Quality Control of Sterile Products

Dr. Prafulla Kumar Sahu Raghu College of Pharmacy

QUALITY CONTROL OF STERILE PRODUCTS

PYROGEN TESTING

Pyrogens produce symptoms of fever, chill, joint pain, malaise, headache and other

complaints following IV injection within 30-120 minutes which may subside within 10-12 hours.

Pyrogens are the heat stable, filterable and soluble substances of 0.05-1.0 micrometer size and

arise from microbial contamination. Chemically these are lipopolysaccharides from the outer cell

wall of the bacteria, thus, the term endotoxins is also used interchangeably but not correct

entirely. Both G+ and G- bacteria produce pyrogens, however, the pyrogens of G- bacteria are

more potent. The pyrogens are heat stable up to some extent, thus, withstand normal

sterilization temperatures.

Depyrogenation

Depyrogenation is the removal of pyrogen. This is achieved by the following methods.

Inactivation - Application of very high dry heat (2500P) for not less than 30 minutes is the

desired method for rendering material pyrogen free.

Removal of pyrogen by distillation

Dr. Prafulla Kumar Sahu

M.Pharm., Ph.D.

Professor

Raghu College of Pharmacy

Dakamarri, Visakhapatnam, AP, India

Sample Questions Define sterile products. What are the features in sterile products? Define clean area. What are the classifications/specifications for clean areas? Write a detailed account of environmental control in sterile area. What do you know about the facilities in clean rooms? What is source of particulate in sterile products? What are several methods for testing of clarity of sterile products? Write detail account of production of sterile products. How liquid sterile products are filled into their containers? What are the in-process quality control tests employed on the sterile products? Discuss two methods for the testing of clarity/particulate matter in parenteral preparations? What is purpose and procedure of LAL test? What are the advantages of LAL test over in-vivo test? What is the purpose and procedure of rabbit test?


Detection and quantification of Pyrogens

1) In-vivo pyrogen (rabbit) test

In-vivo pyrogen test involves the evaluation of the presence of pyrogens in parenteral sample

by quantitative fever response produced in rabbits. The principle is based on the fact that the

human and rabbits are equally responsive to pyrogen injected intravenously on a dose per

weight basis. This test requires the following.

Test animals: healthy adult rabbits (of either sex) weighing not less than 1500 gm

(1.5kg). The animals have been properly maintained in terms of environment and diet prior to

the performance of test. The animals are screened for their temperature. Their control

temperature must not differ more than 1°C from each other. Any individual animal having

temperature 39.8°C or less than 38.0°C is excluded from the test.

The rabbit-retaining boxes are required to house the rabbits. These boxes "hold" the

rabbits so that the temperature can be noted easily during test. The specific directions given in

the individual monograph must be followed for the products.

The sample to be tested is injected with a slower rate to the animals. The dose of the

sample if not specified should be smaller than 10 ml/kg. Special preliminary steps are required

and thus, consideration must be given for the products requiring; 1) dilution, 2) pH adjustment,

and 3) isotonicity adjustment.

PROCEDURE

The control (baseline) temperature of three rabbits is determined. The sample is injected

into the ear vein of each of three rabbits which are held in the retaining boxes. A dose of

10ml/kg of body weight is used unless specified in the individual monograph. The temperature

of each rabbit is determined at 1, 2, and 3 hours subsequent to the injection of sample. The

difference between the initial and final temperatures of each rabbits is noted. Any increase in

temperature is taken to be the response of sample injected.

Interpretation of the results

The material under examination meets the requirements for apyrogenicity if no rabbit

shows an individual rise in temperature of 0.6°C or more above its respective control

temperature OR the sum of the temperature rise of 3 rabbits does not exceed 1.4°C. If the

results are not within the limits, the test is repeated for additional 5 rabbits and the result is

considered for the eight rabbits. After repeating, the material under examination meets the

requirements if not more than 3 out of eight rabbits show individual rise in temperature of 0.6 °C

OR the sum of rise in the temperature in eight rabbits does not exceed 3.7°C.


Sometimes the difference of initial and the final temperature is negative. If the difference

is negative, the result of the rabbit test is counted as zero response and the sample is

considered apyrogenic.

Advantages of Rabbit Test

The human and rabbits are equally responsive to threshold levels of the pyrogens.

2) Limulus Amebocyte Lysate Test

The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP XXI

Specified new test). Officially it is termed as bacterial endotoxin test (BET). The test principle is

based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in

the presence of pyrogens. The extract from the blood cells of horse shoe crab, Limulus

Polyphemus contains an enzyme and protein system called "Limulus- Amebocyte Lysate" (LAL)

which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an

opaque gel is formed.

Amebocyte + Pyrogen ~ Opaque gel

The reaction accomplishes within 15-60 minutes, depending on concentration of

pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick.

Requirements

Limulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart

puncture. The amebocytes are carefully concentrated, washed and lysed by osmotic effects.

Prior to perform the LAL test, lysate assay is carried out with purified endotoxins and are

accepted if it detects 0.001ug/ml or less concentration of the purified endotoxins.

