PRINCIPLE, ADVANTAGES,DISADVANTAGES, APPLICATIONS OF DIFFERENT STERILISATION METHODS AND IN PROCESS CONTROL

PREPARED BY KOSARAJU SAI VIVEK

I M.PHARMACY,

DEPARTMENT OF PHARMACEUTICS

Jss college of pharmacy

contents

1. WHAT IS STERILIZATION2. METHODS OF STERILIZATION

3. MERITS, DEMERITS ANDAPPLICATIONS OF DIFFERENT METHODS OF STERILIZATION

4. PHARMACEUTICAL IMPORTANCE OF STERILIZATION

5. PHARMACEUTICAL IN PROCESS CONTROL

WHAT IS STERILIZATION:

Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses and prions) from a surface, equipment, foods, medications, or biological culture medium.

METHODS OF STERILIZATION

The various methods of sterilization are: 1. Physical Method a. Thermal (Heat) methods b. Radiation method c. Filtration method 2. Chemical Method a. Gaseous method

PHYSICAL METHODS:

1. HEAT STERILIZATION: Heat sterilization is the most widely used and

reliable method of sterilization, involving destruction of enzymes and other essential cell constituents.

This method of sterilization can be applied only to the THERMO STABLE PRODUCTS, but it can be used for MOISTURE-SENSITIVE MATERIALS.

i) Dry Heat (160-1800˚C) Sterilization for thermo stable products

ii) moist heat (121-1340 ˚C) sterilization is used for moisture- resistant materials.

The efficiency with which heat is able to inactivate microorganisms is dependent upon

i) the degree of heat, the exposure time and ii) the presence of water. The action of heat will be due to induction of

lethal chemical events mediated through the action of water and oxygen.

In the presence of water much lower temperature time exposures are required to kill microbe than in the absence of water.

THERMAL (HEAT) METHODS

Thermal methods includes: i) Dry Heat Sterilization Ex:1. Incineration 2. Red heat 3. Flaming 4. Hot air oven ii) Moist Heat Sterilization 1.Dry saturated steam – Autoclaving 2. Boiling water/ steam at

atmospheric pressure 3. Hot water below boiling point

Dry Heat Sterilization

It employs higher temperatures in the range of 160-180˚C and requires exposures time up to 2 hours, depending upon the temperature employed.

The benefit of dry heat includes good penetrability and non-corrosive nature which makes it applicable for sterilizing glass wares and metal surgical instruments. It is also used for sterilizing non-aqueous thermo stable liquids and thermo stable powders.

Dry heat destroys bacterial endotoxins (or pyrogens) which are difficult to eliminate by other means and this property makes it applicable for sterilizing glass bottles which are to be filled aseptically

Moist Heat Sterilization

Moist heat sterilization involves the use of steam in the range of 121-134˚C. Steam under pressure is used to generate high temperature needed for sterilization. Saturated steam acts as an effective sterilizing agent.

Autoclave

Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents. For efficient heat transfer, steam must flush the air out of the autoclave chamber.

Generally the conditions employed are Temperature upto121-134˚C for 15-20 min

under 15 lbs pressure,based on type of metiral used.

Radiation Sterilization

Many types of radiation are used for sterilization like electromagnetic radiation (e.g. gamma rays and UV light), particulate radiation (e.g. accelerated electrons).The major target for these radiation is microbial DNA.

Radiation sterilization with high energy gamma rays or accelerated electrons has proven to be a useful method for the industrial sterilization of heat sensitive products.

Radiation sterilization is generally applied to articles in the dry state; including surgical instruments, sutures, prostheses, unit dose ointments, plastic syringes and dry pharmaceutical products.

UV light, with its much lower energy, and poor penetrability finds uses in the sterilization of air, for surface sterilization of aseptic work areas, for treatment of manufacturing grade water, but is not suitable for sterilization of pharmaceutical dosage forms.

LAMINAR AIR FLOW CHAMBER

Filtration Sterilization

Filtration process does not destroy but removes the microorganisms. It is used for both the clarification and sterilization of liquids and gases as it is capable of preventing the passage of both viable and non viable particles.

The major mechanisms of filtration are sieving, adsorption and trapping within the matrix of the filter material.

Ex:HEPA FILTERS

Sterilizing grade filters are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing

There are two types of filters used in filtration sterilization:

(a) Depth filters:(b) Membrane filters: These are porous

membrane about 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, and polyvinylidene fluoride, or some other synthetic material.

