Validation of the Sterilization Process

Prepared by :

Microbiology and Immunology Department

Objectives of the Lecture 1. Validation of the Sterilization Process:

I.   Monitoring of the sterilization process: §  Physical indicators. §  Chemical indicators. §  Microbiological indicators.

II.   Monitoring of the sterilized product (Sterility testing): §  Culture media for sterility test:

ü Fluid Thioglycolate Medium (FTM). ü Trypticase Soya Broth (TSB).

§  General sterility testing procedure: ü Direct transfer method (DT). ü Membrane filtration method (MF).

§  Limitations and significance of sterility testing. 2. Tests for pyrogen and bacterial endotoxin in pharmaceuticals. 3. Microbial limits for non-sterile products.

1.Validation of the Sterilization Process

Control Devices for Sterilization

Are devices, tests and systems used to insure that a product may be

issued for use with confidence of its sterility status.

They are classified into two categories:

I- By monitoring the sterilization process through the use of physical,

chemical and biological indicators.

II- By monitoring the sterilized product (Sterility testing).

I. Monitoring the sterilization process A) Physical indicators

1)  Dry heat: by thermocouples which record temp at various parts of the load and timer for recording time.

2) Moist heat: Pressure and vacuum gauge and thermocouples. 3) Ethylene oxide: Pressure and vacuum gauge, thermocouples and

hygrometer for humidity. 4) Radiation: Dosimeter to record accurately the absorbed radiation dose. 5) Filtration: by bubble point pressure technique. A solution with

known surface tension is allowed to pass through the filter using gradual increasing pressure. The pressure, at which the first air bubble, is recorded and the following equation is applied.

•  D = 4 φ/P •  D = pore size in microns φ = surface tension P = pressure •  For ! Pore Size Determination; for a given fluid & pore size with a

constant wetting, the pressure required to force an air bubble through the pore is inverse proportion to the size of the hole.

•  The flow rate and the integrity of the filter are also examined.

B) Chemical indicators 1) Browne's tube: Ampoule containing certain chemical which change

in color at certain temp. 2) Witness tube: Sealed tube containing a component. that melts at the

sterilization temp. 3) Hour glass: Two bulbs connected by a capillary tube, when a

powder melts in the upper bulb it flows down through the capillary tube in certain time.

4) Paper strips: Steri tapes or stickers, which change in color during sterilization forming letters, writing the word sterile.

C) Biological indicators By placing a resistant M.O. impregnated in strips e.g. paper or

metal foil at different sites in the sterilizer, and test for its viability at the end of the sterilization process.

Characteristics of the selected M.O.: 1- Stable and reproducible. 2- Easily recovered on nutritive medium. 3- Highly resistant to the method. 4- Non pathogenic.

1) Dry heat: Bacillus subtilis or Clostridium tetani. 2) Moist heat: Bacillus staerothermophillus. 3) Ethylene oxide: Bacillus globigii. 4) Ionizing radiation: Bacillus pumilus or Micrococcus radiodurans. 5) Filtration: Serratia marcescens or Pseudomonas diminuta which are very small Gram negative M.O.

A, B, spore strip and Disc. C:A test with media, D: Chemspor “spore strip in ampoule with media”. E: Plastic carrier with dried spores. F: Brown’s tube. G: Thermalog strip. H: Chemdi strip that changes color. I: Chemspor: 2 ampoule system (biological & chemical)

2. Sterility Testing •  Required for all articles or substances to be introduced into raw tissue

(injectables and ophthalmics). •  It is considered as the final control on sterilization as it provides an

additional mean for checking that all facts of sterilization process were achieved.

•  Sterility test is not an evaluation test for the sterilization process. However, it is of value in checking sterility of subs. sterilized by filtration only.

•  The sterility test is a destructive test i.e. samples tested are not reused.

•  The test show that samples tested were free from living bacteria and fungi but not viruses, since it is impossible to test singular item for different types of M.O.

Batch: group of articles which are exposed to the same conditions from "A" to "Z" i.e. prepared, mixed, packed and sterilized together.

•  Sampling for sterility testing: depends upon –  1) number of units in batch. –  2) volume per container. –  3) method of sterilization. –  4) use of biological indicators during sterilization. –  5) application of good manufacturing practice (GMP).

