USP <71> Sterility Testing http://www.gibraltarlabsinc.com/sterility-testing.html

By: Minal Patel

Proprietary and Confidential © Gibraltar Laboratories, Inc.

• Definition

• Culture Media and Incubation Temperature

• Growth Promotion and Method Validation /

Method Suitability Test

• Number of Articles and volume to be tested

• Test Method

• Observation and Interpretation of Results

• Essential thing to know regarding Sterility

testing

What is sterility Testing?

• A test conducted to ensure the batch of products is sterile or has been

sterilized, meaning free of viable micro organisms ( Bacteria, yeast or

mold).

• Sterility testing is qualitative test which reveals quality of samples

tested.

• Sterility testing of medical devices is required during the sterilization

validation process as well as for routine quality control.

• The test is applied to substance, preparations, or articles which

according to pharmacopeia are required to be sterile. However, a

satisfactory results only indicated that no contaminating microorganism

has been found in the sample examined under the conditions of the test.

• Sterility testing is a very tedious and artful process that must be

performed by trained and qualified laboratory personnel.

Culture Media and Incubation Conditions

→ Fluid Thioglycollate Medium (FTM) and incubation temperature is

30⁰ to 35⁰C for 14 days.

→ Soybean-Casein Digest Medium – SCDB (previously called as

TSB) and incubation temperature is 20⁰ to 25⁰C for 14 days.

Fluid Thioglycollate Medium (FTM) Ingredients Amount Significance

L-Cystine 0.5 g

Reducing Agent, prevent accumulation of

Peroxides which are lethal to some

organism.

Sodium Chloride 2.5 g Osmotic Balance

Dextrose

Monohydrate/Anhydrous 5.5/5.0 g

Energy Source

Agar 0.75 g

Prevent rapid uptake of Oxygen and helps

to maintain anaerobic environment

Yeast Extract (water-soluble) 5.0 g

Growth Factor

Pancreatic Digest of Casein 15.0 g

Growth Factor

Sodium Thioglycollate 0.5 g Reducing agent, remove Oxygen

or Thioglycolic Acid 0.3 mL

Resazurin Sodium Solution (1

in 1000), freshly prepared 1.0 mL

Redox Indicator, red in oxidized state and

colorless under anaerobic conditions

Purified Water 1000 mL pH – 6.9 to 7.3

Soybean-Casein Digest Broth (SCDB / TSB)

Ingredients Amount Significance

Pancreatic Digest of Casein 17.0 gram Provide Nitrogen, Vitamins

& Minerals.

Papaic Digest of Soybean Meal 3.0 gram Provide Nitrogen, Vitamins

& Minerals.

Sodium Chloride 5.0 gram For Osmotic Balance

Dibasic Potassium Phosphate 2.5 gram Buffering Agent to control

pH

Dextrose Monohydrate / Anhydrous 2.5 / 2.3 gram Energy source to promote

organisms growth

Purified Water 1000 mL pH - 7.1 to 7.5

Diluting and Rinsing Fluids

• Fluid A – 0.1% Peptone water; pH 6.9 to 7.3

• Fluid D – 0.1% Peptone Water with 0.1% Tween 80; pH

6.9 to 7.3

• Fluid K ; pH 6.7 to 7.1

Aerobic bacteria

Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518,

NBRC 13276

Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134

Pseudomonas aeruginosa 1 ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275

Anaerobic bacterium

Clostridium sporogenes 2 ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437,

NBRC 14293

Fungi

Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594

Aspergillus brasiliensis

(Aspergillus Niger)

ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455

1 An alternative microorganism is Kocuria rhizophila (Micrococcus luteus) ATCC 9341. 2 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is

desired, is Bacteroides vulgatus (ATCC 8482).

Table: Strains of the Test Microorganisms Suitable for Use in the

Growth Promotion Test and the Method Suitability Test

Growth Promotion Test and Method Validation

USP <71> Sterility Test contains two qualifying Assays which must be

performed prior to sterility testing. They are “ Growth Promotion Test” and

“Validation Test” ( Bacteriostasis and Fungistasis Test)

Growth Promotion Test:

Test each lot of medium prior to use for sterility testing to ensure that the

medium used support the growth of <100 viable micro-organisms.

Inoculate portions of the media with less than 100 cfu of appropriate organisms.