The glassware, such as glass test tubes (10 x 75mm) used in the test must be

thoroughly cleaned, dry and heat sterilized. Abuffer solution of potassium phosphate 2mEq/ml is

used to adjust the pH of test sample at 7. The alcoholic content in sample is to be removed as it

causes precipitation of lysate. If the sample contains proteins, it produces gel thus the proteins

must be diluted to appropriate concentration before the test.

Similarly other interfering substances present in sample must also be removed before

the test.

Procedure

The pH of test sample if specified is adjusted. The test solution and standardized LAL

are separately mixed in equal parts (0.05-0.2ml). The mixture is incubated immediately at 36-

38°C for 1 hour in assay tube. The assay tube must be remained undisturbed completely

because agitation may irreversibly destroy the gel leading to a false negative result. The test

tube is observed after the specified time and is examined for the formation of opaque gel.


Formation of gel represents a positive test endpoint reaction. The test is performed using a

commercial LAL test kit. This kit contains a lyophilized LAL, and E. coli endotoxin and pure

water as standards and these later two are used to check the sensitivity of the test.

Advantage of LAL test

1. It is in-vitro and does not require animal handling, thus is more convenient

2. It is 10 times more sensitive than that of the in-vivo rabbit test

3. It is economical

4. It consume less time, i.e., 1 vs 3 hours required by rabbits test

5. It requires less laboratory facilities and minimum equipments

6. It requires less test volume

7. It is more accurate

INSTRUMENTS FOR EVALUATION OF PARTICULATE MATTER

System Working Principle Remarks

Visual Based Inspection

Autoskan Light Scattering Non-Destructive

Eisai Ampoule Inspection

(AIM) System

Light blockage (Shadow) Non-Destructive

Schering PDS/A-V System Light Scattering Non-Destructive

Electronic Particle Counters

Coulter Counter Change in Electric Resistance Destructive; Large errors in measuring flakes

and fibers;

Not recommended by FDA for parenterals

HIAC (High Accuracy

Instruments)

Light Blockage Destructive; High Efficiency,

Easy calibration;

Recommended by USP;

Expensive

Met-One Climet Particle

Counter

Light Scattering Destructive; Measures 6 particles sizes at a time;

Excellent large particle detection

Climet Instrument Light Obstruction Non-destructive


CLARITY TESTING (DETECTION OF PARTICULATE MATTER)

Particulate matter can be detected in parenteral product by two methods, including

visual inspection and electronic particulate counting.

A) Visual methods

I) Visual inspection by naked eye

In visual inspection, each injectable is inspected visually against white and black

backgrounds. The white background aids in diction of dark colored particles. The light or

reflective particles will appear against the black back ground. Some visual-enhancing aids can

increase the efficiency. A magnifying lens at 2.5 × magnification set at the eye level facilitates

the inspection. Microscopic examination enhances detection of particulate matter in injectables.

Visual inspection gives the qualitative estimation of the particulate matter. Acceptance

Standards is that each container checked must not contain any visible particulate matter.

II) Automated visual inspection

The automatic systems are also called as the electron particles counter. The electronic

particles counter evaluates the particles in injectables automatically. However, this method

requires destruction of the ampoule/container for the particle examination.

Electronic particles counting may be based on any one of the following principles: a)

change in electrical resistance, b) light blockages principle and c) light scattering. Some of the

automated systems for visual particle inspection include Autoskan, Eisai Ampoule inspection

machine, Schering PDS/A-V system, etc. Autoskan System.

The Autoskan system is based on light scattering principle whereby the particle in the

path of a light source causes the scattering of light. The scattered light is measured and

provides the corresponding information regarding the presence of particulate in the sample. This

is a non-destructive test.

EISAI AMPOULE MACHINE SYSTEM

The Eisai ampoule machine (AIM) system is based on the light blockage principle. The

particle size dimensions are determined with the shadow created by the particle under light

source. Assessment of the shadow is the indication of the presence of particulate matter. This is

also a non-destructive test.

Schering PDS/A-V system

The Schering PDS/A-V System is based on light scattering by particle if present in the sample.

This is also a non-destructive test.


B) PARTICLE COUNT METHODS

Particle count methods are the USP specified microscopic methods, which require the

use of optical microscope and automatic microscope.

I) Optical Microscopic Method

The optical microscopic method requires magnification of 100 k.10x. One eyepiece must

be equipped with graticule. A graticule have a series of circles of different diameters, usually in

a “under root 2 progression”. The graticule is in circular diameter used to size the particulate.

The micrometer is graduated in 10 micro meter increments.

A circular diameter graticule

II) Automated Particle Counters

The automated particle counters are based on the light obscuration, light scattering

method and the electrical resistance methods. Coulter Counter counts the particles in a sample

based on the change in the electrical resistance. Particle size detection limit in this instrument is

from 0.1 to 1000 micrometer.

The powder sample requires pretreatment such as dispersion in an electrolyte to form a

very dilute suspension. The Suspension is usually subjected to ultrasonic agitation to avoid

particle agglomerates. A dispersant may also be added to aid particle deagglomeration.

Passage of particle causes the change in electrical resistance in between the electrodes which

is proportional to the volume of particle. The change in resistance is converted into voltage

pulse which is amplified and processed electronically and split into the particle size distribution

into many different size-range.

Glass Tube

Orifice

Principle of coulter counter

This is a destructive test and large errors in measuring flakes and fibers are expected.

This test is not recommended by FDA for parenterals.

