Tuesday, July 22, 2014

Methods of Analysis: 3. Biological methods: 3.1 Microbiological assay of antibiotics

Methods of Analysis: 3. Biological methods: 3.1 Microbiological assay of antibiotics

The potency (activity) of an antibiotic product is expressed as the ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Biological Standard, an International Biological Reference Preparation, or an International Chemical Reference Substance of that antibiotic that produces similar inhibition. Properly validated secondary reference materials may also be utilized in the assay. To carry out the assay a comparison is made between the inhibition of the growth of microorganisms produced by known concentrations of the reference material and that produced by measured dilutions of the test substance. This response can be measured by the diffusion method, as described below, or in a liquid medium by the turbidimetric method.
An International Unit is the specific activity contained in such an amount (weight) of the relevant International Biological Standard or International Biological Reference Preparation as the WHO Expert Committee on Biological Standardization may from time to indicate as the quantity exactly equivalent to the unit accepted for international use. In some cases, when owing to the properties of the material, difficulties are experienced in weighing with adequate accuracy small amounts of the relevant International Biological Standard or International Biological Reference Preparation, International Units are defined on the basis of the total contents of the material in an ampoule or a vial. A defined number of International Units is then assigned to the total contents of an ampoule or a vial; this material has to be carefully removed with an appropriate solvent and the final volume of the solution has to be accurately adjusted.
International Chemical Reference Substances do not have defined units of biological activity. The potency of those products for which biological assays are required are in such cases expressed in terms of an equivalent weight of the pure substance.
Recommended procedure
Use Petri dishes or rectangular trays filled to a depth of 3-4 mm, unless otherwise indicated in the monograph, with a culture medium that has previously been inoculated with a suitable inoculum of a susceptible test organism prepared as described below. The nutrient agar may be composed of two separate layers of which only the upper one may be inoculated. The concentration of the inoculum should be so selected that the sharpest zones of inhibition and suitable dose response at different concentrations of the standard are obtained. When using the inoculum prepared as described below, an inoculated medium containing 1 ml of inoculum per 100 ml of the culture medium is usually suitable. When the inoculum consists of a suspension of vegetative organisms, the temperature of the molten agar medium must not exceed 48-50 °C at the time of inoculation. The dishes or trays should be specially selected with flat bottoms. During the filling they should be placed on a flat, horizontal surface so as to ensure that the layer of the medium will be of a uniform thickness. With some test organisms, the procedure may be improved if the inoculated plates are allowed to dry for 30 minutes at room temperature before use, or refrigerated at 4 °C for several hours.
For the application of the test solution, small sterile cylinders of uniform size, approximately 10 mm high and having an internal diameter of approximately 5 mm, made of suitable material such as glass, porcelain, or stainless steel, are placed on the surface of the inoculated medium. Instead of cylinders, holes 8-10 mm in diameter may be bored in the medium with a previously sterilized borer. Other methods of application of the test solution may also be used. The arrangement on the plate should be such that overlapping of zones is avoided.
