Dry Heat Sterilisation

 

Since the 1970s, an increasing number of compact lines has been developed and used for the mass production of ampoules and bottles in the pharmaceutical industry. These consist of a combination of washing machine/sterilisation tunnel/filling machine in continual operation. In the past, most sterilisation tunnels were tunnels with infrared quartz tubes. In these devices, the temperature was regulated by switching the individual quartz tubes on and off, which caused particles to be emitted on the tubes and on the internal surfaces of the tunnel. This also meant that the air flow in the tunnel was no longer stable, which has a negative effect on the pressure conditions at the transition to the filling equipment's cleanliness grade. This was one reason why these devices were superseded by laminar flow hot air tunnels, which are equipped with terminal particle filters in the hot air and offer an improved, controlled air flow pattern. Laminar flow units also offer secure connections at the entrance and exit of the conveyor belt.

The requirements of the pharmacopoeias for the parameters time/temperature are shown in figure 12.K-1.

It must be possible to maintain these conditions for any chosen length of time. To meet these requirements, sterilisation tunnels are used. These transport the objects on speed-regulated conveyor belts through the hot air zone and thus fulfil the temperature/time conditions of the empty glass container. The phases of dry heat sterilisation in the sterilisation tunnel are listed in figure 12.K-2.

The sterilisation process in a sterilisation tunnel can be described using a time-temperature curve (see figure 12.K-3).

12.K.1 Description of the procedure

The hot air flow must come into contact with the inside and outside of the empty glass containers to be sterilised (ampoules, bottles), or the objects must be brought to the necessary temperature for sterilisation and depyrogenation within the required time by the radiating heat from neighbouring objects. The sterilisation time/depyrogenation time does not begin until the defined temperature is reched within the container. In closed containers, it can take a long time to achieve the required internal temperatures (often several hours). Hot air sterilisation tunnels are usually not suitable for this type of object. In this case, sterilisation or depyrogenation is performed in hot air sterilisation chambers.

When sterilising empty, open glass containers in compact lines with enclosed hot air sterilisation tunnels, much higher temperatures than those given in figure 12.K-1 are often used. The temperature can be >250 °C, mainly to avoid having to provide evidence of the 3-log reduction in endotoxins using bioindicators (spores).

Above 180 °C, the D values of these bioindicators are well below 1 min (at 160 °C 5 mins, at 200 °C under 1 sec, for example ATCC 9372). In the continual process, the temperature in the container also rises constantly without a plateau phase until it reaches the maximum temperature. It is therefore not possible to remove the bioindicator at the appropriate time (or position in the tunnel) without affecting the temperature.

Depyrogenation

Containers for parenterals must be pyrogen-free if they are filled aseptically. From volumes of 15 ml and above, injectable drug products for use in humans must be tested for the presence of endotoxins or pyrogens. In practice, following dry heat sterilisation, the remaining residual quantities in the empty container are below the limit of detection of 0.03 EU/ml. Based on the known effectiveness of the washing process, the initial endotoxin load established by monitoring, and the residual moisture in the final rinse that contains < 0.25 EU/ml, the quantities of endotoxin to be destroyed in dry heat sterilisation are very low. The risk that endotoxins will enter from the solution is several times greater, due to the WFI and active ingredients or components (with the permitted endotoxin content) that they contain. To assess the overall situation, the endotoxin limits for parenterals in accordance with the USP should be observed.

To achieve the deactivation of pyrogens by 3 log takes 1.5 mins at 250 °C. For this reason, in practice, the sterilisation tunnel should aim to achieve object temperatures of > 250 °C and residence times of > 3 mins above this temperature.

12.K.2 Sterilisation kinetics

The same rules apply as described in chapter 12.F Steam sterilisation, but different variables are used for the D and Z values.

n Number of germs, expressed as CFU (colony-forming units), also known as microbial count.

The D value is the decimal mortality rate in minutes at a given temperature, or the time in minutes that is required to kill 90% of the spores or vegetative cells of a specified microorganism at a given temperature. The D value always refers to a specific temperature and germ species, for example D_{170 °C}.

The Z value for hot air is the Z value of a given microorganism defined against different temperatures and is 20 °C (see figure 12.K-4).

FH 170 °C The total amount of heat that acts on the sterilisation goods during a sterilisation process, converted to the relevant temperature (here 170 °C) in minutes or the temperature course of the whole sterilisation curve.