Illustration for a coulter counter

High Accuracy (HIAC) Instrument

High Accuracy (HIAC) Instrument is based on light blockage principle. The test is highly

effective for counting the both solid and liquid suspended particles. The instrument is calibrated

easily and the test is recommended by USP. This is destructive test method and is expensive.

P Light source

Digital Value


Principle of light blockage

Met-One Climet Particle Counter

Met-One Climet Particle Counter is based on light scattering principle. The particles are

assessed and counted in the sample based on the principle of light scattering. The instrument

measures 6 particles sizes at a time and has the excellent ability for the detection of large

particle. The test is destructive.

Depending on the instrument used, the sample may be presented as the liquid as

suspension or air suspension. The light emitted by a helium-neon laser is incident on the

sample particle. Light-particle interaction results in scattering of light. The photo detector

converts the signals corresponding area/volume diameter of the particle.

Instrument based on light scatter principle

BIOLOGICAL HAZARDS REPORTED OF PARTICULATE MATTER

The particles may localize in lungs, liver, spleen and myocardial tissues and may lead to

thrombosis, Particles may lead to myocardial infarction due to embolic fibers. Injection of

solution contaminated with particulate matter causes granulomas and emboli in lungs

Compendial requirements.

Due to having a potential for blockage of the capillaries, an injection must be free from

the visual evidence of particulate contamination. Thus, according to the Compendial

requirements, each final container of the injectable must be inspected individually and the

container must show evidence of contamination with visible foreign material.

The in-process quality control test includes the leak and clarity testing. The quality

control of finished product required the pyrogen and sterility testing.

Leakage test

Leakage test is employed to test the package integrity. Package integrity reflects its

ability to keep the product in and to keep potential contamination out”. It is because leakage

occurs when a discontinuity exists in the wall of a package that can allow the passage of gas

under pressure or concentration differential existing across the wall. Leakage differs from

permeation, which is the flow of matter through the barrier itself. Followings are the leak test

methods.

A) VISUAL INSPECTION

Visual inspection is the easiest leak test method to perform. But this method is least

sensitive. The method is used for the evaluation of large volume parenterals. To increase the

sensitivity of the method, the visual inspection of the sample container may be coupled with the

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application of vacuum to make leakage more readily observable. This method is simple and

inexpensive. However, the method is insensitive, operator dependent, and qualitative.

Sometimes, the method is used in combination with pressure and /or temperature

cycling to accelerate leakage to improve sensitivity.

B) BUBBLE TEST

The test package is submerged in liquids. A differential pressure is applied on the

container. The container is observed for bubbles. Sometimes, surfactant added liquid is used for

immersion of test package. Any leakage is evident after the application of differential pressure

as the generation of foaming in immersion liquid. The method is simple and inexpensive. The

location of the leaks can be observed in this method. However, it is relatively insensitive and the

findings are operator dependent and are qualitative. The optimized conditions can be achieved

using a surfactant immersion fluid along with the dark background and High intensity lighting.

Generation of a differential positive pressure of 3 psi inside the vial and observation of any

leakage using magnifying glass within a maximum test time of 15 minutes.

C) DYE TESTS

The test container is immersed in a dye bath. Vacuum and pressure is applied for some

time. The container is removed from the dye bath and washed. The container is then inspected

for the presence of dye either visually or by means of UV spectroscopy. The dye used may be

of blue, green, yellowish-green color. The dye test can be optimized by use of a surfactant and

or a low viscosity fluid in the dye solution to increase the capillary migration through the pores.

The dye test is widely accepted in industry and is approved in drug use. The test is inexpensive

and is requires no special equipment required for visual dye detection. However, the test is

qualitative, destructive and slow. The test is used for ampoules and vials.

D) VACUUM IONIZATION TEST

Vacuum ionization test is useful for testing leakage in the vials or bottled sealed under

vacuum. This test is used for online testing of the lyophilized products. High voltage, high

frequency field is applied to vials which to cause residual gas, if present to glow.

Glow intensity is the function of headspace vacuum level. The blue glow is the indicative

of vacuum while the purple glow indicative of no vacuum. The sensitivity of the method is not

documented. This test is on-line, rapid and is non destructive test. However, the proteins

present in the test sample may be decomposed. This method is used for the lyophilized vials of

biopharmaceuticals.

CLARITY TESTING

Clarity testing is carried out to check the particulate matter in the sample.


Particulate matter

Matter of biological or non-biological origin and with observable length, width, and

thickness, e.g., bacteria, fungi, dust, dirt, fibers, plastic, rubber, lint etc. It may be any matter,

mixed accidentally during manufacturing in the parenteral product which does not belong to the

product. Particulate mater may be tiny pieces of lint, glass, dust, rubber, metal fibers, hair,

microbes or unidentified and can make the product impure, unclean or unfit for use.

Sources of particulate matter

Particulate contamination particularly of cellulose fibers, dust, cotton fibers, hair, dandruff

and loose skin from human origin as well as microbial contamination may arise from the

following main sources.

1. Material arising from the drug: undissolved substances and trace contaminants etc.

2. Material arising from vehicle or added substances: These may include those material not

filtered out during a clarification process before to filling the final container.

