Sterilisation

Question 1

What is the difference between a terminally sterilised product and an aseptically processed product?

Answer 1

Terminally sterilised is where the final product is heat treated in its final container on a validated sterilisation cycle. Aseptically processed product cannot be heat treated without spoilage of the product, it will undergo a sterilisation step such as sterile filtration as part of the manufacturing process then then any subsequent steps need to be strictly controlled as to not introduce any contamination of the product

Question 2

Can you tell me about the different methods that can be used for sterilisation?

Answer 2

Moist or dry heat, depyrogenation, gamma radiation, Ethylene oxide, VHP, sterile filtration

Question 3

How would you monitor autoclave performance on a batch by batch basis?

Answer 3

I would review the sterilisation trace and look at critical values such as temperature, pressure and time. A master control record can also be used to compare the batch trace is as per expected

Question 4

What other routine tests or checks would you perform on an autoclave?

Answer 4

Leak rate and Bowie dick

Question 5

What does a Bowie-Dick test tell you.

Answer 5

Steam penetration, it should produce an even colour change to the indicator paper located within the pack

Question 6

Validation of an autoclave. Describe.

Answer 6

URS, Commissioning and cycle development, IQ - document machine info including software and firmware versions, drawing walkdown, component installation, calibration, etc, OQ - Leak rate, bowie dick, air detector test, automatic control tests, thermometric small load, PQ - include min and max loads for each required load and includes BIs

Question 7

Tell me how you would test for steam quality and is there a guide?

Answer 7

ISPE baseline guide and BS EN285 should be used for the validation of autoclaves. Steam quality testing includes dryness fraction, superheat and non condensable gases

Question 8

What would you do if, after a routine re validation, a BI was found to be viable?

Answer 8

I would follow the OOS procedure to confirm it was a valid result. If confirmed as valid I would raise a deviation and work through - scope and contain - stop production and release and block anything in my control then quickly look into the root cause as this is a routine re validation I would want assurance that anything released since previous successful validation was not impacted. I would look at the cycle from the failed BI run and see if there was anything unusual from the prep of the load, dry load on completion, load pattern as defined etc. Has there been any maintenance carried out, if so what and when etc and use this information to reduce scope

Question 9

What do you understand by the term SAL

Answer 9

Sterility assurance level is the probability that a single unit that has been subjected to sterilization nevertheless remains nonsterile. It is never possible to prove that all organisms have been destroyed, as the likelihood of survival of an individual microorganism is never zero.

Question 10

Can you differentiate between the environmental conditions of terminal sterilisation and aseptic sterilisation.

Answer 10

Open manipulations and filling etc in aseptic manufacture should be carried out in a grade A zone where as terminally sterilised product such as a sterile nasal spray for example can be carried in is less stringent conditions such as Grade C

Question 11

What do you understand by Fo? What are the units? What is the minimum Fo?

Answer 11

Fo value is the time equivalent to 1 minute at 121oC. F8 is the minium Fo value expected = needs at least 8 minutes at 121oC

Question 12

What would be the effect if the steam was super saturated?

Answer 12

Superheated steam is too dry so doesn’t have the same kill as moist steam - takes a lot longer to achieve the same result

Question 13

What is the D value?

Answer 13

Time taken to see a 1 log reduction

Question 14

What is the Z value?

Answer 14

Temperature increase required to effect 1 log reduction

Question 15

Is there anything you should consider during autoclave validation where the autoclave will be used for both porous and non-porous loads?

Answer 15

Cycle development and validated loads

Question 16

Autoclave validation. Air detector, what design parameters?

Answer 16

An air detector is fitted to certain sterilizers that employ vacuum as a means of removing air from the load before sterilization. It is required for porous load sterilizers. The air detector should cause a fault to be indicated if the amount of air or gas in the chamber at the start of the plateau period is sufficient to depress the temperature in the centre of the load more than 2ºC below the temperature in the active chamber discharge.

Question 17

Air leaking in, what test used to detect?

Answer 17

It is used to determine whether any air or non-condensable gas present in the chamber is sufficient to impair the sterilizing process.

Question 18

If air leaking in two autoclaves, one finished product, one equipment, which most concern and why?

Answer 18

If air leaks into the chamber then the cycle should fail. The finished product would then have not had a successful sterilisation cycle so could not be released. Finished product will be in final container and sealed therefore no risk of additional contamination. For the equipment this could not be used but could be re-prepped and resterilised on a repeat cycle

Question 19

What sort of gauges would you expect to see on an autoclave?

Answer 19

Pressure gauge to monitor the pressure of the chamber

Question 20

Why do you measure temperature and pressure?

Answer 20

Key parameters for a successful autoclave chamber - Need good air removal for even temperature distribution and kill

Question 21

Removal of pyrogens - dehydrogenization can be done in one of two ways:

Answer 21

Inactivation - LPS is detoxified by chemical treatment which breaks the chemical bonds needed for pyrogenic activity, or by totally destroying the molecule - dry heat and dehydrogenisation

Removal - LPS is physically removed from the solution, usually based on the size, molecular weight, electrostatic charge or the attraction of LPS for various surfaces - filtration

Question 22

D value

Answer 22

This parameter is calculated as the time taken to achieve a one log, or 90%, reduction in the number of microorganisms present.

