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Introduction

 

 

Here you will find answers to the following questions:

  • Which requirements are sterile products expected to meet?
  • In which cleanliness grades do the individual products to be sterilised steps take place for products to be sterilised in the final container or for aseptic processing?
  • What are the requirements for particle load and bioburden?
  • What problems can occur in connection with production equipment?

The absence of living microorganisms is a central requirement for a sterile medicinal product.For category 1 preparations, the pharmacopoeia requires that sterility testing is complied with (acc. to PhEur. 5.1.4, part 1). This is defined as a reduction in microbial count of six log factors (10-6). In addition to the absence of microorganisms, the reduction of pyrogens and endotoxins is also required for many products (see figure 1).

Figure 1 Definitions of pyrogens and endotoxins

Definitions

Pyrogens

Any substances (chemical, microbiological) that cause fever on parenteral administration.

Tested using the rabbit test.

Endotoxins

Components of the cell wall in gram-negative bacteria that cause fever on parenteral administration.

Tested using the LAL test (Limulus Amebocyte Lysate test).

Parenterals (products that are administrated to the bloodstream) are also required to be "free from visible particles". For invisible particles, the requirements apply as described in figure 2.

Figure 2 Requirement for invisible particles in preparations for infusion or injection

Requirement for invisible particles in preparations for infusion or injection

Nominal volume > 100ml

Nominal volume of £ 100 ml

Average number* of particles
³10 mm 25 P/ml

Average number* of particles
³ 10 mm, maximum 6 000 per container

Average number* of particles
³25 mm, maximum 3 P/ml

Average number* of particles
³ 25 mm, maximum 600 per container

* The average number is obtained from three measurements using the light blockade test procedure.

Usually, these are aqueous substances that are administrated parenterally (i.m. or i.v. as an injection or infusion), or eye medicines (ophthalmics) for application on the eyeball. Drug substances that are not stable in aqueous solution, or which would not survive the heat treatment for sterilisation without becoming damaged, are often freeze-dried and resuspended with sterile solvents before application.

Since testing for sterility (see chapter 12.H Test for sterility) always destroys the product and can therefore only be carried out on random samples, this test does not provide absolute certainty that each individual container in the manufactured batch is sterile. Validation of the processing steps, in particular of sterilisation, together with detailed batch documentation of the manufacturing and sterilisation parameters, is one method of ensuring the sterility of the whole product batch.

Due to the specific requirements for the manufacture of sterile products, the technical knowledge, behaviour and training of the relevant personnel are key factors in reducing contamination with microorganisms, particles and pyrogens to a minimum. Products must be manufactured according to validated methods and procedures defined in writing. Quality aspects must not be limited to the final manufacturing step or testing of the final product.

Sterile medicinal products are generally manufactured according to one of two different procedures:

1. Manufacturing with sterilisation in the final container (see chapter 12.A.1 Manufacturing products that can be sterilised in the final container)

2. Aseptic processing (see chapter 12.A.2 Aseptic processing)

The crucial processing steps for achieving the ultimate aim of sterility are sterilisation and sterile filtration (see chapter 12.C.4 Sterile filtration). Sterilisation in the final container is the preferred method.Sterile filtration is considered if a heat sterilisation procedure would destroy the properties of the medicinal product.

12.A.1 Manufacturing products that can be sterilised
in the final container

Sterilisation in the final container should be used wherever possible. All processing steps in the run-up to and environment of a sterilisation in the final container must be targeted towards the ultimate aim of sterility by employing sterilisation methods and measures to reduce microorganisms. This includes the need to perform the batch/filtration manufacturing operations in a closed system in cleanliness grade D (EU-GMP-Guideline), and the filling of the final container in cleanliness grade C under LF (Laminar Flow, class 100 US Federal Standard). Annex 1 of the EU-GMP-Guideline also stipulates the following: "Where the product is at a high or unusual risk of microbial contamination, (for example, because the product actively supports microbial growth or must be held for a long period before sterilisation or is necessarily processed not mainly in closed vessels), then preparation should be carried out in a grade C environment. Filling of products for terminal sterilisation should be carried out in at least a grade C environment." (see figure 4).