3. Materials present in the final container: Material already present in container and which

were not removed by rinsing prior to filling

4. Materials falling by chance into the final container during the filling process

5. The container or closures which may be deposited in the produce during sterilization,

e.g. carbon black, whiting, zinc oxide and clay

6. Packaging components: Including glass, plastic, rubber, I/V administration sets, etc.

7. Environmental contaminants: Including air, work tops, insects’ parts

8. Processing equipments: Including glass, stainless steel, rubber, or filter fiber, etc.

9. Personnel: Including skin, hair, and clothing etc.

Particle size

Particles present in injectable are non-reactive, apyrogenic, sterilized. However, by

virtue of their size may biologically hazardous. The particulate matter may be capable of

blocking the blood vessels with severe results on induction into body with injection.

A person with 20/20 vision under inspection conditions is able to detect particles of size

range 40 – 50 μm. However, it is universally accepted that the particles size of 50 μm is

detected visually by an unaided eye.

Particle size greater than 7 μm diameter is considered to be more threatening.

Pulmonary capillary are approximately 7 μm in diameter, thus particle of this much size

entrapped in vascular bed resulting in multiple pulmonary infarction.


SEALING OF STERILE PREPARATIONS

E) Sealing

The container should be sealed in the aseptic area immediately adjacent to the filling

machine. In addition to retaining the content of the sterile product, sealing of containers assures

sterility of its contents. Temper-proof sealing is essential so as the sterility can be ensured until

usage. Different approaches have been used for sealing of ampoules and the bottles.

Sealing of ampoules

The ampoules can be sealed either by tip or bead seal or pull seal. Both of the methods

require heating with high-temperature oxygen flame. During sealing, the heating must be even

and carefully controlled to avoid distortion of the seal. It is sometimes necessary to displace the

air in the space within the ampoule above the product to prevent decomposition. This may be

done by introducing a stream of inert gas, such as nitrogen or carbon dioxide, during or after

filling with the product. Immediately thereafter, the ampoule is sealed before the gas can diffuse

out. The tip seals are made by melting sufficient glass at the tip of the ampoules neck to form a

bead of glass and close the opening. Thus, tip seal is also known as bead seals since a bead is

formed during melting of the neck. Excessive heat of air and gases in the neck cause expansion

against the soft glass with the formation of fragile bubbles at the point of seal. Open capillaries

at the point of seal or cracks result in leakers. Fracture of the neck of ampoule often occurs

during sealing if wetting had occurred at the time of filling, Also wet glass at the neck increases

the frequency of bubble formation and contaminating deposits of carbon or oxides as a result of

the effect of the heat of sealing on the droplet of the product. Pull seals are made by heating the

neck of the rotating ampoule below the tip, then pulling the tip away to form a small, twisted

capillary just prior to being melted closed. Pull sealing is a slower process, but the seals are

more reliable than those from the tip sealing. Powder ampoules or other types having a wide

opening must be sealed by pull-sealing.

With some sensitive products, it may be necessary to seal the ampoules with pull-seals

to prevent combustion produces of the flame from entering the ampoule at the time of sealing,

as might occur with tip-sealing.

Sealing of bottles, cartridges and vials

The closure is to be slide from a rotating or vibrating drum to the bottom of a chute,

where it is positioned over a container ready for insertion by a plunger or some other pressure

device which is followed by stoperring. To facilitate sliding of rubber closures, their surface is

halogenated or coated with silicon which reduces the friction during slipping into the container’s

mouth.

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Aluminum caps are used to hold rubber closures in place. Single caps may have a

permanent center hole or a center that is torn away at the time of use to expose the rubber

closure. When applied, the bottom edge of the aluminium cap is bent (crimped) around and

under the lip of the glass container. It cannot be removed without destroying the cap but

perforation permit tearing away the portions of the cap to be discarded. Crimping can be

achieved using the heavy duty crimping machines.

FILLING OF POWDERS

Sterile solids are more difficult to subdivide accurately and precisely into individual dose

containers than are liquids. The rate of flow of the solid materials tends to be slow and irregular,

particularly if the powder is finally divided. Small granular particles flow most evenly. Uniform

particle size and good flow properties of solids are necessary for uniform and effective filling by

machines. For powder showing poor flow, the containers with a relatively large opening must be

used, even so, the filling rate is slow and the risk of the spillage is ever present. For these

reasons, the tolerances permitted for the contents of such containers must be relatively large.

Relatively freely flowing solids are filled using filling machines. One type of machine for delivery

of measured quantities of solid material employs an augar in the stem of the funnel-shaped

hopper. The size and rotation of the augar can be adjusted to deliver a regulated volume of

granular material from the funnel stem into the container.

In another filling machine, a adjustable cavity in the rim of the filling wheel is filled by

vacuum as the wheel passes under the hopper. The contents are held by vacuum until the

cavity is inserted over the container when a jet of sterile air discharges the solids. This machine

also dispenses dry solid that flow less freely.

D) FILLING PROCEDURE

The filling process has been categorized as the filling of low density and viscosity liquids,

filling of viscous liquids and filling of solids. Filling equipment has a reservoir to hold bulk

product. The reservoir is connected to delivery tube to dispense product into container. A mean

is provided for repetitively forcing a measured volume/amount through the orifice of a delivery

tube. The accuracy and the precision of the machine filling of sterile liquids vary with the

method. Therefore, a method is selected to provide the degree of accuracy and precision

required by the nature of the product. The slightest excess is required in each container to

provide for the loss that occurs at the time of the withdrawal of dose at the time of administration

due to adherence of container content to the wall of container and retention in the syringe.