Solutions of the reference material of known concentration and corresponding dilutions of the test substance, presumed to be of approximately the same concentration, are prepared in a sterile buffer of a suitable pH value. To assess the validity of the assay at least 3 different doses of the reference material should be used together with an equal number of doses of the test substance having the same presumed activity as the solutions of the reference material. The dose levels used should be in geometric progression, for example, by preparing a series of dilutions in the ratio 2:1. Once the relationship between the logarithm of concentration of the antibiotic and the diameter of the zone of inhibition has been shown to be approximately rectilinear for the system used, routine assays may be carried out using only 2 concentrations of the reference material and 2 dilutions of the test substance. Where a monograph gives directions for the initial preparation of a solution of the substance, this solution is then diluted as necessary with the appropriate sterile buffer.
The solutions of the reference material and the test substance are preferably arranged in the form of a Latin square when rectangular trays are employed. When Petri dishes are used, the solutions are arranged on each dish so that the solutions of the reference material and those of the test substance alternate around the dish and are placed in such a manner that the highest concentrations of the reference material and of the test substance are not adjacent. The solutions are placed in the cylinders or holes by means of a pipette that delivers a uniform volume of liquid. When the holes are used the delivered volume should be sufficient to fill them almost completely.
The plates are incubated at a suitable temperature, the selected temperature being controlled at ±0.5 °C, for approximately 16 hours, and the diameters or areas of the zones of inhibition produced by the varied concentrations of the standard and of the test substance are measured accurately, preferably to the nearest 0.1 mm of the actual zone size, by using a suitable measuring device. From the results, the potency of the tested substance is calculated. Suitable publications on the statistics of bioassays are listed below.
Conditions for the assay of individual antibiotics and suitable test organisms are given in the monographs. The choice of an appropriate strain of test organism may be critical for the assay. For easy reference, examples of suitable test organisms for a number of antibiotics are shown in Table 4. The designations of the test strains are as follows:
NCTC - National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, England
NCYC - National Collection of Yeast Cultures, AFRC Food Research Institute, Colney Lane, Norwich NR4 7UA, England
ATCC - American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA
Other suitable strains of test organisms can be used. Additional information regarding sources of suitable strains may be obtained from Quality Assurance and Safety: Biologicals, World Health Organization, 1211 Geneva 27, Switzerland.
Precision of the assay
In order to determine whether or not a substance satisfies the requirements for potency specified in the monograph, the assay should, if necessary, be repeated until the required precision has been attained. This precision is such that the fiducial limits (P = 0.95) of the mean estimated potency, expressed as a percentage of the mean estimated potency, should be within the required range given in the individual monographs.
Calculation of results
The following publications contain suitable methods that may be used to carry out the statistical evaluation of the microbiological assay of antibiotics:
1. Bliss CI.: Statistics of bioassay. New York, Academic Press, 1952.
2. Bliss CI.: Statistics in biology. vol. I, New York, McGraw Hill, 1967.
3. Bliss CI.: Statistics in biology. vol. II, New York, McGraw Hill, 1970.
4. Finney DJ.: Statistical methods in biological assays. London, Griffin, 1964.
5. Hewitt W.: Microbiological assay. New York, Academic Press, 1977.
6. Philippe J.: Les methodes statistiques en pharmacie et en chimie. Paris, Masson, 1967.
The methods of carrying out the statistical evaluation of the microbiological assay of antibiotics are also described in many national and regional pharmacopoeias.
TABLE 4. TEST ORGANISMS AND CONDITIONS OF ASSAY OF INDIVIDUAL ANTIBIOTICS
Antibiotic
Test organism
Culture
medium;
final pH
Phosphate
buffer,
sterile,
pHa, TS
Concentration
(weight or
International
Units per ml)b
Incubation temperature (in °C)
           