In addition to these requirements, the microorganism and endotoxin load (see chapter 12.C.3 Testing the bioburden) of the active and excipient ingredients must be determined before manufacturing and, after filling from the final container, before sterilisation. The loads determined in the solution and substances can be used to calculate the sterilisation probability of the preparation and the remaining endotoxin load in advance, based on the efficiency of microorganism and endotoxin reduction determined and evaluated in the qualification and process validation runs.

In special cases, particles are removed from liquids as a part of steril filtration using membrane filter layers with a pore width of 0.22 m or 0.45 m.

Sterilisation in saturated steam in an autoclave at 121 °C for 15 mins is the method of choice for achieving a sterile aqueous medicinal product (PhEur.) (see chapter 12.F Steam sterilisation). An SAL (Sterility Assurance Level) of at least 10-6 must be achieved. This means that the probability of finding a non-sterile object must be one in 1,000,000 units.

12.A.2 Aseptic processing

When manufacturing a sterile medicinal product that cannot be sterilised in its final container, it is necessary to place higher demands on the personnel, the environment (see figure 3) and the processing equipment, in order to completely exclude the possibility of microbial contamination.

Figure 3 Cleanliness grades for aseptic processing

Cleanliness grades for aseptic processing

Manufacture of the product

Cleanliness grade C

Steril filtration

Cleanliness grade B

Filling in the final container

Cleanliness grade A

Manufacture of non-filterable preparations

Cleanliness grade A

A batch-specific, microbiological and particle monitoring of the environment and personnel is carried out (see chapter 12.G Microbiological monitoring).

The suitability of all processing steps is confirmed twice yearly using a nutrition agar culture fill to ensure <0.1% non-sterile objects (see chapter 12.E.5 Culture medium filling (Media Fill)).

12.A.3 Production areas/premises

Clean areas for the manufacturing of sterile products are graded according to the required environmental characteristics. Each manufacturing process must fulfil the appropriate cleanliness grade for the environment while in process, in order to minimise the risk of contamination of the material by microorganisms or particles. To fulfil the conditions in process, the areas must be arranged to ensure that a certain level of air purity can be achieved at rest. At rest, the technical facility is installed and ready for operation.In process, the technical facility is being operated by staff.

Personnel must only be able to access the clean areas via air locks positioned between one cleanliness grade and the next (see chapter 12.B Air Lock Concepts). These should correspond to the relevant cleanliness grade and be ventilated using filters of an adequate efficiency level (see chapter 3.H Heating Ventilation Air Conditioning (HVAC)). This means that the local separation rate of the HEPA filter (High Efficiency Particulate Air filter) must be at least 99.97 % and must be selected and fitted in accordance with PIC document PH 5/89 from Sept. 89 to the appropriate level for the cleanliness grade.

Cleanliness grade A

H14

99.995 %

separation rate (integral value)

Cleanliness grade B

H14

99.995 %

separation rate (integral value)

Cleanliness grade C

H13

99.95 %

separation rate (integral value)

Cleanliness grade D

H11

95.00 %

separation rate (integral value)

Materials should enter the room via separate material locks (see chapter 12.B.2 Material locks). The various operations such as the preparation of ingredients, formulation and filling should be carried out in separate zones within the clean area.

Manufacturing operations are classified into two categories:

1. Products that can be sterilised in their sealed final containers (cleanliness grade D)

2. Products for which particular stages or all stages of the manufacturing processes are carried out under aseptic conditions (cleanliness grade C).

The cleanliness grades have different names depending on the underlying regional requirements: In the EU-GMP-Guideline A-D, in the USA according to Federal Standard (FS) 209 E in classes 100, 10.000 and 100.000 (no longer valid since November 2001, but can still be found in practice), and according to the ISO guideline no.14644 applicable since 2001, in ISO classes 5-8 (see figure 4). For more information, see chapter 12.G.2 Room classification.

Figure 4 Cleanliness grades

Cleanliness grades

Cleanliness grade A
(FS 209 E class 100 at rest) ISO 5

US class 100 in operation

SI M* 3.5

  • The local zone for operations with a high level of risk, for example in the filling area, assembly of the filling apparatus (pump, filter, etc.), aseptic connections (equipment, tubes, couplings) under a laminar air flow of 0.45 m/s ± 20%.
  • Microbiological limit <1 CFU/m3
  • In the case of faults, controlled, brief intervention by personnel from cleanliness grade B is permitted.