Filling machines should be designed so that the part through which the liquid flows can be easily

demounted for cleaning and for sterilization. These parts also should be constructed of non-

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reactive materials, such as borosilicate glass or stainless steel. Syringes are usually made of

stainless steel, when the pressure required for delivery of the viscous liquid or large volumes

would be useful for glass syringes.

Filling of low viscosity small volume liquid preparations

A liquid may be subdivided from a bulk container to individual dose containers more

easily and uniformly than a solid. Mechanical subdivision of a mobile, low density liquid can be

achieved with light-duty machinery. Certain fundamental features are found on all filling

equipments for liquid preparations. The filling equipment has a reservoir to hold the bulk of the

liquid preparation to be filled into containers. A means is provided for repetitively forcing a

precisely measured volume of the liquid through the orifice of a delivery tube designed to enter

the constricted opening of a container. The size of the delivery tube is governed by the opening

in the container to be used, viscosity and density of the liquid and the speed of the delivery

desired. The tube must enter freely into the neck of the container and deliver the liquid deep

enough to permit air to escape without sweeping the entering liquid into the neck or out of the

container. To reduce the resistance to the follow of the liquid, the tube should have the

maximum possible diameter. Excessive force of delivery causes splashing of the liquid and

troublesome foaming, if the liquid has a low surface tension. The delivery of relatively small

volumes of liquids is usually obtained with pressure obtained from the strokes of the plunger of

a syringe. The stroke of the syringe forces the liquid through a two-way valve that provides for

an alternate filling of the syringe from a reservoir and delivery to a container. A drop of liquid

normally hangs at the tip of the tube after a delivery. When the container to be filed is an

ampoule, withdrawal of the tube without wetting the long restricted neck is almost impossible,

unless the hanging drop of the liquid is retracted. Thus, a retracting device is designated as a

part of the most filling machines.

Filling of low viscosity large volume liquid preparations

Sterile solutions of relatively low potency dispensed in large volumes (up to 1 liter) do

not normally require the precision of filling that is required for small volumes of potent

injectables. Therefore, the liquid is filled into the bottles by gravity, pressure or vacuum filling

devices. Generally, gravity filling is relatively slow, but is accomplished with simpler means. A

liquid reservoir is positioned above the filling line, with a hose connection from the reservoir to a

shut-off device at the filling line. The shut-off device is usually hand operated, and the bottles

are filled to graduations on the bottles.

The pressure pump filler often is operated semi-automatically and differs from the gravity

fillers, principally in that the liquid is under pressure. It is usually equipped with the overflow tube


connected to a receiver to prevent excess flow of the container. Vacuum filling is commonly

used in faster filling lines for large liquid volumes because it is more acceptable for automation.

A vacuum is produced in a bottle when a nozzle gasket makes a seal against the tip of the

bottle to be filled. The vacuum draws the liquid from a reservoir through the delivery tube into

the bottle. When the liquid level reaches to a level of an adjustable overflow tube, the seal is

mechanically loosened and the vacuum is released. Any liquid that had been drawn into the

vacuum line is collected in a trap receiver and then returned to the reservoir.

Filling of high viscosity liquid preparations

The viscous, sticky or high density liquids require much more heavy machines to

withstand the pressure required to dispense them in individual containers. Thus, compared to

the plunger-syringe assembly for filling of low viscosity liquid, for heavy, viscous liquids, a sliding

piston valve provides more positive action. Emulsion, suspensions and semisolid preparations

often require specially designed filling equipments because of their high viscosity. To obtain a

reasonable flow rate of the emulsion and suspension, high pressure must be applied or

container with large openings must be used to permit the entry of large delivery tubes.

Sometimes the jacketed tanks can be used to raise the temperature of the product to facilitate

filling by lowering its viscosity. It is normally necessary to keep suspensions and sometimes

emulsion, constantly agitated in the reservoir during filling so that the product remains

homogeneous and each subdivided unit contains the required amount of drug.

C) FORMULATION OF STERILE PRODUCT

The product formulation is sometimes, just compounding of the ingredients. In other

situations, the parenteral products are formulated as emulsions, suspensions, cream and

ointments. All the process are undertaken in strict aseptic conditions.

Compounding of ingredients

The ingredients should be compounded under clean environmental conditions. A sterile

condition is usually not required since it may not be possible or feasible to sterilize some of the

ingredients or equipment, e.g., large tanks. Whenever possible, however, the equipments and

the ingredients should be sterile to reduce the microbial load.

The compounding process should meet the rigid standards accepted in pharmaceutical

procedures, regardless of the batch size, recognizing that small multiple errors may be additive.

In large batches particular attention must be given to achieving and maintaining homogeneity of

solution, suspensions and mixtures, maintaining a given temperature and accelerating cooling.

The order of mixing ingredients may become highly significant, for example, owing to the

physical problem of disturbing a pH adjusting ingredient throughout a large tank of liquid.


Compounding problems for large batches of product often are different from those of the small

batches.