•  Sample size

–  A) 2% of the batch with maximum 20 articles selected randomly from different parts.

–  B) 0.4 √N which is more efficient statistically. (N = number of articles in the batch).

•  Quantity used per sample

–  -depends on the volume or weight of the sample

Culture media for sterility test

There is no one medium which could detect both bacteria and fungi. The USP recommends two media.

A.  Fluid Thioglycolate Medium (FTM): for detection of both

aerobic and anaerobic bacteria.

B.  Trypticase Soya Broth (TSB): for detection of fungi. TSB replaces Sabouraud's dextrose medium, as it proved to support growth of both fungi and highly aerobic bacteria such as Bacillus spp..

Both media are sterilized by autoclaving.

A. Formulation of "FTM" • Yeast extract ..........………….......... nutrient (growth factors) • Pancreatic digest of casein ………nutrient (nitrogen source) • Dextrose…………...nutrient (carbon source) and antioxidant • L-cysteine...... antioxidant, support growth of clostridia & neutralize metal

preservatives • Sod. thioglycolate……...antioxidant & neutralize metal preservatives. • Agar (low conc.) viscosity inducer to reduce oxygen diffusion • Sod. chloride................................... isotonic agent. • Resazuarin sod ……... Indicator (pink at high O2 tension upper third of

container and yellow in low O2 tension, lower 2/3 of the container) • Distilled water • pH after sterilization 7.1 ±0.1. • The medium is prepared in special tube to maintain the ratio between surface

to depth of the medium. • One disadvantage of FTM is that it does not support the growth of Bacillus

subtilis (highly aerobic) entrapped in solid samples located in the anaerobic lower portion of the medium.

B. Formulation of "TSB"

- Pancreatic digest of casein..... nutrient - Pancreatic digest of soybean..... nutrient - Dextrose………………...... nutrient - Sod. chloride.............. isotonic agent - Dibasic potassium phosphate.. .buffer - Water pH after sterilization 7.3 ± 0.1

General sterility testing procedure

USP specifies 2 basic methods

1) Direct transfer method 2) Membrane filtration method

1) Direct transfer method "DT"

•  The container is cleaned with suitable antimicrobial agent and aseptically opened (in laminar flow cabinet) to avoid contamination and hence false positive result.

• The specified volume of the sample is withdrawn.

• One half is added to the required volume of "FTM" and the other half is added to the required volume of "TSB".

• "FTM" is incubated at 30 -35oC (suitable for bacteria) for at least 14 days.

• "TSB" is incubated at 20 -25oC (suitable for fungi) for at least 14 days.

2) Membrane filtration method “MF" • The filtration unit is properly assembled and sterilized by autoclaving

(0.45 µm pore size membrane filter is used c.f. 0.22 µm membrane filter used in sterilization).

• The contents of the sample are transferred to the filter under strict

aseptic conditions. M.O. ,if present are trapped on the surface of the filter.

• Vacuum pump is usually used to assist filtration.

• The filter is washed several times with sterile distilled water.

• The membrane filter is cut into 2 pieces, one half is transferred into the "FTM" and the other half into the "TSB" under aseptic conditions.

• Incubation and interpretation of results.

Interpretation of Results

•  If no growth is observed after the incubation period the batch pass the test.

•  If visible growth is observed the test is repeated. •  If again growth is observed the material fails the test. •  It is recommended to identify the contaminant and trace it.

Advantages of "MF" over "DT" : 1) Antimicrobial agents (preservatives, antibiotics) are completely

eliminated and so minimize false negative result. 2) In case of large volume parentals, the whole contents are tested. 3) Several containers could be tested at once, hence contaminants

are concentrated, thus increasing the ability to detect containers with low contaminants in short time.

4) Ease of separation of contaminants from oily preparations.

Disadvantages of "MF" over "DT": 1) High probability of contamination throughout the procedure (need highly skilled operators). 2) Needs specific equipment with validated insurance for sterility. 3) Unable to differentiate between the extent of contamination between different items.