Bacitracin Micrococcus luteus
Cm1;
7.0
1-4 IU
35-37

NCTC 7743;
7.0-7.1




ATCC 10240




Micrococcus luteus
Cm1;
6.0
1-4 IU
30-31

NCTC 7743;
6.5-6.6




ATCC 10240



           
Cefalexin Staphylococcus aureus
Cm1;
6.0
10-40 μg
32-35

NCTC 6571;
6.5-6.6




ATCC 9144




Staphylococcus aureus
Cm1;
6.0
10-40 μg
32-35

ATCC 6538-P
6.5-6.6



           
Cefalotin Staphylococcus aureus
Cm1;
6.0
0.5-2 IU
32-35

NCTC 6571;
6.5-6.6




ATCC 9144




Staphylococcus aureus
Cm1;
6.0
0.5-2 IU
32-35

ATCC 6538-P
6.5-6.6



           
Chlortetracycline Bacillus pumilus
Cm1;
4.5
2-20 IU
37-39

NCTC 8241;
6.5-6.6




ATCC 14884




Bacillus cereus
Cm1;
4.5
0.05-0.2 IU
30-33

ATCC 11778
5.9-6.0



           
Cloxacillin Bacillus subtilis
Cm1;
7.0
5-20 μg
37-39

NCTC 8236;
6.5-6.6




ATCC 11774




Staphylococcus aureus
Cm1;
6.0
2-8 μg
32-35

ATCC 6538-P
6.5-6.6



           
Dicloxacillin Staphylococcus aureus




NCTC 6571;
Cm1;
6.0
2.5-10 μg
37-39

ATCC 9144
6.6




Staphylococcus aureus
Cm1;
6.0
2-8 μg
32-35

ATCC 6538-P
6.5-6.6



           
Erythromycin Bacillus pumilus




NCTC 8241;
Cm1;
8.0
5-25 IU
37-39

ATCC 14884
8.0-8.1




Micrococcus luteus
Cm1;
8.0
0.5-1.5 IU
35-37

ATCC 9341
8.0-8.1



           
Kanamycin Bacillus subtilis
Cm1;
8.0
5-20 IU
30-37

ATCC 6633
7.9

Staphylococcus aureus
Cm1;
8.0
10 IU
35-39

ATCC 6538P
7.9
           
Neomycin Bacillus pumilus
Cm1;
8.0
2-14 IU
37-39

NCTC 8241;
8.0-8.1




ATCC 14884




Staphylococcus aureus
Cm1;
8.0
2-20 IU
35-37

ATCC 29737
7.8-8.0




Staphylococcus epidermidis
Cm1;
8.0
0.5-2 IU
35-37

ATCC 12228
8.0-8.1



           
Novobiocin Bacillus subtilis
Cm1;
6.0
1-5 IU
30-33

NCTC 10315;
6.5-6.6




Micrococcus luteus
Cm1;
6.0
10-50 IU
30-35

ATCC 9341
6.5-6.6



           
Nystatin Saccharomyces cerevisiae
Cm3;
-c
25-300 IU
35-37

NCYC 87;
6.0-6.2




ATCC 9763



           
Oxacillin Bacillus subtilis
Cm1;
7.0
2.5-10 μg
37-39

NCTC 8236;
6.5-6.6




ATCC 11774




Staphylococcus aureus
Cm1;
6.0
2-8 μg
32-35

ATCC 6538-P
6.5-6.6



           
Oxytetracycline Bacillus pumilus
Cm1;
4.5
2-20 IU
37-39

NCTC 8241;
6.5-6.6




ATCC 14884




Bacillus cereus
Cm1;
4.5
0.5-2 IU
30-33

ATCC 11778
5.9-6.0



           
Polymyxin B Bordetella bronchiseptica
Cm2;
6.0, TS3
20-100 IU
35-37

NCTC 8344;
7.2-7.3




ATCC 4617




Bordetella bronchiseptica
Cm2;
7.2
50-200 IU
35-37

NCTC 8344;
7.2-7.3




ATCC 4617




Escherichia coli
Cm1;
7.2
5-100 IU
35-37

ATCC 10536
6.5-6.6



           
Streptomycin Bacillus subtilis
Cm1;
8.0
5-20 IU
37-39

NCTC 8236;
7.9-8.0




ATCC 11774




Bacillus subtilis
Cm1;
8.0
3-15 IU
35-37

ATCC 6633
8.0-8.1



           
Tetracycline Bacillus pumilus
Cm1;
4.5
2-20 IU
37-39

NCTC 8241;
6.5-6.6




ATCC 14884




Bacillus cereus
Cm1;
4.5
0.5-2 IU
30-33

ATCC 11778
5.9-6.0



a Phosphate buffers, sterile, of suitable pH. Buffers designated as TS, TS1, or TS2 may be used.
b Range within which suitable concentrations may be found.
c The preparation of the solution of the reference material and of the corresponding dilution of the test substance is done as described in the monograph with the aid of dimethylformamide R and phosphate buffer, sterile, pH 6.0 TS3.
Culture media