B) Sterilization methods

Six sterilization methods are available and are selected based on the item, material of

product to be sterilized. These include; 1) sterilization by steam, 2) sterilization by dry heat, 3)

sterilization by ethylene oxide, 4) sterilization by filtration, 5) Lyophilization, and 6) sterilization

by -radiations. A detail description of the methods has already been given in previous classes.

CLEANING RUBBERY PLASTIC COMPONENTS

The rubber closures are usually washed by mechanical agitation in a tank of hot

detergent solution (such as 0.5% sodium pyrophosphate) followed by a series of through water

rinses, the final rinse being WFI. The objective is to remove the surface debris accumulated

from the molding operation and from handling and leachable constituents at or near the surface.

Part of the debris is attached and held on the surface by electrostatic forces. Similarly, plastic

materials accumulate surface debris.

The multiple objectives for washing closures and other parts include loosening debris,

minimizing abrasion and sweeping away the loosened debris.

CLEANING OF CONTAINERS, GLASSWARE AND METAL WARES

Like instruments, the unused containers are contaminated with dust, fibers, and

chemical films. These are removed by vigorous treatment with hot detergents. The containers

are inverted on spindles in the front of the cleaning machine and are automatically conveyed in

an inverted position. During rotation of the cleaning machine, they are carried through a series

of rigorous, high pressure treatment including hot detergent, hot tap water and final rinses with

distilled water. Because many containers have restricted openings, it is essential that the

treatments in any washer be introduced though tubes into each container with smooth outflow.

For ampoules or containers with a markedly constricted opening that makes water drainage

incomplete, the final treatment is usually a blast of clean air to blow out remaining water.

After cleaning, it is essential that the clean containers be protected from dust and other

particulates that might be present in the environment. Therefore, the clean containers are often

removed from the rinser and placed in clean stainless steel boxes for sterilization under the

protection of HEPA filtered airflow. Glassware and metal ware (small) may also be

contaminated from previous use and can also be mechanically like containers in their converted

position during automatically conveying through a series of rigorous, high pressure treatment

including hot detergent, hot tap water and final rinses with distilled water. In cleaning new

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glassware, the detergent treatment is usually eliminated, and with it the risk of the detergent

residue.

With rinsing alternatively by hot (preferably clean steam) and cold treatments should be

used to lessen the debris. Final rinses should be done with filtered WFI.

A) CLEANING OF EQUIPMENTS, CONTAINERS AND GLASSWARE (1)

Cleaning of Equipments

The equipments to be used in the processing of the sterile products must be thoroughly

cleaned. The new and unused equipments are contaminated principally with dust, fibers, and

chemical films which usually are relatively easy to remove often by rinsing only.

Debris that is more dangerous and more difficult to remove may be present as residue

from the previous use. These are removed by vigorous treatment with hot detergents.

Whenever possible, large equipments should be disassembled so that each part can be

thoroughly scrubbed and clean with particular attention given to screw threads, joints and other

dirt collecting structures. After cleaning, the equipments should be rinsed several times with final

rinse with water for injection (WFI). Just prior to re-use, large cleaned tanks and similar

equipments should be rinsed thoroughly with WFI. Reserving of the equipment for use with only

one type of the product reduces the cleaning problems.

A new method for large tanks, pipe lines and associated equipments that can be isolated

and contained within a process unit has been developed and identified as clean in place (CIP)

system. Under this system, cleaning of the dedicated instruments for specific products is

accomplished primarily with high pressure rinsing treatments, delivered automatically within the

equipment. This is usually followed by steam sanitization through the same system.

STERILE PRODUCTION PROCESS

The general production process started from the accumulation of raw materials,

ingredients and packaging components and then combining of the ingredients of the formula

into a product. The prepared product is enclosed in the individual containers for distribution.

Sterilization during production and/or after production is also employed.

During production, in-process quality control and the finished product quality control is

also performed. The required equipments are cleaned prior to the start of the sterile production

process.

STANDARD OPERATING PROCEDURES

To enhance the assurance of the successful manufacturing operations, all process steps

must be carefully written. The written process steps are often called standard operating

procedures (SOP). No extemporaneous changes are permitted in the SOP. Any change in SOP

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must go through the same approval steps as the original written SOP. According to the FDA

guidelines on the good manufacturing practices, extensive records must be kept to assure, at

the end of the production process in that all steps have been performed as prescribed.

Such in-process control is essential to assure the quality of the product. Since this

assurance is even more significant than those from product release testing. Production of

quality product is a result of continuous, dedicated efforts of the quality assurance, production

and quality control personnel within the plan in developing, performing and confirming effective

SOP.

The process equipments and the components of the containers, cleaned thoroughly

according to the required specifications are assembled in a clean environment. The assembled

components are preferably sterilized and depyrogenated prior to use. All equipments and the

supplies, introduced into the aseptic filling areas should be sterilized.

The outer surfaces of the boxes, packages, or equipments should be wiped with a

disinfectant solution as they are transferred to clean room. All the supplies must be introduced

into the aseptic filling room in such a manner that the aseptic state of these room is maintained,

thereby, preventing the introduction of environmental contamination into the product while it is

being subdivided into individual containers. After sealing of containers, contamination cannot

enter into the container and the product. Thus the product is sealed in its final container within

the aseptic room from where it is transported to packaging area. This area is maintained clean

but need not meet the standard imposed for the aseptic room or for the sterile compounding

room. Packaged products are placed in quarantine storage until all tests have been completed

and in-process control records have been evaluated.

