Application Note – Cleanroom N.05

The evolution of the Cleanroom

The Cleanroom is relatively modern development. It was the need for a clean environment  for industrial manufacturing during the 1950s that led to the Clean Room as we know now.

Font: Lab Manager

Application Note – Cleanroom N.04

Investigation about microbiological environmental monitoring excursions

The first step for investigating environmental monitoring excursion is to ensure that the excursion is not due to operator error. Many times a lot of energy is expended in investigating excursions that are not due to environment being out of control but due to analyst or laboratory errors. It is therefore important to identify true and false excursions.

The main points to investigate could be:

• Verify it is not an error

• Validate the microbial identification

• Separate excursions from Out of Specifications

• Developing and action plan when an excursion occurs:

(a) Are the counts correct?

(b) Is the identification correct?

(c) Is it an objectionable?

• Planning and investigation: Information to be collected

(d) Estimated causes

(e) Probable causes

(f) Root causes

• What are the corrective actions and how effective are they.

Application Note – Cleanroom N.03

Education of staff in Cleanroom

We report the Chapter “Personnel” on pages 7 and 8 of the Annex I of the Eudralex document “The rules governing medicinal products in the European Union” 25 November 2008 (rev.)

Personnel

36. Only the minimum number of personnel required should be present in clean areas; this is particularly important during aseptic processing. Inspections and controls should be conducted outside the clean areas as far as possible.

37. All personnel (including those concerned with cleaning and maintenance) employed in such area should receive regular training in disciplines relevant to the correct manufacture of sterile products. This training should include reference to hygiene and to the basic elements of microbiology. When outside staff who have not received such training (e.g.: building or maintenance contractors) need to be brought in, particular care should be taken over their instruction and supervision.

38. Staff who have been engaged in the processing of animal tissue material or of cultures of microorganisms other than those used in the current manufacturing process should not enter sterile products areas unless rigorous and clearly defined entry procedures have been followed.

39. High standards of personnel  hygiene and cleanliness are essential. Personnel involved in the manufacture of sterile preparation should be instructed to report any condition which may cause the shedding of abnormal numbers or types of contaminants; periodic health checks for such conditions are desirable. Actions to be taken about personnel who could be introducing undue microbiological hazards should be decided by a designated competent person.

40. Wristwatches, make-up, and jewellery should not be worn in clean areas.

41. Changing and washing should follow a written procedure designed to minimize contamination of clean area clothing or carry-through of contaminants to the clean areas.

42. The clothing and its quality should be appropriate for the process and the grade of the working area. It should be worn in such a way as to protect the product from contamination.

43. The description of clothing required for each grade is given below:

Grade D

Hair and, when relevant, beard should be covered. A general protective suit and appropriate shoes or overshoes should be worn. Appropriate measures should be taken to avoid any contamination coming from outside the clean area.

Grade C

Hair and where relevant beard and moustache should be covered. A single or two-pieces trouser suit, gathered at the wrist and with high neck and appropriate shoes or overshoes should be worn. They should shed virtually no fibres or particulate mater.

Grade A/B

Headgear should totally enclosed hair and, where relevant, beard and moustache; it should be tucked into the neck of the suit; a face mask should be worn to prevent the shedding of droplets. Appropriate sterilized, non-powdered rubber or plastic gloves and sterilized or disinfected footwear should be worn.

Trouser-legs should be tucked inside the footwear and garment sleeves into the gloves. The protective clothing should shed virtually no fibres or particulate matter and retain particles shed by the body.

44. Outdoor clothing should not be brought into changing rooms leading to Grade B and C rooms. For every worker in Grade A/B area, clean sterile (sterilized or adequately sanitized) protective garments should be provided at each work session. Gloves should be regularly disinfected during operations. Masks and gloves should be changed at least for every working session.

45. Clean area clothing should be cleaned an handled in such a way that it does not gather additional contaminants which can later be shed. These operations should follow written procedures. Separate laundry facilities for such clothing are desirable. Inappropriate treatment of clothing will damage fibres and may increase the risk of shedding of particles.