Controls for sterility test A) Control for Sterility of the media (negative control): Carried out by incubating uninoculated tubes (FTM and TSB) under the same conditions, they should show no growth at the end of the incubation period. All materials used t o c a r r y o u t t h e t e s t s h o u l d p a s s t h e s a m e c o n t r o l . B) Control for Growth promoting ability of the medium (positive control): Carried out by separately inoculating the media with the following M.O. " F T M " 1 - B a c i l l u s s u b t i l i s . . . . . . . . . . . . . . . . . . . . . . a e r o b i c a l l y 2 - B a c t e r o i d v u l g a r i s . . . . . . . . . . . . . . . . . . . a n a e r o b i c a l l y " T S B " 1 - C a n d i d a a l b i c a n s . . . . . . . . . . . . . . . . . . . . a e r o b i c a l l y 2 - B a c i l l u s s u b t i l i s . . . . . . . . . . . . . . . . . . . . . . . . a e r o b i c a l l y

All tubes should show visible growth within the incubation period at the appropriate temp. For successful sterility test, all tubes used for testing a batch of m a t e r i a l s h o u l d c o m e f r o m t h e s a m e b a t c h o f m e d i a . The media could be stored in sealed containers for one year in dark area at not more than 25oC during which are tested for growth promoting ability every 3 m o n t h s .

Problems created during sterility testing

1)Products which gave turbidity with the medium: 2) Immiscible liquids (oily): e.g. progesterone 3) Creams and ointments: 4) Surgical dressing and sutures: 5) Sterilized devices: small syringes, needles and similar articles .

6) Products with antimicrobial activity: Antibiotics and multidose injections or eye drops that contain preservative need special treatment before testing for sterility. The antimicrobial agent should be eliminated by one of the following procedures” a- Dilution (for compounds with high conc. coef like phenol) b- Antagonism by constituents of the medium (ex: thioglycolate neutralizes Hg compounds). c- Antagonism by addition of sterile materials to the medium Examples:

- Penicillin with penicilnase. - Streptomycin with cysteine - QAC by lecithin and non ionic surfactants. - Sulphonamides by p-ABA

d- Membrane filtration: used when the above three methods are non effective i.e. the antimicrobial agent is active in very low conc. e.g. most antibiotics or when the product is not easily antagonized.

Limitation of Sterility Test

1) Uncertainty that the small number of samples used represents

the whole batch.

2) Inability of one culture medium to support growth of all

known M.O.

Significance of sterility test

The test is not a control on whether the whole batch is sterile or

not but a control on whether the designed theoretical conditions

for sterilization have been in action during the process or not.

It also detects unusual sources of contamination. It is more

significant in sterilization by filtration.

Complete knowledge of the entire history of the product is very

essential in sterilization.

Tests for pyrogen and bacterial endotoxin in pharmaceuticals

Pyrogen: “Substance that produces fever”, usually a bacterial endotoxin or other microbial product.

1)  Measures the rise in temperature of standardized rabbits upon intravenous injection of a sterile solution of the substance.

2)  LAL test. (precise) Limulus Amebocyte Lysate reagent, aqueous extracts of the circulating blood cells (amebocytes) of the horseshoe crab Limulus polyphemus.

-  A series of dilutions of the standard endotoxin and a series of dilutions of the preparation being tested are placed in test tubes, and to each the LAL reagent is added.

-  The contents of each tube are mixed and incubated, undisturbed - with positive and negative controls - for 60 ± 2 minutes.

-  A positive reaction is obtained when a gel is observed that remains firm when inverted 180o. Any other reaction is negative.

-  The test may be carried out on slide with incubation for 20 minutes, protected from loss of moisture.

   -  on a  

Microbial Limits for non-sterile products

Microbial limit tests are applied to all raw materials and preparations not

required to be sterile.

They include

1)  Total microbial viable aerobic count (TC) which is quantitative. -The TC must not exceed a certain value. The TC limit ranges between 100 and

10,000 cfu per ml or g. -It is rare for pharmacopoeias to specify anaerobic bacterial counts, fungal counts,

and counts for specified species

-Internal limits of as little as 5 are often enforced by many producers who have found that such a limit is technically feasible with current GMP and technology and is a good measure of GMP and TQA.

2) Test for absence of pathogens which is qualitative -For topical products Ps. aeruginosa and S. aureus should be absent. -For oral products E. coli and Salmonella spp. - rarely to enterotoxin-producing S. aureus should be absent.