Cleanliness grade B
(FS 209 E class 100 at rest) ISO 5

US class 10.000 in operation

SI M* 5.5

  • The presence of appropriately dressed personnel is permitted.
  • Turbulent air flow is permitted.
  • Microbiological limit 10 CFU/m3 (action limit)

Cleanliness grade C
(FS 209 E class 10 000 at rest) ISO 7

US class 100.000 in operation

SI M* 6.5

  • The presence of appropriately dressed personnel is permitted.
  • Turbulent air flow is permitted.
  • Microbiological limit 100 CFU/m3 (action limit)

Cleanliness grade D
(FS 209 E class 100 000 at rest) ISO 8

US class: not classified

  • The presence of appropriately dressed personnel is permitted.
  • Turbulent air flow is permitted.
  • Microbiological limit 200 CFU/m3 (action limit)

* SI M represents the classification on the basis of ³0.5mm particles

Limits for the microbial contamination of surfaces in these cleanliness grades are generally tested with purchased nutrition agar contact plates with a diameter of 55 mm (corresponds to a surface area of 23.75 cm2). You therefore need to consider the specifications per cm2 and include them in standard operating procedures (SOPs). To be on the safe side, you can comply with the required limit (see figure 5 and figure 12.G-3) for a contact area of > 23.75 cm2. Normally, specifications are provided per plate with a diameter of 55 mm or per 25 cm2 (figure 5).

Figure 5 Recommended residue values for microbiological contamination (according to the EU-GMP-Guideline, Annex 1)

Recommended limits for microbial contamination
(EU-GMP-Guideline, Annex 1, USP 25)

Grade

air sample
cfu/m3

settle plates
(diam. 90mm)
cfu/4 hours

contact plates
(diam. 55m)
cfu/plate

glove print
(5 fingers)
cfu/glove

A

<1
<3

<1

<1
3

<1
3 glove
5 clothes

B

10
<20

5

5
5
10 floor

5
10 Gloves
20 clothes

C

100
<100

50

25

-

D

200

100

50

-

For absolutely necessary interventions in cleanliness grade A, the hands covered by the gloves should be disinfected to ensure that the limit of <1 CFU/5 fingertips is not reached. However, since individual values of 1 and >1 cannot be excluded (<1 is an average value), in this case, the average value of the last ten quality controls should be used. This average value must be <1 CFU/5 finger tips (measure limit value/action limit) (see figure 5). In the presence of correct personnel behaviour and operational cycle, a bioburden on the finger tips of >3 CFU/5 finger tips is extremely rare. If operating procedures are not adhered to, the values are considerably higher, i.e., a value of 2 CFU/5 finger tips can be an indication of systematic failures and should be regarded as a warning sign. The alert limit could therefore be determined at 2 CFU/5 finger tips.

Particle contamination in the air is measured at the start and end of the procedure at 3 points below the LF at object height.

1. Start of LF in direction of transport

2. At the filling point of the objects

3. Before the sealing apparatus (stoppering or tip sealing for ampoules)

The contamination levels must be measured at the beginning and end of production.

Figure 6 Max. permitted number of particles/m3 (greater than or equal to) according to the EU-GMP-Guideline, Annex 1

Max. permitted no. of particles/m3 (equal to or above)
from the EU-GMP-Guideline, Annex 1

Cleanliness grade

At rest

In operation

 

0.5 mm

5 mm

0.5 mm

5 mm

A

3,500

1

3,500

1

B

3,500

1

350,000

2,000

C

350,000

2,000

3,500,000

20,000

D

3,500,000

20,000

not defined

not defined

This enables you to prove and document, if you are using the alert system (audio signal or red light if the value drops below 0.35 m/s) of the LF box, that the laminarity of the air flow has been maintained throughout the processing period.In automatic systems, selected points are recorded; usually two points per square metre. The action limits according to the EU-GMP-Guideline, Annex 1, are shown in figure 6.