Inoculate FTM with less than 100 cfu of Pseudomonas aeruginosa,

Staphylococcus aureus and Clostridium sporogenes . Inoculate SCDB / TSB

with less than 100 cfu of Bacillus subtilis, Candida albicans and less than 100

spores of Aspergillus brasiliensis. Incubate for not more than 3 days in case of

bacteria and not more than 5 days in the case of fungi.

The media are suitable if a clearly visible growth of the microorganisms occurs.

Method Validation / Suitability Test /

Bacteriostasis and Fungistasis Test

• The Validation Test is used to determine if the test sample will inhibit the growth of microorganisms in the test media.

• In terms of microbiology, is defined as the inability of a microorganism to grow and proliferate in microbiological media.

• The Validation Test must be performed on each product prior and / or during sterility testing.

• Microbiological growth in the presence of the test samples is compared to controls without test samples. If microbial growth is present in the sample and control, then the test is valid.

• The microorganisms used for suitability test are same as Growth Promotion Test.

Test Methods

1. Membrane Filtration

2. Direct Inoculation

Membrane Filtration

After transferring the content of the container or containers to be tested to the

membrane, add an inoculum of a small number of viable microorganisms (not more

than 100 cfu) to the final portion of sterile diluent used to rinse the filter.

Direct Inoculation

After transferring the contents of the container or containers to be tested (for catgut and

other surgical sutures for veterinary use: strands) to the culture medium, add an

inoculum of a small number of viable microorganisms (not more than 100 cfu) to the

medium.

In both cases use the same microorganisms as those described above under Growth

Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as

a positive control. Incubate all the containers containing medium for not more than 5

days.

Quantity per Container

Minimum Quantity to be Used

(unless otherwise justified and authorized)

Liquids

Less than 1 mL The whole contents of each container

1–40 mL Half the contents of each container, but not less than 1 mL

Greater than 40 mL, and not greater than 100 mL 20 mL

Greater than 100 mL 10% of the contents of the container, but not less than 20 mL

Antibiotic liquids 1 mL

Insoluble preparations, creams, and ointments to be suspended

or emulsified

Use the contents of each container to provide not less than 200 mg

Solids

Less than 50 mg The whole contents of each container

50 mg or more, but less than 300 mg Half the contents of each container, but not less than 50 mg

300 mg–5 g 150 mg

Greater than 5 g 500 mg

Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long)

Surgical dressing/cotton/gauze (in packages) 100 mg per package

Sutures and other individually packaged single-use material The whole device

Other medical devices The whole device, cut into pieces or disassembled

Table : Minimum Quantity to be Used for Each Medium

Number of Items in the Batch*

Minimum Number of Items to be Tested for Each

Medium (unless otherwise justified and authorized)**

Parenteral preparations

Not more than 100 containers 10% or 4 containers, whichever is the greater

More than 100 but not more than 500 containers 10 containers

More than 500 containers 2% or 20 containers, whichever is less

For large-volume parenterals 2% or 10 containers, whichever is less

Antibiotic solids

Pharmacy bulk packages (<5 g) 20 containers

Pharmacy bulk packages ( 5 g) 6 containers

Bulks and blends See Bulk solid products

Ophthalmic and other noninjectable preparations

Not more than 200 containers 5% or 2 containers, whichever is the greater

More than 200 containers 10 containers

If the product is presented in the form of single-dose containers,

apply the scheme shown above for preparations for parenteral

use.

Catgut and other surgical sutures for veterinary use 2% or 5 packages, whichever is the greater,

up to a maximum total of 20 packages

Not more than 100 articles 10% or 4 articles, whichever is greater

More than 100, but not more than 500 articles 10 articles

More than 500 articles 2% or 20 articles, whichever is less

Bulk solid products

Up to 4 containers Each container

More than 4 containers, but not more than 50 containers 20% or 4 containers, whichever is greater

More than 50 containers 2% or 10 containers, whichever is greater * If the batch size is unknown, use the maximum number of items prescribed. ** If the contents of one container are enough to inoculate the two media, this column gives the number of containers

needed for both the media together.

Table : Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch

Things to know Regarding Membrane Filtration

►This test is method of choice for pharmaceutical products.

►Use membrane filters having a nominal pore size not greater than 0.45 µm, in

which the effectiveness to retain microorganisms has been established.

►Cellulose nitrate filters are used for aqueous, oily, and weakly alcoholic

solutions.

