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What is a clean room?

An area with defined environmental control of particulate and microbial contamination
It is constructed and used in a way as to reduce the introduction, generation and retention of contaminants within the area
It's important to remember that a clean room is a controlled area.


In clean rooms, things can contaminate, what 3 things are we trying to minimise the risk of contamination by?

Microbials (including pyrogens)


Particulates are non living, non viable organisms, why is it important to remove them from clean rooms?

Need to remove particulates as non viable articles will form a base for viable organisms to then grown on.


What guide has been published By the MHRA that sets standards of clean rooms?

Orange guide
Annex1, manufacture of sterile products
Standards of clean rooms set out in here


Three main standards/ principles to focus on with clean rooms?

Air quality; air needs to be as pure as possible
Layout of room: need to make sure eg the work station is not right next to the door
Access to room: want to minimise particle contamination when people walk in and out, need to minimise number of people walking in and out when a procedure is taking place.


How do you determine active and passive microbial counts in clean rooms?

Active: number per measured volume
Passive: number during whole session


What are pressure differentials all about in clean rooms?

The pressure of the air in the clean room means that when people walk out of the clean room, dirty air will follow out of the room, the pressure lets dirty air out but doesn't let any air in.


What is grade A activity of sterile products?

Aspeptic preparation of the product and filling into final containers


What is grade A activity of non sterile products?

Filling of products at particular risk of contamination into their final container.


What is grade C and D activity of sterile products?

C is preparation of solutions to be filtered.
D is The storage of ingredients and components.

Remember grade D is activity of a low critical level, ie not as much care has to be taken to ensure sterility, whereas grade A is the opposite!


What is grade C and D activity of non sterile products?

Grade C : preparing solutions at risk of contamination & filling of all products
Grade D: preparation of solutions and components for filling.


There are certain limits for particulate contamination in grade A-D environments, grade D allowing the largest number to be present and grade A allowing the smallest number.
For microbial contamination, what kind of organisms are there limits for?

For bacterial contamination only.
Not viral
Assesses colony forming forming units on air plate, settle plate, contact place and glove plate.


How is air filtered in clean rooms? Name of filter?

Via a HEPA filter
This maintains a positive pressure
And removes 99.997% of particles


What is the pressure differential between rooms of different grades?

10-15 Pascals between rooms of different grades (grades A to D)
Lowest pressure will be grade D room


What's the warning system in clean rooms for?

Air supply, this system checks air flow to make sure air is clean.


What should surfaces in a clean room be like? What other things in the clean room should be considered?

Smooth. No pores. No broken / cracked surfaces.
Minimise amount of ledges, ceiling should contain no seal ie just be smooth all along.
Minimal number of drawers and cupboards
No wood!
Stainless steel trolleys
No sinks allowed in grade A and B areas


Things to consider with equipment in clean rooms?

Should sterilise after reassembly of equipment
Should be easily removable from the clean room so they can be cleaned
Equipment should use a clean water supply that is circulated above 80 degrees.


What does HEPA, as in HEPA filter, stand for?

High efficiency particulate air flow


What are laminar flow cabinets?

They're like a mini clean room with a HEPA filter and air supply
Can get vertical or horizontal laminar flow cabinets!


What are HORIZONTAL laminar flow cabinets for? Which direction does the air flow?

Air flows TOWARDS your operator (safest way, don't want air flowing in your cabinet and brushing past your operator)
They're used for TPN (total parenteral nutrition)
Used for any non harmful substance ( as air flows towards operator)
Used in grade B rooms
Involve a pre-filter to protect your HEPA filter.


What do vertical laminar flow cabinets involve?

Used for HAZARDROUS substances!
Operator must keep hands in cabinet at all times
Curtain of air produced to protect the operator
Siting of these cabinets is important; should not be near the door as this disrupts air flow


Where should vertical laminar air flow cabinets be sited?

In a Seperate, dedicated room
Away from any drafts or ventilation from air conditioning
Away from doors
Away from pedestrian corridors
Away from other cabinets
Present in grade B clean rooms


What are isolators/ barrier technology?

Totally enclosed work spaces
But they're not ABSOLUTE barriers, as product still has to go into them to reach the barrier.
Still some contamination can get let in
Found in grade A environments


Some advantages of isolators?

Cheaper to run
Disturbances in air flow not as critical as with laminar flow cabinets
Better for operator protection due to negative pressure


What are the differences between positive and negative pressure?

Leaks of pressured air less critical in positive pressure
Negative pressure is required to handle hazardous substances (such as cytotoxics, radiopharmaceuticals, certain antibiotics)


Difference between rigid vs flexible film?

Flexible film isolators only suitable for positive pressure
Also problems with cleaning and care of PVC film with flexible film
But flexible will offer less limited workspace.

The rigid design is more robust (sturdy in construct)


Unidirectional (laminar) vs turbulent air flow?

Laminar will minimise air contamination
Air is changed round continuously: high air change rate

Turbulent flow; results in some 'dead spots' ie where no flow is present.
Requires a smaller air filter


What are gauntlets and are they sterile?