The flow rate of the air is measured at 0.45 m/s approx. 10-30 cm below the filter surface and is usually displayed on the digital or analogue display of the relevant LF box.The control measurement should then be performed at the level of the opening on the object to be filled, at least at the filling point, since in general this is where the strongest movements take place which can affect the air flow. The lower limit of 0.35 m/s can be easily complied with if there are no serious mechanical problems. The flow measurement must also be performed at the beginning and end of production.

12.A.4 Production equipment

Requirements for premises and equipment are to be taken from Annex 1 of the EU-GMP-Guideline. Considering the variation in technical, physical, chemical and microbiological requirements of the materials to be processed and the procedures used, it is understandably only possible to make very general statements, such as "exposed surfaces should be smooth, unbroken and impervious ... air-conditioned ... cleaned, disinfected and/or sterilised where appropriate ... subject to validation and planned maintenance" (see chapter 4 Facilities and Equipment).

Production equipment must be qualified, regularly serviced and, in the event of repairs and modifications, subject to a change control procedure. Incalculable risks in drug product safety almost always originate from incorrect procedures in technical systems (short-term defects are particularly dangerous) related to insufficient personnel understanding of the influence on product quality resulting from a change in the general conditions of a sequence of operation.

Example:

A machine stop due to breakage of glassware (bottles, ampoules, etc.), switching errors and the resulting intervention can mean that the machine is not stopped in the end position of a cycle (compressed air release, spraying with water, pressure maintenance of pumps, time switch points of cycles, etc.). Since nowadays, compact lines are most commonly used (washing machine, sterilisation tunnel, filling machine in one), these faults have an impact across several levels of processing. When eliminating faults, it must be guaranteed that objects which have not reached the end of the process are disposed of, or that they are allowed to reach the end of the process uninfluenced.

Examples of serious faults:

The interruption of a dosing pump movement consistently leads to dosing variations due to the change in pressure conditions in the system cylinder - piston - suction pressure - pressure line and switching.

In switching mechanisms employed in compact lines (for example, spraying pressure of water or compressed air), no fault must be allowed to occur that does not lead to immediate stopping of the machine and that can be "overridden". This may mean that objects are not adequately rinsed or that, for example, partially filled WFI objects reach the sterilisation tunnel and are not properly sterilised.

In order to exclude problems resulting from faults as much as possible, limits must be defined in relation to the time period of the machine cycles, pressures and temperatures of media, the revolution speed of drives, end positions of machine levers and cams, and the mechanical process flow must be recorded in the electronic parallel program. This ensures that when deviations occur, error displays are generated, the machine is stopped appropriately, and both technical and pharmaceutical measures can be implemented.

Microbiological influences are nearly always a consequence of physical or technical changes.The deciding factor when selecting production equipment is the necessity of a requirement from a technical and pharmaceutical perspective, together with the technical feasibility. Maximum requirements are only reasonable in cases in which clear values must be fulfilled. For example: In the procurement of prototype machines and filling systems that have not actually performed any development steps, their performance can only be imagined, but not backed up with practical experience.In this case, clear maximum requirements must be set and enforced in terms of parameters (such as flow rates and particle counts in the air in accordance with the cleanliness grade, sterilisation temperatures, temperature control limits and time specifications). All other requirements are secondary and should be amicably deleted in advance between the technical and pharmaceutical operators. "Overloading" with automatic logs, controls and networking leads to an inflation of potential fault sources, increased costs and prolonged timeframes. The common understanding, comprehension and processing of the requirement in the form of compromises (for example price, capacity, performance, current costs) lead to sustainable, reasonable regulations that comply with the demand for "state-of-the-art" and "legally conformant drug product production".

Summary

Sterile products must be free from any organisms that can survive in the product, and the levels of particles and endotoxins must remain within the prescribed limits. Sterility is achieved either through steam sterilisation in the final container (preferred), sterile filtration, or aseptic environmental conditions.

Permissible limits for particles and microorganisms for production areas in grades A-D are specified in Annex 1 of the EU-GMP-Guideline. Each individual operation is assigned to a cleanliness grade. The main source of deviations and values that exceed the limits is machine faults.



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