STERILITY TESTfor Pharmaceutical product

Marlia Singgih Wibowo

School of Pahrmacy ITB

OBJECTIVE

To determine whether a pharmaceutical product which claimed “sterile” is fulfill the requirement of sterility test based on their monography

STERILITY TEST based on : (FARMAKOPE INDONESIA IV/ FI IV 1995) Indonesia

USP/BP/EP the latest edition GUIDELINES FOR STERILITY TESTING OF

THERAPEUTIC GOODS (THERAPEUTIC GOODS ADMINISTRATION/ TGA 2006)

INTRODUCTION

Provide guidance for sterility testing of sterile therapeutic drugsand devices supplied in Australia for human use.

The official (legal) requirements for sterility tests in Australia British Pharmacopoeia (BP), European Pharmacopoeia (Ph Eur)

These Guidelines are based on the superseded documentAustralian Code of Good Manufacturing Practice for TherapeuticGoods - Medicinal Products

PRECAUTIONS AGAINST MICROBIAL CONTAMINATION

Tests for sterility are to be carried out by trained personnel Conducted in a clean-room environment the standard of

clean-room

Personnel should wear sterilized over-garments All equipment may come into contact in the course of the testing should

be sterilized prior to use. All substances added to the goods tested, should be sterilized prior to

use by heat All vessels, substances or outer clothing to be used should be

appropriately packaged or closed, The outer surfaces of all packages of equipment which are introduced

into the aseptic testing environment should be free of contamination

TEST METHODS VALIDATION (TGA 2006)

Test method validation Before tests for sterility for any product are initially carried out, it

is necessary to demonstrate the validity of the test method used Validation should mimic the test proper in every detail Validation is to be performed when the test for sterility has to be

carried out on reformulated or new product All validation procedures should be carried out by personnel who

are responsible for the routine testing of the product

MEDIA, DILUTION AND RINSING SOLUTION MEDIA LIQUID THIOGLYCOLLATE MEDIUM ALTERNATIVE THIOGLYCOLLATE MEDIUM SOYBEAN-CASEIN DIGEST MEDIUM

DILUTING AND RINSING SOLUTION FLUID A FLUID D FLUID K

LIQUID THIOGLYCOLLATE MEDIUM and ALTERNATIVE THIOGLYCOLLATE MEDIUM for an-aerobic bacteria and some aerobic bacteria

SOYBEAN-CASEIN DIGEST MEDIUM for fungi and some aerobic bacteria

MEDIUM composition according to FI IV and TGA 2006

Medium 1 (Fluid Thioglycollate Medium)Pancreatic Digest of Casein 15.0 g

Yeast Extract (water-soluble) 5.0 g

Glucose monohydrate/anhydrous 5.5 g/5.0 g

Sodium chloride 2.5 g

L-Cystine 0.5 g

Sodium thioglycollate 0.5 g

0.1% Resazurin Sodium Solution (freshly prepared) 1.0 mL

Granulated Agar (moisture not more than 15%) 0.75 g

Purified Water 1000 mL

Polysorbate 80 (optional) 5.0 mL

pH after sterilisation (measured at room temperature): 7.1 0.2

MEDIUM (FI IV)Alternative Thioglycollate Medium (for devices with

small lumen) : Pancreatic Digest of Casein 15.0 g

Yeast Extract (water-soluble) 5.0 g

Glucose monohydrate/anhydrous 5.5 g/5.0 g

Sodium chloride 2.5 g

L-Cystine 0.5 g

Sodium thioglycollate 0.5 g

Purified Water 1000 mL

pH after sterilization (measured at room temperature): 7.1±0.2

MEDIUM (FI IV/TGA 2006)Medium 2 (Soybean-Casein Digest Medium)

Pancreatic Digest of Casein 17.0 g

Papain Digest of Soybean Meal 3.0 g

Glucose monohydrate/anhydrous 2.5 g/2.3 g

Sodium chloride 5.0 g

Dipotassium hydrogen phosphate 2.5 g

Purified Water 1000 mL

Polysorbate 80 (optional) 5.0 mL

pH after sterilisation (measured at room temperature): 7.3±0.2

DILUTING AND RINSING FLUID (FI ed. IV/TGA 2006)Fluid A :

1 g of peptic digest of animal tissue

Ad 1L water

Filter/ centrifuge until the solution clear

Adjust to pH 7.1± 0.2

Dispense into containers @100 mL

Sterilization using autoclave 121ºC, 15 min.