The formulae for the culture media (Cm) referred to in Table 4 are described in Reagents, test solutions and volumetric solutions. In each instance the final pH is adjusted to that stated in the table.
Preparation of inoculum
Bacillus cereus; Bacillus pumilus; Bacillus subtilis. The test organism is grown for 7 days at a temperature of 37-39°C on the surface of culture medium Cm1 (pH 6.5-6.6 after sterilization) to which has been added 1 μg of manganese sulfate R per ml. Using sterile water, the growth, which consists mainly of spores, is washed off, heated for 30 minutes at 70 °C, and suitably diluted - for example, to give between 107 and 108 spores per ml. The spore suspension may be stored for long periods at a temperature not exceeding 4 °C.
Bordetella bronchiseptica. The test organism is grown overnight on culture medium Cm2 (pH 6.5-6.6 after sterilization) at a temperature of 35-37 °C. A suspension is prepared by washing off the growth and diluting with sterile water or saline TS to a suitable opacity, for example, such that a 1-cm layer transmits 50% of the incident light when examined at 650 nm. The suspension may be stored for up to 2 weeks at a temperature not exceeding 4 °C.
A freshly prepared inoculum may be replaced by a suitable suspension of the inoculum in a suitable vehicle, such as sterile peptone (1 g/l) TS2 that has been stored frozen at -70 °C and subsequently thawed.
Micrococcus luteus. The test organism is grown overnight on culture medium Cm1 (pH 6.5-6.6 after sterilization) at a temperature of 35-37 °C. A suspension is prepared by washing off the growth and diluting with saline TS to a suitable opacity, for example, such that a 1-cm layer of a 1 in 50 dilution transmits 80% of the incident light when examined at 650 nm. The suspension may be stored for up to 2 weeks at a temperature not exceeding 4 °C.
A freshly prepared inoculum may be replaced by a suitable suspension of the inoculum in a suitable vehicle, such as sterile peptone (1 g/l) TS2 that has been stored frozen at -70 °C and subsequently thawed.
Saccharomyces cerevisiae. The test organism is grown overnight on culture medium Cm3 (pH 6.0-6.2 after sterilization) at a temperature of 35-37 °C. A suspension is prepared by washing off the growth with saline TS and diluting to a suitable opacity, for example, such that a 1-cm layer transmits 50% of the incident light when examined at 650 nm. The suspension may be stored for up to 2 weeks at a temperature not exceeding 4 °C.
A freshly prepared inoculum may be replaced by a suitable suspension of the inoculum in a suitable vehicle, such as sterile peptone (1 g/l) TS2 that has been stored frozen at -70 °C and subsequently thawed.
Staphylococcus aureus. The test organism is grown overnight on culture medium Cm1 (pH 6.5-6.6 after sterilization) at a temperature of 35-37 °C. A suspension is prepared by washing off the growth with saline TS and diluting to a suitable opacity, for example, such that a 1-cm layer transmits 50% of the incident light when examined at 650 nm.
A freshly prepared inoculum may be replaced by a suitable suspension of the inoculum in a suitable vehicle, such as sterile peptone (5 g/l) TS that has been stored frozen at -70 °C and subsequently thawed.

