STERILE

PREPARATIONS
STERILE PRODUCTS
Dosage forms of therapeutic agents that are free of
viable organisms

Are unique because they are injected through the skin or

mucous membranes into internal body compartments

All component and processes are selected and designed

to eliminate contamination of all types whether physical,
chemical or microbiological origin
 Classification
(based on route of administration)
1. Parenteral preparations
intended for injection under or through one or
more layers of the skin or mucous membranes
5 Most common routes
a. Intravenous
b. Intramuscular
c. Subcutaneous
d. Intracutaneous (Intradermal)
e. Intraspinal
2. Ophthalmic preparations
for the eye

3. Otic
for the ear

4. Nasal preparations
for the nose and throat

5. Irrigating solutions
for washing wounds or abraded mucous
membrane
COMPONENTS:
Components used in the compounding of sterile
preparations must be of highest quality and should have the
following characteristics:

1. Therapeutically effective (when used as active ingredient)

2. Provide maximum safety
3. Function efficiently (when used as excipient)
4. Free from contamination
5. Physically and chemically stable even after thermal
sterilization
6. Produce little or no tissue irritation at site of
administration

On the basis of their functions, components are

classified on their therapeutic or active ingredient, vehicle
and additives.
VEHICLES
- WATER most frequently employed vehicle for sterile
products

- WATER FOR INJECTION superior quality

- Water of highest quality is prepared by distillation or

reverse osmosis
TOTAL SOLIDS CONTENT
one of the most inclusive tests for the quality of
water
officially permitted amount for total solids is 10 ppm

-A less time consuming test, the ELECTROLYTE

MEASUREMENT OF CONDUCTIVITY OF THE WATER, is
the one most frequently used.

- WATER FOR INJECTION normally should not have a

conductivity of more than 0.1 ppm NaCl
PYROGENS
lipid substances associated with a carrier molecule, w/c is
usually a polysaccharide but may be a protein

Product of the metabolism of micoorganisms such as

most bacteria, many molds and viruses

Any raw material, container or facility that is

contaminated with microorganisms is a possible source of
pyrogens
 Produce febrile reactions about an hour after injection
into man this is accomplished by chills, body aches,
cutaneous vasoconstriction and a rise in arterial blood
pressure

Antipyretics eliminate the fever, but not the other

systemic effects of pyrogens
2 Official Tests for Detecting and Measuring
Pyrogens (USP XXI):

1. BACTERIAL ENDOTOXINS TEST

2. PYROGEN TEST
1. BACTERIAL ENDOTOXINS TEST

test for estimating the concentration of bacterial

endotoxins that may be present in a sample using Limulus
Amebocyte Lysate (LAL) which has been obtained from
aqueous extracts of the circulating amebocytes of the
horseshoe crab, Limulus polyphemus, and w/c has been
prepared and characterized for use as a LAL reagent for
gel-clot formation
 Where the test is conducted as a limit test, the specimen
is determined to be positive or negative to the test judged
against the endotoxin concentration specified in the
individual monograph

Where the test is conducted as an assay of the

concentration of endotoxin, with calculation of confidence
limits of the results obtained, the specimen is judged to
comply with the requirements if the result does not exceed.
a. The concentration limit specified in the individual
monograph
b. The specified confidence limits for the assay
 In either case, the determination of the reaction end-
point is made with dilutions from the material under test in
direct comparison with parallel dilutions of a reference
endotoxin and quantities of endotoxin are expressed in
defined ENDOTOXIN UNITS (EU)

The procedures include incubation for a preselected

time of reacting endotoxin and control solutions with LAL
Reagent and reading of the spectrophotometric light
absorbance at suitable wavelengths.
2. PYROGEN TEST

Designed to limit to an acceptable level of the risks of

febrile reaction in the patient to the administration, by
injection, of the product concerned

The test involves measuring the rise in temperature of

rabbits following the IV injection of a test solution

This is designed for products that can be tolerated by

the test rabbit in a dose not to exceed 10 mL/kg injected IV
w/in a period of nmt 10 mins.
If no rabbit shows an individual rise in temp. 0.6C or
more above its respective control temp., and if the sum of
the three individual maximum temp. rises and does not
exceed 1.4C, the product meets the requirements for the
absence of pyrogens.
Pyrogens may be destroyed or eliminated through
DEPYROGENATION METHOD:

1. Adequate washing with detergent treatment followed by

dry-heat sterilization is recommended for glasswares and
equipment.
Optimum temp. is 250C for 45 minutes or 180C
for three to four hrs.
Autoclaving temperatures will not destroy
pyrogens during a normal cycle.
2. DISTILLATION is the most reliable method of eliminating
pyrogens from water.
Pyrogenic substances are not volatile and will
thus remain in the distill.