* For Specimen contains penicillin/cephalosporin, a sterile penicillinase was added into Fluid A to inactivate residue of antibiotic

DILUTING AND RINSINGFLUID (FI IV/TGA 2006)Fluid D:

Used if the specimens contain lecithin or oil,or for sterile medical devices which labelled as “sterile pathway”

Fluid A + 1mL polysorbat 80 per L Adjust to pH 7.1± 0.2 Dispense into several flasks Sterilization using autoclave, 121ºC, 15 min.

DILUTING AND RINSING FLUID(FI IV/TGA 2006)Fluid K : Peptic Digest of animal tissue 50 g Beef Extract P 3,0 g Polysorbate 80 P 10,0 g Water 1000 mL Adjust to pH 6.9 ± 0.2 Sterilization using autoclave

HOW TO AVOID FAKE NEGATIVE RESULT? Based on FI IV FERTILITY TEST For MEDIUM

BACTERIOSTATIC TEST AND FUNGISTATIC For Samples to be tested

FERTILITY TEST (FI IV)

Should be done for every lot of medium from each autoclave used

Inoculation of any media separately 2 times duplo with 10 -100 mikroba viabel from every species

The fertility test will pass if : there are viable growth in every media container within 7 days

Storage: in the dark place (2-25ºC) In vacuum container : not more than 1 month

Tested for 7 days before its usage, colored indicator Vacuum container : not more than 1 year

Tested for 3 months, colored indicator should fulfilled the requirement

Monitoring the efficacy of test media at the end of the incubation period/stasis test (TGA 2006)

not mandated by the Pharmacopoeias but is recommended as part of Good Laboratory Practice (GLP)

to demonstrate that the media inoculated with the test preparation will support growth for the full incubation

Acceptable challenge organisms are listed in Table 5 the number of organisms in the inoculum is to be not more than 100 CFU should all show growth of the added organisms within 48 hours If conspicuous growth is not apparent within 5 days for both bacteria and fungi

the test is considered invalid Invalid stasis tests may be repeated once If conspicuous growth is not obtained at the second attempt the test method

should be modified and revalidated. If the media are found to support growth of the test micro-organisms then this

test need not be applied to every sample. It should be repeated periodically on the relevant categories of products or when product is reformulated. Every 12 months is recommended.

Medium Tested microorganism INCUBATION

Temp. Condition

LiquidThioglycolate

Bacillus subtilis (ATCC No. 6633)

30 -35

AEROBICCandida Albicans(ATCC No. 10231)

30 -35

Bacteriodes vulgatus(ATCC No. 8482)

30 -35

AlternativeThioglycollate

Bacteriodes vulgatus(ATCC No. 8482)

30 -35 ANAEROBIC

Soybean-Casein Digest

Bacillus subtilis (ATCC No. 6633)

20 -25AEROBIC

Candida Albicans(ATCC No. 10231)

20-25

BACTERIOSTATIC DAN FUNGISTATIC (FI IV)

Bacteria and fungi : 10 - 100 viable microbes

Sample tested

INCUBATION min 7 days

BAKTERIOSTATIC AND FUNGISTATIC (FI IV) If sample has bacteriostatic/fungistatic properties : use

neutralizing agent If neutralizing agent is not available :

Repeat the test with bigger volume to determine the ratio of sample/medium for suitable growing condition

If 250 mL of medium still showed bacteriostatic/ fungistatic activity : reduce the volume of sample

Solution/suspension ≤ 1 mL & solid substances which is not soluble/ dispersed : add more medium solution

BACTERIOSTATIC AND FUNGISTATIC (FI IV) Sample + dilution and rinsing solution Rinse the membrane 3 x with 100 mL of

Dilution and Rinsing solution Inoculate microbes into the last dilution and

rinsing The growth of microbes on sample should be

equal to the reference

Initial validation of the test method - testing for sterility activity (TGA)

Sample

specified aerobic bacteria, anaerobic bacteria and fungi.