PERSONNEL FOR STERILE PREPARATIONS

F) Personnel

The most ideally planned processes can be rendered ineffective by personnel who do

not have the right attitudes or training. The personnel who produce sterile products usually are

non-professional person, supervised by those with professional training. To be effective

operators, they must inherently neat, orderly, reliable and alert and have good manual dexterity.

They should be appreciative of the vital role that every movement lies in determining the quality

of final product, it its freedom from contaminants.

All employees should be in good health and should be subjected to periodic physical

examinations. They should understand their responsibility to report the developing symptoms of

cold, sore throat or other infectious diseases, so that they can be assigned to a less critical area

until they have fully recovered.

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The attire won by personnel in the aseptic areas usually consists of sterile coveralls,

hoods, face masks and show covers. Sterile rubber gloves also may be required. Personnel

entering the aseptic areas should be required to follow a definite preparatory procedure. This

should include removing at least outside street clothing, washing the hands and arms

thoroughly with a disinfectant soap, and donning the prescribed uniform.

A full body water and soap shower would be essential n most biologic products

processing plants – usually, both when entering and leaving the area to control contaminations

in both directions, between personnel and the product. Since people are continually shedding

viable and non-viable particulate matter from body surfaces, uniform are worn to help to control

this emission. The uniform should, preferably be of coverall type and made of synthetic fibers

such as Dacron. Dacron cloth is made of a continuous fiber, which makes it essentially lint-free

and in air conditioned room, is acceptably comfortable.

ANOTHER SYSTEM OF CLASSIFICATION FOR CLEAN ARE

ANOTHER SYSTEM OF CLASSIFICATION FOR CLEAN AREA

Class Number of Particle Diameter (um)

0.1 0.3 0.5 5

1 35 3 1

10 350 35 10

100

300 100

1000

1000 7

10,000

10,000 70

100,000

100,000 700

Federal Standard 209 (FED STD 209) For Clean Area

1. Class 100,000: Particle count not to exceed a total of 100,000 particles per cubic foot of

a size 0.5μ (micron) and larger or 700 particles per cubic foot of a size 5.0μ (micron) and

larger.

2. Class 10.000: Particle count not to exceed a total of 10,000 particles per cubic foot of a

size 0.5μ (micron) and larger or 65 particles per cubic foot of a size 5.0μ (micron) and

larger.

3. Class 1,000: Particle count not to exceed a total of 1000 particles per cubic foot of a size

0.5μ (micron) and larger or 10 particles per cubic foot of a size 5.0μ (micron) and larger.

4. Class 100: Particle count not to exceed a total of 100 particles per cubic foot of a size

0.5μ (micron) and larger.

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(The Clean Room class is generally achieved in the "at rest" state when there are no people in

the room)

British Standard 5295 For Sterile Area

British Standard 5295

Class 1: The particle counts shall not exceed a total of 3000 particles/m3 of a size of

0.5μ (micron) or greater. The greatest particle present in any sample shall not exceed 5μ

(micron).

Class 2: The particle count shall not exceed a total of 300,000 particles/ m3 of a size

0.5μ (micron) or greater: 2000 particles/m3 of a size 5μ (micron) or greater: 30 particles of a

size 10μ (micron) or greater.

Class 3: The particle count shall not exceed 1,000,000 particles of a size of 1 micron or

greater: 20,000 particles/m3 of a size 5μ (micron) or greater: 4000 particles/m3 of a size 10μ

(micron) or greater; 300 particles/m3 of a size 25μ (micron) or greater.

Class 4: The particle count shall not exceed a total of 200,000 particles/m3 of a size 5μ

(micron) or greater: 40,000 particles/m3 of a size 10μ (micron) or greater: 4000 particles/m3 of a

size 25μ (micron) or greater. (3000 particles/m3 at 0.5um converts to about 85 particles/Ft3)

PRODUCTION FACILITIES FOR STERILE PRODUCTS (Continued...)

A) Environmental control

Effective environmental control, both physical and biologic is essential but the level

achievable is related to the characteristics of the facility. Further rigid standards from plant to

plant and from geographic location to another are not appropriate. Allowance also must be

made for variation in control associated with the seasonal conditions.

The standards of environmental control vary depending on the area involved (cleanup,

packaging, compounding or filling) and type of product being prepared. Unquestionably, the

entire area used for the preparation of a product prepared aseptically (without terminal

sterilization) must be maintained under the most rigid control that the existing technology

permits. If the product is to be terminally sterilized, somewhat less rigid biologic control of the

compounding and filling areas may be acceptable. However, rigid standard of cleanliness must

be maintained. High standards of cleanliness, excluding daily use of disinfecting procedures are

usually acceptable for the cleanup and packaging areas.

B) Traffic control

Carefully designed arrangement to control and minimize traffic, particularly ‘in’ and ‘out’

of the aseptic areas is essential. Access by personnel to the aseptic corridor and aseptic

compound and filling rooms will be only possible through an airlock. Pass-through openings and

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double ended sterilizers are provided to permit controlled passage of supplies from non-aseptic

to aseptic area.