Question 23

Z value

Answer 23

This parameter calculates the increase in temperature for pressurized steam or dry heat, or the dose for radiation sterilization required to produce a one log, 90%, reduction in D value for a particular microorganism. This parameter is used to compare the heat, or dose, resistance of different biological indicators following alterations in temperature or radiation.

Question 24

Inactivation factor

Answer 24

The total reduction in the number of viable microorganisms brought about by a defined sterilization process. This parameter can be calculated from the D value but only if the destruction curve follows the linear logarithmic model. To overcome problems caused by variations from this model, a “most probable effective dose” value can be used. This is the dose needed to achieve n decimal reductions in the number of microorganisms.

Question 25

F value

Answer 25

The measure of total lethality of a heat sterilization process for a given microorganism and is used to compare the lethality of different heat sterilization processes. A reference value, Fo, of Geobacillus stearothermophilus spores at 121 °C is often used with a Z value of 10 °C. The total Fo of a process includes the heating up and cooling down phases of the sterilization cycle.

Question 26

SAL

Answer 26

Sterilization Assurance Level is the probability that a single unit that has been subjected to a sterilization process remains non-sterile. A SAL of 106 is generally accepted for pharmacopeia sterilization procedures, meaning, a probability of not more than one viable microorganism in one million sterilized units of the final product.

Question 27

Moist Heat Sterilization

Answer 27

• Protein inactivation by breaking amino acid cross links
  
  • Lower temperature 115-134°C (239-273°F)
  • Relatively short time 30-3 minutes
  • The basic design of an autoclave is simply a chamber able to withstand the necessary steam pressure, normally 2-3 bar absolute, together with a valve to allow steam in and one to remove condensate.

Question 28

Porous Loads:

Answer 28

• Steam penetrates package
  
  • Steam directly contacts product
  • Instant heat up
  • Sterilization medium is steam
    Porous loads will normally be wrapped to protect them from contamination after they have been sterilized. In these loads it is essential that air is removed, and steam is allowed to penetrate all surfaces.

Question 29

Cycle effectiveness is demonstrated by following the principles set out in what standard?

Answer 29

EN285 to ensure effective air removal or by Biological Indicators to demonstrate cycle lethality.

Question 30

Moist heat sterilisation cycle effectiveness is confirmed by one of the following:

Answer 30

• The Bowie –Dick Test
  
  • The Lantor Cube (3M)
  • DART (Daily Air Removal Test) by AMSCO
  • The Browns TST pack
    It is also important to ensure that air does not leak back into the chamber during vacuum pulsing, and it is normal to control this by carrying out a chamber leak test once per week.
  • For most autoclaves a leak rate of less than1.3 mBar/minute at 50 mBarA is considered satisfactory.

Question 31

Contained Process (fluids/terminal sterilization)

Answer 31

Steam condenses on bottle surface
* Heat transferred to fluid
* Delay in heat up
* Sterilization medium is fluid

The necessary steam atmosphere is generated inside the container by energy transfer from the autoclave steam through the container walls.

The removal of air is thus not as critical; indeed, in some circumstances air is deliberately added to the chamber to balance the pressures inside and outside of the containers being sterilized to prevent their deformation.

It is essential to ensure that loads are adequately cooled before opening the autoclave; otherwise, the residual pressure inside the containers and the thermal shock as the door is opened may result in the containers exploding.

Question 32

For steam sterilization, two approaches may be used:

Answer 32

• The Overkill Approach is exemplified by the pharmacopeia where various time/temperature combinations are identified as: 121oC (250°F) for 15 minutes 134oC (273°F) for 3 minutes. These time/temperature combinations can be expected to kill large populations of even the most resistant organisms and provide adequate levels of sterility assurance. Therefore, the overkill approach would not normally require further justification.
  
  • The Bioburden Approach may be used, for example, for materials with limited thermal stability. In this case, it is necessary to control the pre-sterilization bioburden to ensure that the cycle will deliver sufficient lethality to provide adequate sterility assurance, or SAL.

Question 33

Dry Heat Sterilization

Answer 33

• Oxidation
• Higher temperatures 160-180°C (320-356°F)
• Longer times 2 hours – 30 minutes Dry heat ovens are used to sterilize items that might be damaged by moist heat or that are impenetrable by moist heat.

A modern oven is supplied with heated, filtered air, distributed uniformly throughout the chamber by convection or radiation using a blower system with devices for sensing, monitoring, and controlling the critical parameters. The heat is absorbed by the outside surface of the item, then passes towards the center of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place.

Dry heat does most of the damage by oxidizing molecules. The essential cell components are destroyed and the organism dies. The temperature is maintained for almost an hour to kill the most resistant spores. The minimum operating temperature in not less than 250°C, or 482°F. Dry heat is frequently employed to sterilize and depyrogenate glassware as part of an integrated continuous aseptic filling and sealing system. Heat distribution may be by convection or by direct transfer of heat from an open flame. This type of system usually requires a much higher temperature because of a much shorter dwell time.