+

the number of micro-organisms ≤ 100 CFUshould be apparent within 48 hours, growth does not occur within 5 days, the test procedure is not valid and must be modified

GENERAL PROCEDURE (FI IV)

How to open the container : Decontaminate the surface of vial/ampule

using alcohol 70% or other liquid Sampling the content aseptically If the vial’s content is packed by vacuum

system : add into the vial a sterile air. Sterile cotton, dressing, bandage, etc : open

the container aseptically

Volume sampled for Liquid (FI IV)Volume of

sampleV min

For sampleV min for each sample Amount of

container per mediaDirect

innoculationmembrane

≤ 10 1ml or the whole content if the vol.≤ 1mL

15 100 20(40 if vol of each container is not enough for both medium

10 - ≤ 50 5 ml 40 100 2050 - ≤ 100 10 mL 80 100 2050 - ≤ 100 (iv)

Seluruh isi - 100 10

100-500 Seluruh isi - 100 10≥ 500 500 mL - 100 10

SAMPLING OF LOTS (TGA 2006)

a batch of product is defined as a homogeneous collection of sealed containers prepared

Any samples used for a sterility retest should reflect the original samples in terms of sampling locations or process times.

For aseptically prepared products, the samples should be taken at regular intervals during the filling operations

For terminally sterilized products the samples should be made up from units drawn from various sites throughout the sterilizer load.

The testing of products which are intended for use in clinical trials and which are dispensed in small volumes may be combined with the filling procedure , e.g. a sterile membrane filter is incorporated in the filling line

SAMPLING SCHEDULE - MINIMUM NUMBER OF ITEMS TO BE TESTED FROM EACH BATCH (TGA 2006)

Type of product Number of items in the batch

Minimum number of items to be tested for each medium2

Injectable pharmaceuticals Not more than 100101-500More than 500

10% of batch or 4 containers,10Containers2% of batch or 20 containers

Non-injectable pharmaceuticals

Not more than 200More than 200

5% of batch or 2 containers,10 containers

Bulk solid products Not more than 45-50More than 50

Each container20% of batch or 4 containers,2% of batch or 10 containers,

Pharmacy bulk packages of antibiotics

Less than 5gGreater than or equal to 5g

20 containers6 containers

SELECTION OF TEST SPECIMEN AND INCUBATION TIME (FI IV)If only stated in the monography : Incubation :14 hari: Liquid thioglycollate Medium : 30-35 oC Alternative Thioglycolatte : 30-35 oC Soybean-Casein Digest medium : 20-25 oC Observation : day 3/4, 7/8, and 14

INCUBATION & EXAMINATION OF STERILITY TEST (TGA 2006)

Medium 1 30 - 35°C

Medium 2 20 - 25°C

Incubation period ≥ 14 days

Examination Every 2 working days

PROCEDURE for TEST (FI IV & TGA 2006)

Direct Inoculation

Membrane Filtration

Pharmaceutical product (FI IV &TGA 2006)

LiquidOintments and oil which is not soluble in isopropyl miristateZat padatCotton, dressing, perban, catgut Steril medical devicesSyringe

Liquid and soluble or dispersible solids

Solid preparations

Ointments and oily preparations

FLUIDS (FI ed.IV)

Sample ≥

300 mg • Dispense with specific media

• Incubation ≥ 14 days

• Observe visually (day 3/4/5, 7/8 and 14)

Sample + media : turbid place into fresh media (min 1x between day 3 and day 7) incubate the media up to day 14

using pipette / syringe aseptically

ZAT PADAT (FI IV)

≥ 300 mg

Cairan pengencer steril

Larutan/ suspensi

40 mL Media SCD / TC

INKUBASI

Liquid and soluble or dispersible solids (TGA)

Sampel ≥ 10% volume medium

Incubation:Medium 1 at 30 - 35 C Medium 2 at 20 - 25 C

soluble or dispersible solids + Purified Water / a suitable sterilized diluents or solvent the solid material → transferred directly to the test media

PURIFIED COTTON, DRESSING, NAPKINS, CATGUT, ETC

Cotton, dressing, napkins : Two parts : 100 –500 mg

Individually package of cotton , catgut : 250 mg -500 mg / entire article

STERILE MEDICAL DEVICES If the article can be immersed and disassambled :

immersed in 1000 mL of media

Devices with pipes/ lumens : rinse each lumen from 20 units with medium 1 dan 2 . Each 15 mL of the rinsing fluids + 100 mL of media