CHEMICAL STERILIZATION METHOD

GASEOUS METHOD The chemically reactive gases such as

formaldehyde, (methanol, H.CHO) and ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colorless, odorless, and flammable gas.

The mechanism of antimicrobial action of the two gases is assumed to be through alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and amino groups of nucleic acids.

The concentration ranges (weight of gas per unit chamber volume) are usually in range of 800- 1200 mg/L for ethylene oxide and 15-100 mg/L for formaldehyde with operating temperatures of 45-63°C and 70-75°C respectively.

Both of these gases being alkylating agents are potentially mutagenic and carcinogenic. They also produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa

MERITS, DEMERITS AND APPLICATIONS OF DIFFERENT METHODS OF STERILIZATION

S.no METHOD MECHANISM

MERITS DEMERITS APPLICATIONS

1 Heat sterilization

Destroys bacterial endo toxins

Most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents

Can be applied only to the thermo stable

products

Dry heat is applicable for sterilizing glass wares and metal surgical instruments and moist heat is the most dependable method for decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents.

S.no METHOD MECHANISM

MERITS DEMERITS APPLICATIONS

1

2

Gaseous sterilization

Radiation sterilization

Alkylation

Ionization of nucleic acids

Penetrating ability of gases.

It is a useful method for the industrial sterilization of heat sensitive products

Gases being alkylating agents are potentially mutagenic and carcinogenic.

Undesirable changes occur in irradiated products,an example is aqueous solution where radiolysis of water occurs.

Ethylene oxide gas has been used widely to process heat-sensitive devices.

Radiation sterilization is generally applied to articles in the dry state; including surgical instruments, sutures, prostheses, unit dose ointments, plastics

S.no

METHOD

MECHANISM

MERITS DEMERITS APPLICATIONS

1 Filtration sterilization

Does not destroy but removes the microorganisms

It is used for both the clarification and sterilization of liquids and gases as it is capable of preventing the passage of both viable and non viable particles

Does not differentiate between viable and non viable particles

This method is Sterilizing grade filters are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas

Pharmaceutical Importance of Sterilization

• Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment, Containers, Closures, Aqueous injections, Ophthalmic preparations and Irrigation fluids etc.

• Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture impermeable pharmaceutical and medicinal. These include products like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc.

• Gaseous sterilization is used for sterilizing thermolabile substances like; hormones, proteins, various heat sensitive drugs etc.

• U.V light is perhaps the most lethal component in ordinary sunlight used in sanitation of garments or utensils.

• Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures, plastics and catheters etc.

Filtration sterilizations are used in the treatment of Heat sensitive injections and ophthalmic solutions, biological products, air and other gases for supply to aseptic areas.

They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing.

PHARMACEUTICAL IN PROCESS

CONTROL

In-process controls (IPC) are checks that are carried out before the manufacturing process is completed.

The function of in-process controls is monitoring and – if necessary – adaptation of the manufacturing process in order to comply with the specifications. This may include control of equipment and environment, too.

The In-Process Quality Control system lays emphasis on the responsibility of manufacturers processors in ensuring consistency in quality during all stages of production by adopting quality control drills and exercising control on raw materials and bought-out components, manufacturing process, packing and final testing.

In-Process Quality Control tests of tablets

• SIZE & SHAPE• TABLET THICKNESS• COLOUR• HARDNESS• FRIABILITY• POTENCY & CONTENT UNIFORMITY• WEIGHT / WEIGHT VARIATION• DISINTEGRATION TIME

BLENDING:Blend Uniformity: Not less than 90.0 % and not more

than110.0 % of the labeled claim of drug. Relative standard deviation (RSD) is not more than 5.0 %.

HARDNESS – Stokes (Monsanto), Strong Cobb, Pfizer, Erweka, Hebelein or Schleuniger tester, units: kg/sq inch of kg/sq cm,

FRIABILITY –Roche , limit less tan 1%

UNIFORMITY OF CONTAINER CONTENTS. Tablets comply with the test for contents of

packaged dosage forms. Select a sample of 10 containers and count the

number of capsules, pessaries, suppositories or tablets in each container. The average number of the contents in the 10 containers is not less than the labelled amount and the number in any single container is not less than 98 per cent and not more than 102 per cent of the labelled amount.