Question 34

Advantages of Dry Heat Sterilization are:

Answer 34

• High Lethality
  
  • No residue
  • Depyrogenation Disadvantages of Dry Heat sterilization are:
    ○ Extended Time
    ○ Many materials are incompatible

Question 35

What is terminal sterilisation?

Answer 35

the process of sterilizing a product in its final container. It is the preferred method for drug products because sterilization takes place after the product has been filled into the primary packaging. There are no further opportunities for contamination due to human intervention and are subjected to a sterilization process where the microbiological lethality can be quantified.

Terminal sterilization offers the benefit of parametric release of product. When the mode of sterilization is well understood and the physical parameters of processing are well defined, and the predictability, measurability, and the lethality of the cycle has been microbiologically validated, the terminally sterilized batches can be parametrically released without having to perform the requirements under Sterility Tests (based on regulatory approvals).

Question 36

Sterilization by Radiation

Answer 36

As with heat, ionizing radiation kills by denaturing or destroying essential molecules in the cell, either through direct or indirect action.
* Direct action relies on interaction between the radiation and a vital cellular component such as DNA.
* Indirect action is based on the fact that the major component of the cell is water.
The radiation will therefore interact with water to produce radicals and peroxides which can act both as oxidizing and reducing agents. These active species then initiate chemical reactions which damage the cell causing death.

Spores which have a low water content are resistant to this form of sterilization, as are virus particles. Other radiation frequencies can be used for sterilization, for instance electron beams or UV light.
Note: radiation can be used to kill microorganisms, but it is also capable of chemically altering the article undergoing sterilization, which may be a problem.

Question 38

Gamma Radiation

Answer 38

The most common source of Gamma Radiation is from the radioactive isotope of cobalt, 60Co. The gamma radiation can penetrate a product with a density equal to water to a depth of approximately 30cm. The half-life of the isotope is 5.25 years which means that it loses approximately 10% of its activity each year, and the source usually has a strength of between 1 and 2 x 106Ci. In large scale processes, the cobalt 60 is kept in a concrete cell with 6 ft. thick walls and the items to be sterilized are moved around the source on a conveyor system. The exposure period will be dependent upon the power of the cobalt source, the dose required and the density of the product to be sterilized. Typically, the product exposure time is 1 - 3 hours. Irradiation Processes use Electron Beams which are generated in a cylindrical acceleration chamber, in which electrodes are fed with a high frequency voltage. A narrow beam, approximately 1 cm in width, is produced. The power is extremely concentrated and exposure time is very short - a fraction of a second. Penetration is very shallow; however, and most containers need to be passed under the beam twice to get an even dosage. Dosimeters are used to measure and map penetration on a routine basis.

Question 39

Ultraviolet Light

Answer 39

Ultraviolet Light at around 254nm, can kill microorganisms by damaging DNA, and interfering with its replication. Its practical use for sterilization is very limited however, since UV is line-of-sight dependent and readily absorbed by many materials, including glass and plastics, which makes sterilization of materials in containers difficult. In addition, microorganisms possess enzymes which can repair the damage produced by UV irradiation, leading to reduced lethality and is much more suited to sanitization vs. sterilization.

Question 40

Chemical Sterilization

Answer 40

• Ethylene Oxide (EtO): Relies on the physical diffusion of the gas and water vapor into the article or container to be sterilized. This requires that the container or packaging of the product is permeable to both materials. The time required for sterilization is reduced if the product is at the correct relative humidity before exposure to the EtO. Even under ideal conditions diffusion takes a prolonged time and EtO sterilization procedures can take up to several days to complete. Ethylene Oxide is suitable for the sterilization of equipment, instruments, and articles made from plastic, rubber and metal. It is of no use for the sterilization of solutions or medicines.
  
  • Vaporized Hydrogen Peroxide (VHP): A sporicidal agent in high concentrations and long been used as a surface disinfectant. Recently Vaporized Hydrogen Peroxide has been shown to kill a wide range of bacteria, fungi and spores at low temperature and concentration - typically less than 5mg/L. Hydrogen peroxide is also relatively safe and easy to handle, and it decomposes to water and oxygen. Major applications include sterilization of surfaces in isolators, freeze dryers, and other equipment in aseptic environments.
  • Formaldehyde: Historically used as a surface sanitizing agent in cleanrooms. Its usage has generally declined with improvements in standards of cleanroom design and management. It is toxic and unpleasant to handle, leaves corrosive residues and the process is difficult to monitor.

Question 41

Filtration

Answer 41

• Porous substrate
  
  • Sterilization grade filters are rated at 0.22 or 0.2 microns
  • Pre-fill bioburden samples
  • LRV of not less than 7
  • Allows for a high flow rate
  • Membrane Integrity Testing or Bubble Point pre and post fill
    B Diminuta used as challenge organism due to its small size