EUROPEAN PHARMACOPOEIA 5.0


EUROPEAN PHARMACOPOEIA 5.0
2.9.1. Disintegration of tablets and capsules
2.9. PHARMACEUTICAL
TECHNICAL PROCEDURES
01/2005:20901
2.9.1. DISINTEGRATION OF TABLETS
AND CAPSULES
The disintegration test determine
s whether tablets or
capsules disintegrate within the prescrib
ed time when placed
in a liquid medium in the experimental conditions pr
escribed
below.
Disintegration is considered to be achieved when:
a) no residue remains on the screen, or
b) if there is a residue, it consists of a soft mass having no
palpably firm, unmoistened core, or
c) only fragments of coating (tablets) or only fragments of
shell (capsules) remain on the screen; if a disc has been
used (capsules), fragments of shell may adhere to the lower
surface of the disc.
Use apparatus A for tablets and capsules that are not
greater than 18 mm long. For larger tablets or capsules use
apparatus B.
TEST A - TABLETS AND CAPSULES OF NORMAL SIZE
Apparatus
. The main part of the apparatus (Figure 2.9.1.-1)
is a rigid basket-rack assembly supporting 6 cylindrical
transparenttubes77.5±2.5mmlong,21.5mmininternal
diameter, and with a wall thickness of about 2 mm. Each
tube is provided with a cylindrical disc 20.7 ± 0.15 mm in
diameter and 9.5 ± 0.15 mm thick, made of transparent
plastic with a relative density of 1.18 to 1.20 or weighing
3.0 ± 0.2 g. Each disc is pierced by 5 holes 2 mm in diameter,
1 in the centre and the other 4 spaced equally on a circle
of radius 6 mm from the centre of the disc. On the lateral
surface of the disc, 4 equally spaced grooves are cut in such
a way that at the upper surface of the disc they are 9.5 mm
wide and 2.55 mm deep and at the lower surface 1.6 mm
square. The tubes are held vertically by 2 separate and
superimposed rigid plastic plates 90 mm in diameter and
6 mm thick with 6 holes. The holes are equidistant from the
centre of the plate and equally spaced. Attached to the under
sideofthelowerplateisapieceofwovengauzemadefrom
stainless steel wire 0.635 mm in diameter and having mesh
apertures of 2.00 mm. The plates are held rigidly in position
and 77.5 mm apart by vertical metal rods at the periphery,
a metal rod is also fixed to the centre of the upper plate to
enable the assembly to be attached to a mechanical device
Figure 2.9.1.-1. –
Apparatus A
Dimensions in millimetres
GeneralNotices(1)applytoallmonographsandothertexts
225
2.9.1. Disintegration of tablets and capsules
EUROPEAN PHARMACOPOEIA 5.0
capableofraisingandloweringitsmoothlyataconstant
frequency between 29 and 32 cycles per minute, through a
distance of 50 mm to 60 mm.
The assembly is suspended in the specified liquid in a
suitable vessel, preferably a 1 litre beaker. The volume of
the liquid is such that when the assembly is in the highest
position the wire mesh is at least 15 mm below the surface
of the liquid, and when the assembly is in the lowest position
thewiremeshisatleast25mmabovethebottomofthe
beaker and the upper open ends of the tubes remain above
thesurfaceoftheliquid.Asuitabledevicemaintainsthe
temperature of the liquid at 35-39 °C.
The design of the basket-rack assembly may be varied
providedthespecificationsforthetubesandwiremeshare
maintained.
Method
. In each of the 6 tubes, place one tablet or capsule
and, if prescribed, add a disc; suspend the assembly in
the beaker containing the specified liquid. Operate the
apparatus for the prescribed p
eriod, withdraw the assembly
and examine the state of the tablets or capsules. To pass the
test, all the tablets or capsules must have disintegrated.
TEST B – LARGE TABLETS AND LARGE CAPSULES
Apparatus
. The main part of the apparatus (Figure 2.9.1.-2)
is a rigid basket-rack assembly supporting 3 cylindrical
transparent tubes 77.5 ± 2.5 mm long, 33.0 mm ± 0.5 mm in
internal diameter, and with a wall thickness of 2.5 ± 0.5 mm.
Each tube is provided with a cylindrical disc 31.4 ± 0.13 mm
indiameterand15.3±0.15mmthick,madeoftransparent
plastic with a relative density of 1.18 to 1.20 or weighing
13.0 ± 0.2 g. Each disc is pierced by 7 holes, each
3.15±0.1mmindiameter,1inthecentreandtheother
6 spaced equally on a circle of radius 4.2 mm from the centre
of the disc. The tubes are held vertically by 2 separate and
superimposed rigid plastic plates 97 mm in diameter and
9 mm thick, with 3 holes. The holes are equidistant from
the centre of the plate and equally spaced. Attached to
theundersideofthelowerplateisapieceofwovengauze
made from stainless steel wire 0.63 ± 0.03 mm in diameter
andhavingmeshaperturesof2.0±0.2mm.Theplatesare
held rigidly in position and 77.5 mm apart by vertical metal
rods at the periphery, a metal rod is also fixed to the centre