At a constant temperature, therefore:

• F_H = Dt x 10 ^{(T - 170 )/Z}
• t = Sterilisation time (mins)
• T = Sterilisation temperature (°C)
• Z = 20 °C

The following example is based on real-life settings on the basis of 255 °C and 3.5 mins. F_H value of the process for an actual 3-min. sterilisation time for the container at 251 °C.

• F_H = 3 x 10 ^{(251 - 170 )/20}
• F_H = 3 x 10 ^(81)/20
• F_H = 3 x 10 ^4.05
• F_H = 33660
• Calculation of the F value (total lethality)

According to the formula F = n x D, at D _{180 °C} = 30 secs, Z = 20 °C, an assumed original germ count of approx. 10³ and expected contamination probability of 10 ^-6 results in:

• F_{180 °C} = 9 x 30 secs = 270 s = 4.5 mins

Based on existing experimental data, the F value for an equivalent procedure can be calculated according to the following formula:

• F_{Z 180 °C} /F_T = 10^{(T - 180 )/Z}; F_T = F_{Z 180 °C} /10^{(T - 180 )/Z}

Inserting the values F _{180 °C} = 4.5 mins and Z = 20 °C into the above equation results in:

• F _{200 °C} = 4.5/10 ^{(200 - 180)/20}
• F _{200 °C} = 4.5/10^20/20 = 4.5/10 = 0.45 mins.

For an equivalent procedure at 200 °C, this results in an equivalent value of 0.45 mins.

12.K.3 Qualification of a sterilisation tunnel

The operation of a sterilisation tunnel must ensure that the objects or solutions treated in the system achieve the required end result - i.e. they are free from living microorganisms. Furthermore, it should also be possible to perform depyrogenation of the objects by selecting a suitable temperature. The fulfilment of this task can be ensured by the technical layout and the qualification (see figure 12.K-5).

When selecting the design of tunnel, it is essential to decide whether you only want to use the tunnel for sterilisation, or also for depyrogenation. This affects the temperature of the tunnel in operation and also the design, which later will not permit any temperature increase.

12.K.3.1 Installation qualification

The execution of the tests is described in detail to ensure that the same tests can be accurately replicated in the future. Measured values must be recorded in test records and documents (printouts, diagrams, etc.) must be included as attachments.

Calibration of measuring points

The scope of installation qualification includes performing an initial calibration of the measuring equipment before start-up of the sterilisation tunnel. The measuring points within IQ, their measurement type, operating range, adjustment points, calibration intervals and degree of accuracy must all be incorporated in the initial calibration. Quality-relevant measuring points in the sterilisation tunnel are as follows:

• Temperature (sterilisation air, cool air)
• Belt speed
• Time
• Air velocity (LF hot air, LF inflow, LF cool air)
• Pressure differentials (LF air currents/exhaust)
• Particle measurement in the LF in accordance with class 100

Measured values for calibration must be compared with reference systems from a higher class. For values outside the acceptable measured value tolerance, the measuring chain must be adjusted and recalibrated. The operational qualification cannot take place until a successful initial calibration has been performed.

12.K.3.2 Operational qualification

In the operational qualification, all the operating states of the sterilisation tunnel are simulated to their limits (worst case), and the results documented (see also figure 12.K-8 and figure 12.K-6).

Testing heat distribution

During operational qualification, a heat distribution test must be performed on the empty conveyor belt. To do this, three measuring points are fixed on the conveyor belt across the direction of transport (see figure 12.K-7).

The temperature difference at the heat registering points between one side of the tunnel and the other should not exceed 5 °C.

12.K.4 Validation of the sterilisation process

The validation protocol (see chapter 7 Process Validation) states the objective to provide evidence that a reproducible process can lead to a 3-log reduction in bacterial endotoxin content.

In accordance with the EP, when using hot air at >220 °C for sterilisation, the evidence of a 3-log reduction in endotoxins is acceptable instead of evidence from bioindicators. The validation protocol is used to describe, structure, monitor and document the validation activities (figure 12.K-9).

Prerequisite for executing the validation is the employment of qualified personnel, and proof that the systems and test equipment are qualified for the intended purpose, and the measuring equipment is calibrated.

12.K.4.1 Description of the device

Example

The hot air sterilisation tunnel made by XYZ is installed in building 000 in cleanliness grade XY. The sterilisation tunnel is filled with washed containers (ampoules/bottles) from a washing machine automatically or by hand. A test point for rejecting objects with more than approx. 100 mg residual moisture in the container is located upstream of the entrance to the sterilisation tunnel.

The sterilisation tunnel consists of the inlet LF box, a hot air part, the cool air LF, and the passage sector to cleanliness grade C, A or B (see figure 12.K-10).