Persons should be permitted to enter aseptic areas only after following rigidly prescribed

procedures for removing street clothing, washing their hands and putting on gowns, hats, shoes,

facemasks, gloves and other prescribed attire. Once they have entered the aseptic area, they

should not be permitted to move in and out of the area with out regowning.

Personnel assigned to cleaning and packaging should be restricted to these areas.

Unauthorized personnel should never be permitted to enter the aseptic area.

C) House keeping

All equipment and the surrounding work area must be cleaned thoroughly at the end of

the working day. No contaminating residues from the concluded process may remain.

The ceiling, walls, and other structural surfaces must be cleaned with a frequency which

is most appropriate. All cleaning equipments should be selected for its effectiveness and

freedom from lint-producing tendencies. It should be reserved for use in the aseptic areas only.

D) Surface disinfection

After through cleaning all surfaces should be disinfected, at least in the aseptic areas.

An effective liquid disinfectant should be sprayed or wiped on all surfaces. Irradiation from

ultraviolet lamps that are located provide adequate radiation intensity on the maximum extent of

surfaces in a room and that are maintained free from dust and films further reduces the viable

microorganisms present on the surface and in the air.

Ultraviolet rays may be particularly useful to irradiate the inside exposed surfaces of the

processing tanks, surfaces under hoods. The surface of the conveyor belts and the similar

confined surfaces those are otherwise, difficult to render aseptic. However they cannot reach

unexposed surfaces such as pipe connections to tanks, the undersides of conveyors and the

inside of containers.

The UV lamps must be kept clean and care must be taken to check for a decrease in

effective emission, a natural occurrence due to a change in the glass structure with aging.

E) Air control

In any area occupied by personnel, air must be exchanged at frequent intervals. Fresh

outside or recycled air must first be filtered to remove gross particulate matter. A spun glass,

cloth, or shredded polyethylene filter may be used for this preliminary cleaning operation.

At times, more than one pre-filter may be used in series, the first one is quite large and

the next somewhat smaller pore size to provide a gradation of particle size removal from heavily

contaminated air. To remove finer debris down to the submicron range, including


microorganism, a high efficiency particulate air (HEPA) filter is used. The HEPA filter has been

defined as at least 99.97% efficient in removing particles of 0.3um size and larger and

composed of glass fibers and filters or electrostatic precipitators may be employed. Air passing

though these units can be considered virtually free from foreign matter. Another air cleaning

system washes the air with a disinfectant and controls the humidity at the same time.

Blowers should be installed in the air ventilation system upstream to the filters, so that all

the dirt producing devices are ahead of the filter. The clean air is normally distributed to the

regulated areas by means of metal (preferably stainless steel) ducts. Since it is practically

impossible to keep these ducts as clean as required, it is normally preferred to install HEPA

filters at the where the clean air enters the controlled room. Alternatively, the ducts may be

replaced with a room (a plenum) usually above the production area, into which clean air is

blown and then distributed through opening into each of the process rooms. The entire plenum

can be kept clean and aseptic.

The clean and aseptic air is distributed in such a manner that it flows into the maximum

security room at the greatest volume flow rate, thereby producing a positive pressure in these

areas. This prevents unclean air from rushing into the aseptic area though cracks, temporarily

opened doors or other openings. The pressure is reduced successively so that the air follows

from the maximum security area to other less critical areas for return to the filtration system. At

the intake end of the system, fresh air usually about 2% is continually introduced for the comfort

and needs of the personnel. Further, the air is usually conditioned with respect to the

temperature and humidity for the comfort of the personnel and sometimes to meet the special

requirements of a product.

Horizontal laminar flow hood Vertical laminar flow hood

A relatively new air control system, based on laminar flow principle, has greatly improved

the potential for environmental control of aseptic areas. Currently, it is the only means available

for achieving a class 100 clean room. A class 100 clean room is defined as a room in which the

particle count in the air is not more than 100 per cubic feet of 0.5um and lager in size. The air

filtered through HEPA filter is blown evenly out of the entire back or top of the work bench or

entire side or from ceiling of a room. The air flow must be uniform in velocity and direction

throughout any given cross-section of the area, being exhausted from the opposite side. The air

velocity employed should be 100 k 20 ft/min.

Contamination is controlled because it is swept away with the airflow.

Although class 100 work environments are normally specified for the most critical aseptic

and or clean operations associated with the parenteral preparations, achieving such levels of


cleanliness is expensive and requires effective maintenance and monitoring. It should be

recognized that not all operations associates with parenteral medication require such an

environment. To such an end other classes are defined. For example, a class 10,000 room is

one in which the particle count is not more than 10,000 per cubic feet of 0.5 um and large size.

Such a cleanliness level is usually considered suitable for buffer areas around class 100

worksites in which operations such as handling of pre-cleaned containers, process filtration and

aseptic gowning of personnel may be performed. Still less stringent requirements would be

applied to laboratories, stock staging areas, and finish packaging where a class 100,000 or

similar cleanliness levels would be considered suitable.

Different classes and standards of clean rooms:

The determination of how clean an area is, depends on the classification that it has been

designed with different standards. Four different classes are described according to British

Standard system 5295 and Federal Standard 209 (FED STD 209).

Documents

Quality Control of Sterile Products