Big thick gloves used for handling hot glassware.
They're NON sterile
They don't get changed as often as normal plastic gloves, and the plastic gloves are sterile, and changed before each session


There is a type of isolator called a transfer isolator
It has interlocking doors
different types will effect these isolators siting

When cleaning an isolator it must not effect the integrity of the isolator (ie must not effect it's actions)
You disinfect using liquids
Sterilise using gas such as H2O2


Clothing of operators of these processes will be appropriate to the process they're carrying out and for the grade of the working area.



Grade C and D environments both require the operator to have:
Hair covered
Protective suit/ overcoat on
Gloves (grade C sterile, grade D can be non sterile)

Grade B environment requires the operator to have:
Sterile headgear
Sterile face mask
Sterile boiler suit
Sterile booties
Sterile gloves

Everything sterile!!!


Remember cleaning of clean rooms will require rotation of cleaning material to stop microbes becoming resistant.
Cleaning requires trained staff.
Sterile disinfectants and detergents required in grade A/ B
Cleaning is a written and validated procedure



What do we need to MONITOR in the clean room?

Air flow
Particulate monitoring by air sampling
Microbiological monitoring by passive air sampling, also active air, surface and operator sampling


How to / what to validate in clean rooms?

Transfer (from people and goods)
Container washing
Aseptic processes by manipulation of tryptamine soya broth


The British and European pharmacopeia test for microbial contamination

Raw materials:
Tested for absence of certain organisms (BP)
Limit for total viable count (EP & USP)

Finished Product: sterility test, endotoxins, pyrogens, limit for total viable count


When assessing for contamination, what is the limit for total viable count (Cfu/g or ml) of oral and topical preps, for both bacteria and fungi?

Oral preparations: must be less than 10^3 to 10^4 bacteria, and must be less than 10^2 fungi.

Topical preparations: must be less than 10^2 bacteria and less than 10^2 fungi.


One type of sterility test is filtration. What do you need to use/ do with filtration? Pore size? What products is it used for?

Membrane filtration:
Make sure you choose an appropriate filter membrane, pore size should be no greater than 0.45 microns, diameter usually 50 mm.
Product then filtered through membrane
Wash the filter, using isotonic salts solution
Place filter in a suitable media and incubate.

You use filtration for products/ drug that have antimicrobial activity.


One type of sterility test is direct inoculation. Tell me abit about this?

Direct inoculation:
A Sample of your product is transferred to media and is incubated (for ~14 days)
Neutralise anti microbials
May need to plate out if your product is turbid (cloudy)
Used for: surgical sutures (stitches), suspensions, creams, ointments.


What kind of media is used for sterility tests?

Must be capable of supporting growth of likely contaminants
Must also promote recovery of damaged survivors

Examples of medium used:
Fluid thioglycollate medium
(supports growth of anaerobic organisms ~30 degrees)
Soya bean casein digest medium
(supports growth of aerobic bacteria ~30 degrees and fungi ~20 degrees)


What types of control are there for sterility tests?

Test media for sterility: negative control, checking the medium itself is sterile before placing any product on it.
Test media for nutritive support (positive control, as you're checking that your medium will support bacterial growth)

Test organisms used; staph aureus, candida albicans, CI sporogenes


What do sterility tests tell us?

That a sample is free from all viable bacteria and fungi
Indicates the rest of the batch is sterile
Once tested that product must be disposed of as even testing can introduce some bacteria, that's why we don't test the whole batch!


What statistics must be calculated when sterility tests have been done?

In one batch of product:
P= proportion contaminated
Q= proportion not contaminated
P+Q= 1
If you sample n containers:
Probability of all uncontaminated= Q^n


When to reject the batch after sterility test has been done?

Product fails sterility test if at least one container shows signs of microbiological growth.
Probability of rejecting a batch= 1-q^n
q= proportion not contaminated

Eg if a sample of 20 containers (n) are takes from a batch of which 1% of containers are contaminated (p)
n=20, p=0.01, q=0.99(1-0.01)
Probability of rejecting batch= 1-q^n; 1-(0.99^20)=0.18
Therefore you can accept the batch 1-0.18=0.82= 82% of the time!
remember how to do this: potential multiple choice calc!


Sterility tests can only test for bacteria, yeasts and moulds

Sterility tests are destructive: once a products been tested it must be disposed of, can't be used.


What are the BP guidelines for failing a sterility test?

Must be able to prove that the growth is due to the testing procedure.
You can retest using the same sample size.
Any growth on the retest= batch will fail.


Why do we need to neutralise antimicrobial agents for sterility tests?

Neutralising them will inactivate them.
We want to inactivate them as these agents act to destroy bacteria. This means we won't know if the product was contaminated when it comes to the sterility test.


How do we test for endotoxins?

Endotoxins: LAL test
LAL is an aqueous extract of blood cells (amoebocyte lysate) from a horseshoe crab
Endotoxins are from gram negative bacteria (LPS)
There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic


How do we test for pyrogens?

Rabbit pyrogen test
Inject product into rabbit and see if they get fever or not


Total viable count is important for testing for microbial contamination.

To do a total viable count you need to incubate with an appropriate broth:
Salmonella, e coli, pseudomonas, staph aureus, clostridia, candida
Identity the smallest quantity that gives a positive result (growth), and the largest quantity that gives a negative result (no growth).


If one batch fails a sterility test...

Batch recalls occur!
Drug alerts forms filled out
Products recalled from all hospitals and clinics back to department of manufacture

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