of the upper plate to enable the assembly to be attached
Figure 2.9.1.-2. –
Apparatus B
Dimensions in millimetres
226
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
2.9.2. Disintegration of suppositories and pessaries
to a mechanical device capable of raising and lowering it
smoothly at constant frequency between 29 and 32 cycles
per minute, through a distance of 55 ± 2 mm.
The assembly is suspended in the specified liquid medium in
a suitable vessel, preferably a 1 litre beaker. The volume of
the liquid is such that when the assembly is in the highest
position the wire mesh is at least 15 mm below the surface
of the liquid, and when the assembly is in the lowest position
thewiremeshisatleast25mmabovethebottomofthe
beaker and the upper open ends of the tubes remain above
thesurfaceoftheliquid.Asuitabledevicemaintainsthe
temperature of the liquid at 35-39 °C.
The design of the basket-rack assembly may be varied
providedthespecificationsforthetubesandwiremeshare
maintained.
Method
. Test 6 tablets or capsules either by using
2 basket-rack assemblies in parallel or by repeating the
procedure. In each of the 3 tubes, place one tablet or
capsule and, if prescribed, add a disc; suspend the assembly
in the beaker containing the specified liquid. Operate the
apparatus for the prescribed p
eriod, withdraw the assembly
and examine the state of the tablets or capsules. To pass the
test, all 6 of the tablets or capsules must have disintegrated.
01/2005:20902
2.9.2. DISINTEGRATION OF
SUPPOSITORIES AND PESSARIES
The disintegration test determines whether the suppositories
or pessaries soften or disinteg
rate within the prescribed
time when placed in a liquid medium in the experimental
conditions described below.
Disintegration is considered to be achieved when:
a) dissolution is complete,
b) the components of the suppository or pessary have
separated: melted fatty substances collect on the surface of
the liquid, insoluble powders fall to the bottom and soluble
components dissolve, depending on the type of preparation,
the components may be distributed in one or more of these
ways,
c)thereissofteningofthesamplethatmaybeaccompanied
byappreciablechangeofshapewithoutcompleteseparation
of the components, the softening is such that the suppository
or pessary no longer has a solid core offering resistance to
pressure of a glass rod,
d) rupture of the gelatin shell of rectal or vaginal capsules
occurs allowing release of the contents,
e) no residue remains on the perforated disc or if a residue
remains, it consists only of a soft or frothy mass having
no solid core offering resistance to pressure of a glass rod
(vaginal tablets).
Apparatus
. The apparatus (Figure 2.9.2.-1) consists of a
sleeve of glass or suitable transparent plastic, of appropriate
thickness, to the interior of which is attached by means of
threehooksametaldeviceconsistingoftwoperforated
stainless metal discs each containing 39 holes 4 mm in
diameter; the diameter of the discs is similar to that of the
interiorofthesleeve;thediscsareabout30mmapart.
The test is carried out using three such apparatuses each
containing a single sample. Each apparatus is placed in a
beaker with a capacity of at least 4 litres filled with water
maintained at 36-37 °C, unless otherwise prescribed. The
apparatuses may also be placed together in a vessel with a
capacity of at least 12 litres. The beaker is fitted with a slow
stirrer and a device that will hold the cylinders vertically not
less than 90 mm below the surface of the water and allow
them to be inverted without emerging from the water.
Method
. Use three suppositories
or pessaries. Place each
oneonthelowerdiscofadevice,placethelatterinthesleeve
and secure. Invert the apparatuses every 10 min. Examine
the samples after the period prescribed in the monograph.
To pass the test all the samples must have disintegrated.
Figure 2.9.2.-1. —
Apparatus for disintegration of
suppositories and pessaries
Dimensions in millimetres
METHOD OF OPERATION FOR VAGINAL TABLETS
Use the apparatus described above, arranged so as to rest
on the hooks (see Figure 2.9.2.-2). Place it in a beaker of
suitable diameter containing water maintained at 36-37 °C
with the level just below the upper perforated disc. Using
a pipette, adjust the level with water at 36-37 °C until a
uniform film covers the perforations of the disc. Use three
vaginal tablets. Place each
one on the upper plate of an
apparatus and cover the latter with a glass plate to maintain
appropriate conditions of humidity. Examine the state of the
samples after the period prescribed in the monograph. To
pass the test all the samples must have disintegrated.
GeneralNotices(1)applytoallmonographsandothertexts
227