►Cellulose acetate filters are used for strongly alcoholic solutions.

Specially adapted filters may be needed for certain products (e.g., for antibiotics).

►Do not exceed a washing cycle of five times 100 mL per filter.

Use the same volume of each medium as in the Method Suitability Test.

►If the product has antimicrobial properties, wash the membrane not less than

three times by filtering through it each time the volume of the chosen sterile

diluent used in the Method Suitability Test.

►Transfer the whole membrane to the culture medium or cut it aseptically into

two equal parts, and transfer one half to each of two suitable media.

Things to know Regarding Direct Inoculation

►This test is method of choice for medical devices because the device is in direct

contact with media through out the incubation period.

►Transfer the quantity of the preparation to be examined directly into the culture

medium so that the volume of the product is not more than 10% of the volume of the

medium, unless otherwise prescribed.

►If the product to be examined has antimicrobial activity, carry out the test after

neutralizing with a suitable neutralizing agent or by dilution in a sufficient quantity of

culture medium.

►For extremely large devices, immerse those portions of the device that come in

contact with the patient in a volume of medium sufficient to achieve complete

immersion of those portions.

►Articles can be immersed intact or disassembled.

Product Membrane Filtration

Method

Direct Inoculation Method

Oil & Oily

Solution

Low Viscosity – Can be

filtered directly without

Dilution

Viscous – Dilute in Diluent

such as IPM and wash with

Fluid K

Add Emulsifying agent to Medium

such as Tween 80

Ointment and

Creams

IPM – can be warm up to

40C but not more than 44C

Filter rapidly

Dilute Product in Emulsifying

agent containing Medium. Add to

medium, shake every day but

minimum for FTM to keep

Anaerobic Condition

Antibiotics for

Solution -<5g

20 containers and 300 mg

from each into 200 mL of

Diluent or constitute as label

Antibiotics for

Solution -≥5g

6 container and 1 gram from

each into 200mL Diluent

Product Membrane Filtration

Method

Direct Inoculation

Method

Antibiotics Solids, Bulk &

Blends

Equivalent to 6 grams of

solids in 200 mL of Diluent

Aerosol Freeze in Alcohol – dry ice

mixture at -20C for 1 hour

and then remove the

content and add 100 mL

of Fluid D

Syringes Remove the content,

pooled in vessel

Immerse the whole

product

Devices pathway labeled

sterile

Flush with Fluid D, not

less than 10 pathway

volume

Fill the lumen.

OBSERVATION AND INTERPRETATION OF RESULTS

• Technician must be trained as to how to detect growth during the incubation

period.

• At intervals during the incubation period and at its conclusion, examine the

media for macroscopic evidence of microbial growth.

• After 14 incubation if the presence or absence of microbial growth can not

be determined because of the product nature which cause turbidity in the

media transfer portions (each not less than 1 mL) of the medium to fresh

vessels of the same medium, and then incubate the original and transfer

vessels for not less than 4 days.

• If no evidence of microbial growth is found, the product to be examined

complies with the test for sterility.

• If evidence of microbial growth is found, OOS should be conducted. This

investigation should include: Clean Room environmental test data, media

sterilization records, technician training record, the relative difficulty of the

test procedure, media controls data, personal monitoring data, bioburden

data.

OBSERVATION AND INTERPRETATION OF RESULTS – CON’T

The test may be considered invalid only if one or more of the following conditions are

fulfilled:

• Error in test procedure used for the test.

• Microbial growth is found in the negative controls.

• After determination of the identity of the microorganisms isolated from the test,

the growth of this species (or these species) may be misunderstanding to faults

with respect to the material and or the technique used in conducting the sterility

test procedure.

• If the test is declared to be invalid, it is repeated with the same number of units as

in the original test.

• If no evidence of microbial growth is found in the repeat test, the product

examined complies with the test for sterility.

• If microbial growth is found in the repeat test, the product examined does not

comply with the test for sterility.

The four pillars of Sterility testing

•Personnel training & monitoring

•Environmental monitoring

•Facilities design

•Aseptic Technique

If people are a major source

of contamination how do we

avoid contaminating the

product while testing?

Gowning

Personnel: Gloves Aseptic technique of wearing gloves

How often should gloves be examined and sanitized?

Gowning Qualification

•Written ( photographic) procedures describing

methods used to do each gown component in an

aseptic manner and the creation of barriers by

overlapping gown components.