3. Removal of pyrogens by select adsorbents has limited

use because of the concurrent phenomenon of adsorption
of solute ions of molecules.
it is of this interest in the production of antibiotics
where heavy contamination results from the
fermentation process
NONAQUEOUS SOLVENTS
Used because of solubility factors or hydrolytic reactions

Must not be toxic, irritating or sensitizing and must not

exert an adverse effect on the ingredients of the formulation

Frequently used non-aqueous solvents: PEG,

propylene glycol and fixed oils
ADDITIVES
essential for almost every product to enhance its stability

MUST exhibit the ff. characteristics:

1. Perform its function throughout the useful life of the
product
2. Must be non-toxic and non-irritating
3. Must not exert any adverse effect on the product, i.e.
must be compatible with all components of the formulation
4. Must not interfere with:
a. therapeutic efficacy
b. assay of the active therapeutic compound
 Such substances include (but not limited to):
- solubilizers - anti-oxidants
- chelating agents - buffers
- antimicrobial agents - tonicity contributors
- hydrolysis inhibitors - antifoaming agents
 ANTIBACTERIAL/ANTIFUNGAL
AGENTS
USP states that antimicrobial agents in bacteriostatic or
fungistatic concentrations must be added to preparations
contained in multiple-dose containers

Must be present in adequate concentration at the time of

use to prevent the multiplication of microorganisms
inadvertedly introduced into the preparation while
withdrawing portion of the contents w/ a hypodermic needle
and syringe
 Commonly used:
- 2 Mercurials
1. phenylmercuric nitrate
2. thimerosal
- 4 Homologous esters of:
1. p-hydroxybenzoic acid
2. phenol
3. benzyl alcohol
4. chlorobutanol
ANTI-OXIDANTS
OXIDATION is one of the pathways of degradation w/c can
be accelerated during thermal sterilization.

To protect a therapeutic agent susceptible to this

reaction, antioxidants are required
ANTI-OXIDANT CLASSIFICATION

1. Reducing agents
Function by being preferentially oxidized
- ascorbic acid - sodium bisulfite
- metabisulfite - sodium formaldehyde
- sulfoxylate - thiourea
2. Blocking agents
anti-oxidants w/c block an oxidative chain reaction in w/c
they are not usually consumed
- ascorbic acid esters
- butyl hydroxytoluene (BHT)
- tocopherols

3. Synergists
Compounds w/c increase the effectiveness of anti-
oxidants, particularly those blocking oxidative reactions
- ascorbic acid - citric acid
- citraconic acid - phosphoric acid
- tartaric acid
4. Chelating agents
Those that complex with catalysts w/c otherwise would
accelerate the oxidative reaction
- ethylenediaminetetraacetic acid salts

5. Inert gases
Used to displace oxygen from a solution and reduce the
possibility of oxidative changes in the formulation
- nitrogen
- carbon dioxide
BUFFERS
Added to maintain the
required pH for many products;
a change in pH may cause
significant alterations in the rate
of degradation reactions

Principal buffer systems used

to stabilize pH are the
ACETATES, CITRATES and
PHOSPHATES
CHANGES IN pH MAY OCCUR DURING STORAGE AS A
RESULT OF:

1. Dissolving of glass constituents in the product

2. Release of constituents from rubber closures or plastic

components in contact with the product

3. Dissolving of gases and vapors from the air space in the

container or by diffusion through the rubber or plastic
component

4. Reactions w/in the product

TONICITY CONTRIBUTORS
Compounds contributing to the isotonicity of a product
reduce the pain of injection in areas with nerve endings.

Buffers may serve as tonicity contributors as well as

stabilizers for the pH.
CONTAINERS AND CLOSURES
Containers for sterile products are made of glass or
plastics.

Glass is still preferred for injectable products.

 Glass
COMPOSED PRICIPALLY OF SILICON
DIOXIDE, WITH VARYING AMOUNTS
OF OTHER OXIDES SUCH AS SODIUM,
K+, CA+. Mg, Al, boron and iron

Basic structural network of glass is

formed by the silicon oxide tetrahedron

Like ampule cartridges and vials, glass

containers may be manufactured from
glass tubings or by blow molding
 TYPES
TYPE I
Borosilicate glass
Chemically resistant glass (low leachability, low thermal expansion)
TYPE II
Soda lime treated glass
Less resistant
TYPE III
Soda lime glass
Type 2 and 3, melts at lower temps & easier to mold into various
shapes, has higher thermal expansion
NP (nonparenteral)
- general purpose soda-lime glass
 Type I: suitable for all products
Type II: suitable for solution that is buffered has a pH
below 7 and not reactive with the glass
Type III: suitable for anhydrous liquids or dry
Determination of glass types (USP)
1. Powdered glass test
Challenges leaching potential of the interior structure