For catheters where inside and outside lumen are need to be sterile , either cut them into pieces and immersed into media, or : fill in the lumen, and then soak entire article in 1000 mL of media

STERILE EMPTY SYRINGE OR PREFILLED SYRINGE

Pre-filled syringe : Expel the content into vessel through membrane

filter Empty syringe : Aspirate the media through the needle , and then

expel the media into vessel as indicated above method

SOLID ARTICLES (TGA) solid articles tested by immersion in or filling with culture media Aseptically dismantle all articles as completely as possible Immerse all parts of each article in sufficient medium contained

in one vessel to completely cover all parts Incubate the test Medium 1 at 30 - 35°C and Medium 2 at 20 -

25°C As a minimum, portions of 100-500 mg should be cut from that

part of the dressing that is most inaccessible to sterilant Care should be taken to ensure that entrapped air does not

prevent the medium from making contact with all parts of the internal surfaces of an article. To facilitate this contact a surfactant agent is included in Medium 1 and Medium 2; Medium 1 may also be modified by the omission of agar

OINTMENTS AND OIL WHICH IS NOT SOLUBLE IN ISOPROPYL MIRISTATE

20

10

10

@10 mg

@10 mg

100 mL sterile water

10 mL

80 mL medium

100 mL sterile water

INCUBATION

Ointments and oily preparations (TGA)Membrane Filtration is not feasible

Direct Transfer

oily liquids + media emulsifying agent

ointments and creams

emulsifying agent

+ Maximally contact

INTERPRETATION OF RESULTS STERILITY 1ST STAGE

Within interval and end of incubation

Observe all containers for tubidity or growth on surface

If no growth observed

The product complies with the test of sterility

INTERPRETATION OF RESULTS STERILITY – STAGE I IF THERE ARE MICROBIAL GROWTH ,

CHECK: Facilities for sterility test Raw material used Test Procedure Negative Control Aseptic Technique

NOT FULFILL THE REQUIREMENT

PROVED NOT PROVED

STAGE I STAGE II

INTERPRETATION OF RESULTS STERILITY – STAGE IINumber of test specimens 2 x stage I

Minimum Volume, media, incubation period

= stage I

No evidence of microbial growth

The article if complies to test for sterility (ACCEPT)

Evidence of microbial growth Fault

Stage II is not valid Repeat Stage II

NEGATIVE PRODUCT CONTROL TEST (TGA 2006) Negative product control tests

interpretation of sterility test results To declare a test invalid because of

contamination in the negative product controls

The negative control contamination rate should be calculated and recorded

Negative product

• a terminally sterilized item of undoubted sterility: equivalent of 2 sterilization cycles by autoclaving or by dry heat sterilization, or 50 kGy of gamma irradiation

• should be similar in type and container to the product under test

•at least ten negative product control containers should be tested•an aqueous product:

distilled water • an ointment :

liquid paraffin or ointment base

• disposable devices : a section of glass or plastic tubing packaged in a manner similar to the device

NEGATIVE PRODUCT CONTROL TEST (TGA 2006)

Examination of sterility tests

a suspension, flocculation or deposit microbial growth??mixed by gentle swirling or inversion

After 14 days incubation : ≥ 1 mL + fresh same medium

Incubate ≥ 4 days at the same temperature

Examination of sterility tests

turbidity, precipitate, or other evidence of microbial growth

Gram staining

Single colony

Colonial and cell morphology Identification of isolates

RECORDS (TGA 2006)

should contain at least the following information:• description and number of product units tested;• batch/lot number;• stage of manufacture (finished product, intermediate or final bulk);• personnel performing tests;• dates of testing;• test methodology (volume tested, diluents/solvents used, media, media batch numbers, temperature and time of incubation);• results in full

RECORDS (TGA 2006)

Records should also be maintained of:• details of validation of the sterility test method;• periodic stasis testing;• details of any product contamination irrespective of whether the test was valid or invalid;• the negative control contamination rate;• results of environmental and personnel monitoring.

Results of sterility testing for test samples and negative controls should be presented in a format that allows for easy recognition of trends.

These records should be appropriately stored and readily available as defined in Chapter 4 of the Australian Code of Good Manufacturing Practice for Medicinal Products, 2002.