor

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

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

(b) Name two more basic rules of aseptic working.

• Always perform aseptic procedures from the side, do not work above the product

• Do not speak in critical areas if possible

• Only touch sterile materials with sterile equipment

• Keep sterile equipment under Class A conditions & protect it from contamination; exchange it in case of contamination

• Disinfect sterile gloves regularly or change them

8) The larger the F value (holding time), the slower is the SAL value achieved

With a D value of 1 min, an F value of 5 min results in 5 log levels of germ reduction.

a) The surface quality is usually described by the Ra value. What is the Ra value or does the Ra value express?

Roughness = Ra-value = gets measured perpendicular to grinding direction

= 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

= 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

- 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

(a) List four different non-economic requirements that should be included in a user requirement specification (URS) for an autoclave

1) Underlying regulatory requirements for the system

·   Standards to be complied with, guidelines

2) Location / Environment / Dimensioning

·   Place of installation/ Environment / zone requirements

·   Constructional conditions, e.g. available area, floor load

·   Dimensions (chamber volume)

3) 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)

·   Performance / energy consumption / energy efficiency

4) Monitoring / Measurement technology

·    Sensors/ Accuracy/  Additional features

(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?

5) PQ = Performance Qualification = Verification of sterilization success based on relevant loads -> actual process validation

= provide evidence that equipment works according to specified criteria (reliable sterilization)

= include physical & biological tests

Requirements for PQ (Verification of sterilization process)

Inspections must verify that:

1) the documentation confirms a successful IQ & OQ -> IQ/ OQ & cycle development should be successfully finished & working

2) the test load of the sterilizer contains products which are routinely handled -> loading patterns must be defined and [...] represent product families which present the greatest difficulty for the sterilisation -> include worst case conditions

3) the packaging system must be identical to the packaging used for routine production

4) the preconditioning = storage before sterilization must correspond to criteria

5) the loading configuration & amount of load has to bedefined -> size and/or the mass of the sterilizer load

b) What should be considered when validating a steam sterilisation process with regard to loading?

= worst case conditions -> coltest spot

Cycle development = based on relevant loads -> Optimization of steam penetration, venting etc. for different loads

= establishment of sterilization cycles for routine manufacturing for different loads

PQ = Performance Qualification = Verification of sterilization success based on relevant loads -> actual process validation

= provide evidence that equipment works according to specified criteria (reliable sterilization)

= include physical & biological tests

Requirements for PQ (Verification of sterilization process)

Inspections must verify that:

1) the documentation confirms a successful IQ & OQ -> IQ/ OQ & cycle development should be successfully finished & working

2) the test load of the sterilizer contains products which are routinely handled -> loading patterns must be defined

  and [...] represent product families which present the greatest difficulty for the sterilisation -> include worst case conditions

3) the packaging system must be identical to the packaging used for routine production

4) the preconditioning = storage before sterilization must correspond to criteria

5) the loading configuration & amount of load has to bedefined -> size and/or the mass of the sterilizer load

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”

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 et.

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?

= microbial tests

c) DQ = Design Qualification = Determination of requirements for equipment

  URS = user requirements specifications & functional specification document

(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.

DQ = Design Qualification = Determination of requirements for the equipment ->(URS = user requirements specifications & functional specification document)

Ionizing radiation sterilization -> E-beam tunnel

= its applicabel for tube & E-beam tunnel is long, has enough space for 2 m logn tube

(b) What is the name of the document in which the user demands on a sterilization system are recorded as part of the DQ?

URS = user requirements specifications & functional specification document

(c) What is checked during thermoelectric testing with an empty sterilization chamber as part of the PQ of a steam sterilization process?

1) Thermo-electric test with empty chamber

= temperature and pressure sensors get distributed inside empty chamber to check uniformity of parameters (°C/ ∆p)

 -> minimal deviations (+/- 2°C; +/-10 kPa) allowed -> even coldest spot must fulfil criteria

 -> must be performed for minimum & maximum cycle times and temperatures

9)