•Initial training and periodic assessment.

•Annual requalification.

Qualifying Personnel

• Assess after gowning/gloving

• Microbiological surface sampling of several locations

• Glove fingers

• Facemask

• Zipper and Head cover interface

• Boot tops

Periodic requalification is necessary

Personnel: Behavior

Minimize movement: Work slowly and purposefully

Active Air Monitoring

RCS Plus

Passive Air Monitoring

Settling Plates

Environmental Monitoring:

→Viable Microbial Air Monitoring

→Non Viable Air Monitoring

Environmental

Monitoring: Surface Monitoring

Contact plates

- RODAC Plates

Swabs

Horizontal airflow

Unidirectional airflow The operator should

never come between the

air source and the product.

Minimize movement: Work

slowly and purposefully

Facilities: General Cleanroom Design

•HEPA filters on ceiling

•Airlocks and interlocking doors to control air balance

•Seamless and rounded floor to wall junctions

•Readily accessible corners

•Floors, walls, and ceilings constructed of smooth hard surfaces that can be easily cleaned

•Limited equipment, fixtures and personnel

•Layout of equipment to optimize comfort and movement of operators

Facilities

Establishing and Maintaining an aseptic

environment • Use clean-rooms of various classes to establish an aseptic area

• Clean rooms use combinations of filtration, air exchange, and positive

pressure to maintain “clean” environment

• Lower quality clean areas should not be placed next to high quality

areas

Facilities: Cleanroom Classification

FS209

Cleanroom

classification

ISO 14644-1

Cleanroom

classification

≥0.5um

particles/m3

Viable

Microbes

(cfu/m3)

Ave Airflow

Velocity

(fpm)

Air

changes/hr

100,000 8 3,520,000 100 5-10 5-48

10,000 7 352,000 10 10-15 60-90

1000 6 35,200 7 25-40 150-240

100 5 3,520 1 40-80 240-480

Facilities: HEPA Filters

http://people.deas.harvard.edu/~jones/lab_arch/nano_facilities/hepa.gif

High Efficiency Particulate Air

Minimum particle collection efficiency:

99.97% for 0.3µm diameter particles.

Disposable

Filter made of pleated borosilicate glass

Facilities: Pressure Differentials

• Used to maintain airflow in the direction of higher

cleanliness to adjacent less clean areas

• Differential pressure between rooms should be more

than 0.020 inches of water and 0.050 inches of water

between the cleanroom and external environment

http://news.thomasnet.com/images/large/451/451402.jpg

Facilities: Air Lock

• Permits the passage of objects and people

into a cleanroom.

• Consists of two airtight doors in series

which do not open simultaneously.

• Spray down materials with using a

validated disinfectant before placing in the

airlock

•Fiber-shedding materials such as cardboard and paper

•Cardboard packaging must be removed and items placed into

non-cardboard containers.

•Wood (i.e. wooden pallets)

•Undesignated charts

Facilities: Material NOT permitted in a Cleanroom

Aseptic Technique

Contact sterile materials only with sterile

instruments:

•Sterile instruments should be held under Class 100

conditions between uses and placed in sterile

containers

•Operators should not contact sterile products,

containers, closures, or critical surfaces with any part

of their gown or gloves

Which is correct aseptic technique?

Wrong Correct

Aseptic Technique

• Keep the entire body out of the path of unidirectional airflow

• Unidirectional airflow design is used to protect sterile equipment

surfaces, container-closures, and product. Disruption of the path of

unidirectional flow in the critical area can pose a risk to product

sterility.

Aseptic Technique

•Approach a necessary manipulation in a manner that does

not compromise sterility of the product

• Proper aseptic manipulations should be approached from the side

and not above the product (in vertical unidirectional flow

operations).

• Operators should refrain from speaking when in direct proximity

to the critical area.

Which is correct aseptic technique?

Wrong Correct

Which is correct aseptic technique?

Correct

43 43 43

CONTACT US Gibraltar Laboratories is conveniently located

GIBRALTAR

LABORATORIES 122 Fairfield Road

16 Montesano Road (Shipping/Receiving)

Fairfield, New Jersey 07004

(973) 227-6882

kkohan@gibraltarlabsinc.com

www.gibraltarlabsinc.com

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Proprietary and Confidential © Gibraltar Laboratories, Inc.

www.gibraltarlabsinc.com / (973) 227-6882