Monday, July 21, 2014

Preparing for cleanroom certification and validation

By Karen von Holtz, CEO of CSI Testing

How important is it to know what you are getting into? A good portion of new clients that call me say they have just finished building their cleanroom and need it tested. It is rare that they understand what kind of testing is needed beyond particle counting. Often, we find that they do not have a cleanroom that will meet the needs of their product, and fixing a cleanroom can be more costly than building it correctly in the first place.
In order to build the best environment for your product, the product must be analyzed during the process development stage. What kinds of contamination controls are necessary to produce the quality product you have designed? It probably should have a controlled environment, but what controls should your controlled environment have? The Institute of Environmental Sciences and Technology (IEST) has two important documents on design/build/validation of cleanrooms. One is the IEST RP-CC-012 Considerations in Cleanroom Design, and the other is ISO 14644-4 Design, Construction and Start-up of Cleanrooms and associated controlled environments. (See chart on page 36.)
From those documents, and those of the FDA, you will understand that, at minimum, you need: an environment that has a controlled access; a clean air supply through HEPA/ULPA filters, which will provide you with a low contamination environment; an air barrier that consists of the correct amount of positive or negative air pressure; and the correct number of air changes per hour. Beyond that, there may be specific conditions for your product, such as a need for a regulated temperature, relative humidity, special lighting, silicone- or latex-free, and so forth. Furthermore, the documents list items that should be considered and/or specified when designing your clean environment.
Once the environmental controls are decided on, the next step is controlling the operating environment. This includes personnel practices (RP-CC-027), operations (ISO 14644-5 and RP-CC-026), housekeeping (RP-CC-018), garments (RP-CC-003), wiping materials (RP-CC-004), gloves (RP-CC-005), and more. These documents provide a wealth of information to guide the company in the setup of operations, writing the SOPs that control the hiring and training of personnel, the cleaning and maintenance of the cleanroom, and the evaluation of cleanroom supplies.
Prior to full-scale operation, the validation master plan should be finalized. Most often, this should contain three sections: strategy, protocols, and evaluation.
Strategy is the design phase that sets the objectives and specifies the results to be achieved. The results are specified as permitted residual limits. These may include class level such as ISO Class 7, amount of room pressure, how many filters, amount of room air changes, lighting, HVAC needs, etc.
Protocols cover the implementation stage and will specify how these goals will be achieved.
Evaluation describes the means and methods-the tests that will be used to analyze the performance of the cleanroom.
According to the ISO 14644-2 standard, anytime a cleanroom is put into operation or changes its intended use, a “validation” must be performed. First-time validation testing takes place over a specified period of time (usually at least one seasonal cycle) to ensure the cleanroom is functioning as intended and specified. When changes are made to the cleanroom, an assessment of how the changes will affect the cleanroom is performed, and a decision is made of how extensive the revalidation will be. Each time a testing is performed, it is called a “certification,” meaning that the cleanroom is “certified” as meeting the stated standards, SOPs, and/or design criteria.
Each RP or standard uses a variety of terms for each phase of the testing performed.
Phase 1 Installation Qualification, As-Built Testing, or Construction Approval. This is testing that proves the environment was correctly installed and that it meets the intended design specifications.
Phase 2-Operational Qualification, At-Rest Testing, or Functional Approval. This testing is performed with all the equipment present and operating, but without personnel present. It is necessary to determine that all parts of the installation operate together to achieve the required conditions. Also, if something happens to the environment, this testing phase provides baseline data to prove that the environment is back to a “normal condition” prior to restarting production.
Phase 3-Performance Qualification, Operational Testing, or Operational Approval. This is a series of tests/certifications that are carried out to determine that the completed installation achieves the required operational performance with the specified process or activity functioning, and with the specified number of personnel present working in the agreed manner. From this testing data, which is accumulated and trended, the alert and action limits will be set.
Given all this information, how does one determine which tests to perform? Basically, the room, the HVAC system, and all the process equipment must be tested to prove they can continually produce the quality product that was intended and that the process equipment is performing within the intended specification. However, this article only addresses the cleanroom and its related systems.
There are documents that detail tests, methodology, and recommended equipment for cleanroom testing. ISO 14644-3 Test Methods recommend test apparatus, test procedures, and acceptance criteria for determining performance testing. And, IEST has RP-CC-006 Testing Cleanrooms, RP-CC-007 Testing UPLA Filters, RP-CC-034 HEPA and ULPA Filter Leak Tests, and RP-CC-013 Test Equipment Calibration or Validation Procedures. Wading through these documents can be a daunting experience. A qualified independent testing and certification company can help outline an appropriate testing program for each cleanroom or support room.
Unless your product has special needs, a primary testing program might include:
HEPA/ULPA filter installation leak test. This test is performed to verify that the filters have been properly installed and are free of leaks.
Airflow volume, velocity, and uniformity tests. This test is performed to check the uniformity of the clean air and to verify that there is enough air supply to provide the proper room pressures.
Airborne particle count test. This testing verifies that the cleanroom, the personnel, and process equipment is performing to the intended clean level.
Room pressurization test. This test is to verify that there is a proper amount of positive/negative room air pressure.
Microbial testing of air, surfaces, and measurement of the efficiency of the cleaning process (ISO 14698, Parts 1, 2, and 3).
The newest recommended practice from IEST is RP-CC-019 Qualifications for Organizations Engaged in the Testing and Certification for Cleanrooms and Clean-Air Devices. This document defines recommended qualifications for organizations engaged in testing and certification by establishing levels of competence to be demonstrated by testing personnel. It is intended to provide guidance to equipment owners when they are assessing the credentials of a prospective testing vendor.
Basically, if you don’t know or understand the way to specify the design aspects of your cleanroom, know how to specify disposables and cleanroom service or testing, or even know the right questions to ask service providers, you may not get the information or quality of information that is needed.
One place to go for reliable help and information is the IEST. The IEST is a not-for-profit association whose members are internationally recognized for their contributions to the environmental sciences in the area of contamination control. The IEST is a global leader in the development of recommended practices and standards; and is recognized as an international resource for information on controlled environments through education and the development of recommended practices and standards. Without an organization such as this, it is almost impossible to track the most updated international standards.