The heating register is usually located on the side of the hot air part. This aspirates air from the room atmosphere via a prefilter, heats it, and passes it on through a HEPA filter. The partial amount of air that is exhausted or pushed from the hot air part into the inlet LF and the cold air stream is replaced by atmospheric air from the room via the prefilter.

At an air velocity of 0.7-1 m/s, when the air makes contact with the containers and the conveyor belt, the air decelerates to approx. 0.4 m/s. The residual moisture from the containers also contributes to this effect by forming a steam cushion against the air current of the hot air part. To minimise the loss of hot air to the LF units and to maintain the stability of the flow conditions in the sterilisation tunnel, the isolation between inlet LF/hot air part/cool air LF/cleanliness grade filling must be adjusted to permit the minimum clearance above the containers at the passages.

When equipping a sterilisation tunnel with control sensors calibrated in an oil bath at 140 °C (three Fe-Co thermocouples), and running parallel test containers loaded with endotoxin immediately adjacent to the sensors, the whole width of the conveyor belt should be covered.

12.K.4.2 Preparation of the endotoxin test objects

Depyrogenated containers are used as validation samples for measuring endotoxin reduction. A container (ampoule or bottle) of approx. 0.5 ml, is filled with endotoxin solution corresponding to between 4000 and 6000 EU, distributed, and dried for 24 hours under a vacuum over silica gel at room temperature. Three out of twelve samples prepared in this way are subsequently tested for endotoxin recovery (usually approx. 50-100%) and used as a basis for calculating the endotoxin reduction after depyrogenation of the other samples. To interpret the results, all individual values are compared to the lowest and highest recovery rates and calculated. This results in the log reduction in values from ... to ....

12.K.4.3 Description of the process

The three validation runs follow the normal process flow of continual dry heat sterilisation.

12.K.4.4 Position of the heat sensors

The internal surface temperature of the containers is measured by fixing the thermocouple at the neck of the ampoule or bottle to the floor of the container under tension, and the thermocouple junction makes contact with the floor of the glass. The containers loaded with endotoxin are positioned immediately adjacent to the containers equipped with the thermocouples and transported through the sterilisation tunnel.

12.K.4.5 Determining the endotoxin reduction

The success of sterilisation is tested using the evidence of a 3-log reduction in gram-negative bacteria endotoxins (lipopolysaccharides) . To do this, the containers to be sterilised are loaded with endotoxin, subjected to processing in the sterilisation tunnel, and the decrease in the amount of endotoxin is determined.

Determining the sterilisation time

To check the microbiological effectiveness, the duration of the container temperature ³250 °C > 2 minutes is determined based on recordings of the thermocouple temperatures.

12.K.4.6 Executing the validation

The particular configuration (size, internal construction of the tunnel and inlets and outlets) is used to determine the minimum and maximum (worst case) loads based on the routine loading configurations. The sterilisation temperature is above 250 °C. Since the actual sterilisation temperatures are not recorded until the sterilisation process itself, for temperatures >250 °C, set temperature values above 250 °C are specified in order to correspond to the USP 265 °C.

The documentation includes the following documents:

• Time-temperature record of the measurement sensors installed in the sterilisation tunnel
• Time-temperature record of the control heat sensors
• Results of the evaluation of endotoxin reduction
• Certificates of the endotoxins and LAL used

Once the sterilisation process is complete, the data is evaluated. The evaluation in terms of temperature is based on measured data after the temperature of 250 °C is reached, taking into account the correction factors of the thermocouples.

Requirement: Temperature is ³250 °C >2 minutes (temperature, time in the objects).

• Assessment of the measurement results from the device measuring equipment compared to the control measuring equipment. Determination of the difference between the set temperature and the actual container temperature.
• Assessment of effectiveness based on physical data (F_H)
• Assessment of the sterilisation process (in accordance with PDA monograph no. 3)

Reproducibility

The reproducibility of the following setting conditions is tested:

• Temperature: 250 °C (lower permitted limit)
• Time: 2 mins
• Endotoxin reduction: 3 log

The following requirements must be fulfilled:

• The assessment should be carried out using measured data determined after the required temperature was reached.
• Time: ³2 mins
• Temperature: 250 °C - 280 °C

Assessment of the evaluation (in accordance with company SOP) and control of the recorder chart.

• Documentation: Time-temperature record from routine sterilisations.

When the validation process has been successfully completed and all results are positive, the sterilisation tunnel can be released in the described scope for one year. After this time, requalification/revalidation is required.