Application Note – Cleanroom N.02

“Physical  laws” that  regulate the diffusion and deposition of particles in the air

Brownian motion

As the particles migrate through a body of air, random impacts from individual molecules will cause them to veer from course.

Drag coefficient

It is the ratio of the force of gravity to the inertial force on a particle in fluid. It indicates how a particle will resist any force that could cause a change in the particle velocity. Smaller particles have smaller drag coefficients due to their lesser masses.

Deposition velocity

It is also called “settling velocity” and it is the ratio of particle flux (distance per unit time for sedimentation to occur) relative to the ambient particle concentration.

Diffusion forces

This force on a particle varies inversely with the particle’s radius. Therefore smaller particles are more prone to interaction due to diffusion.

Electrostatic forces

It varies with the particle’s electrical charge and the strength of the electrical field in which the particle is located. Electrostatic charge can develop as a particle slips through the air stream.

Gravitational forces

It varies with particle mass and the difference between particle and air density. The larger the particles, the greater the interaction.

Relaxation time

It is the time for a particle initially in equilibrium with a moving fluid to match a change in fluid velocity. Large particles have a long relaxation time.

Stopping distance

A related term of “relaxation time” is “stopping distance” which is defined as the distance for a particle initially moving with a gas stream to come to a stop when the gas flow is halted, as by an obstacle.

Stokes number

It is the ratio of a particle’s ratio to the dimension of an obstacle in fluid flow. This is an important factor in determining when a particle in motion will be collected by any obstacle or will pass around it. An obstacle could be a filter fiber or the sample inlet.

Temperature gradient

The temperature gradient is another factor to be considered in the movement of the small particles.

Viscous forces

The fluid dynamic force from a moving fluid stream or the viscous nature of an air stream will “pull” particles along that flow path. In a unidirectional laminar flow, other forces act upon the larger particles encouraging settling and deposition; smaller particles remain buoyant on a laminar flow. In a turbulent flow stream, the larger particles are re-entrained back into the air flow and the smaller particles are more prone to additional forces acting upon them.

•  Comments

Complexity of particles behaviour

As you may understand from this multitude of physical factors, the behavior  of the particle diffusion in a confined space is quite complex.  The phenomenon is more complicated and difficult to understand when viable particles like micro-organisms, that are not following the “math rules”, are involved.

Importance of the capture of “viable micro-organisms” and not “dead particles”

In this contest, the “capture capacity of micro-organisms” for a microbiological air sampler should be evaluated  in function of its capacity of capturing and guarantee the multiplication of viable germs and not “dead particles”.

Air sampling with vacuum

Considering the microbiological air samplers, if the vacuum is used to transfer the air from the confined space (e.g.: Clean Room or isolator) to an external  central control unit – and the consequent need to use long tubing , the particle behavior is posing doubts on the true analytical value of the system. The official documents 2009 EC GMP Annex 1 point out how important and difficult is the evaluation of the correct length, bending, diameter  to transfer air into tubings from a closed environment to a counter.

Application Note – Cleanroom N.01

Out of Specification (OOS) in Cleanroom

“Investigation for “OOS” results

If there are significative variation in the trend, the following investigations are necessary:

• (a) Identification of isolated environmental micro-organisms
• (b) Revision of the trend during the last 6-12 months in the specific area
• (c) Particle monitoring
• (d) Monitoring and Servicing of the air conditioning or HVAC System
• (e) Sampling improvement
• (f) Revision of the sterilization processes
• (g) Revision of the cleaning and disinfection processes
• (h) Revision of staff training about cleaning, disinfection, sterilisation

Corrective actions in case of “OOS”

• (i) Environmental Sampling procedure revision
• (l) Re-environmental monitoring of the considered area
• (m) Use of other specific microbiological media
• (n) Revision of production procedures
• (o) Revision of cleaning and disinfection procedures
• (p) Challenge of isolates vs products
• (q) Challenge of isolates vs disinfectants
• (r) More appropriate staff training
• (s) Further controls on products