If this requirements is not met, count the number of the contents in 10 additional containers. The average number in the 20 containers is not less than the labelled amount, and the number in not more than 1 of the 20 containers is less than 98 per cent or more than102 per cent of the labelled amount.

Parenteral Preparations

Parenteral Preparations

1 Particulate matter2 Uniformity of content 3 Extractable volume 4 Sterility5 Pyrogens 6 Uniformity of weight7 Clarity of solution8 Leakage

Particulate matter

Parenteral preparations including solutions constituted from sterile solids are

expected to be free from particles of approximately 50 μm or more that can be observed byinspection with the unaided

eye

.

Particulate matter

SterilityCulture Media

MEMBRANE FILTRATION. After transferring the contents of the container or

containers to be tested to the membrane add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the final portion of sterile diluent used to rinse the filter.

After filtration, aseptically remove the membrane(s) from the holder, transfer the whole membrane or cut it aseptically into 2 equal parts. Transfer one half to each of two suitable media.

Direct Inoculation. After transferring the contents of the container or containers to be tested to the culture medium add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the medium

Quantity in each container

Minimum quantity to be used

Less than 1 ml Total contents of a container

40 ml or more but less than 100 ml 20 ml

1 ml or more but less than 40 ml Half the contents of a container

100 ml or more 10 per cent of the contents of a container but not less than 20 ml

Incubate the inoculated media for not less than 14 days.

• Observe the cultures several times during the incubation period. Observe the containers of media periodically during the 14 days of incubation. If the test specimen is positive before 14 days of incubation, further incubation is not necessary.

• For products terminally sterilised by a validated moist heat process, incubate the test specimen for not less than 7 days.

• Observation and Interpretation of Results If no evidence of microbial growth is found, the preparation

under examination complies with the test for sterility. If evidence of microbial growth is found, the preparation under examination does not comply with the test for sterility

Pyrogens

The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 minutes

Test Animals:Use healthy, adult rabbits of either sex, preferably of the same variety,

weighing not less than 1.5 kg MAIN TEST:

Carry out the test using a group of three rabbits.

PREPARATION OF THE SAMPLE:

Dissolve the substance under examination in, or dilute with, pyrogen-free saline solution orother solution prescribed in the monograph. Warm the liquid under examination to approximately 38.5º before injection.

 

Pyrogens

INTERPRETATION OF RESULTS: If the sum of the responses of the group of three

rabbits does not exceed 1.4º and if the response of any individual rabbit is less than 0.6º, the preparation under examination passes the test. If the response of any rabbit is 0.6º or more, or if the sum of the response of the three rabbits exceeds 1.4º, continue the test using five other rabbits.

If not more than three of the eight rabbits show individual responses of 0.6º or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7º, the preparation under examination passes the test

Uniformity of content

Determine the content of active ingredient(s) of each of 10 containers taken at random, using the method given in the monograph or by any other suitable analytical method of equivalent accuracy and precision.

The preparation under examination complies with the test if the individual values thus obtained are all between 85 and 115 per cent of the average value.

The preparation under examination fails to comply with the test if more than one individual value is outside the limits 85 to 115 per cent of the average value or if any one individual value is outside the limits 75 to 125 per cent of the average value

.

If one individual value is outside the limits 85 to 115 per cent but within the limits 75 to 125 per cent of the average value, repeat the determination using another 20 containers taken at random.

The preparation under examination complies with the test if in the total sample of 30 containers not more than one individual value is outside the limits 85 to 115 per cent and none is outside the limits 75 to 125 per cent of the average value

Clarity of solution

Constitute the injection as directed on the label.

a) The solid dissolves completely, leaving no visible residueas undissolved matter.

b) The constituted injection is not significantly less clear

than an equal volume of the diluent or of water for

injections contained in a similar container and examined in the same manner.

Leakage

A LEAKERS TEST : It is a useful method for evaluating the

efficiency of the sealing process. The test consists of immersing completely

the sterile sealed ampoules in an aqueous dye bath (0.5 to 1.0% of methylene blue) within a vacuum chamber.

Negative pressure of 27 inches Hg or more is created, a tiny drop of dye solution can penetrate an opening of an incompletely sealed ampoule.

the colored ampoule are sorted out during washing and 100% inspection that follows after.