2. Water attack test

Performed on powdered glass, w/c exposes internal
surfaces of the glass compound
Results are based on the amount of alkali titrated by
0.02N H2SO4 after autoclaving
PLASTIC CONTAINERS
Established as packaging material for sterile preparation
such as large volume parenteral, ophthalmic and small
volume parenterals
3 Principal Problems:
(1) PERMEATION OF VAPORS in either direction through
the walls of the plastic

(2) LEACHING of constituent from the plastic into the

product

(3) SORPTION of drug molecule or ions on the plastic

material
Advantages
They are not breakable
There is a substantial weight reduction
No air interchange is required
Flexible wall simply collapses as the solution
flows out of the bag

Disadvantages
Not clear
Will soften or melt on thermal sterilization
Rubber closures
Permit introduction of a needle from a hypodermic syringe
into multiple dose vial and provide for releasing
Elastomer: made of latex natural rubber
Vulcanizing (Curing agent) sulfur, peroxide
PRODUCTION
Steps:
1. Compounding
2. Filtration
3. Filling
4. Sealing
5. Sterilization
1. COMPOUNDING

Processing of sterile prepns follow normal

manufacturing procedures w/c must be done in aseptic
conditions

i.e. All conditions must be carefully designed and

controlled to prevent the entrance of
microorganisms to a product (to achieve this,
there must be good environmental control)
1. COMPOUNDING

Personnel implementing this must understand and

practice the particular SOPs from preventing
contamination

All equipments and materials used whenever possible

must be sterile
2. FILTRATION

MEMBRANE FILTERS are used for clarification when a

highly polished soln is desired

The process removes particulate matter down to at least

3 microns in size.

STERILIZATION BY FILTRATION is achieved when

viable microorganisms and spores of approx. 0.3 microns
are removed.
2. FILTRATION

Membranes w/ porosity ratings of 0.22 or 0.45 microns

are usually specified for sterile filtrations.

The larger size has a faster flow rate (3 times) but would
require the use of a prefilter to remove some colloidal
matter w/c cause rapid clogging and thus reduce the
filtration cycle.
2. FILTRATION

BUBBLE TEST tests the efficiency of membrane

filters

Each membrane has a characteristic bubble point w/c is

a function of the porosity rating.
- This is the pressure required to push air through a
liquid-saturated filter.
- Until this point is reached, the filter process retain
and unless there is a rupture larger than the pore
diameter, air pressure will hold indefinitely
 Bubble Point
In order to determine the largest pore size of a
membrane, the pressure at which bubble is reached is
required. In order to reach bubble point, sufficient gas
pressure must be applied to overcome the capillary forces
of the pores.
The smallest pore size can also be calculated by
increasing the gas pressure till all pores has been
emptied and gas flow through the membrane is that of a
dry membrane profile.
At pressures below the Bubble point, gas can only pass
through the membrane through diffusion.
3. FILLING

BULK PREPARATIONS are subdivided into unit dose

containers during filling.

This process forces a measured volume of the prepn

through the orifice of a delivery tube designed to enter the
constricted opening of a container by means of gravity,
vacuum or w/ the aid of a pressure pump.
3. FILLING

The method selected for filling sterile preparations

should provide the degree of accuracy and precision
required by the nature of the product.

A slight excess is required in each container to provide

loss that will occur at the time of administration by
adherence to the wall of the container and retention in the
syringe and hypodermic needle lumen.
4. SEALING

SEALING will retain the contents of a sterile product and

will assure a tamper-proof presentation.

Containers should be sealed in an aseptic area adjacent

to the filling machine.
 AMPULES are sealed by heating w/ a high temp. gas-
oxygen flame to form.
1. TIP SEALS
those made by melting sufficient glass at
the tip of the ampule neck to form a bead
of glass and close the opening
2. PULL-SEALS
those made by heating the neck of a
rotating ampule below the tip, then pulling
away the tip to form a small, twisted
capillary just prior to being melted closed
 Sometimes, it is necessary to displace the air inside the
space w/in the ampule above the product to PREVENT
DECOMPOSITION.

A stream of INERT GAS, such as NITROGEN or

CARBON DIOXIDE is introduced during or after filling with
the product and the ampule is sealed immediately before
the gas can diffuse to the outside.
LEAKERS TEST

Useful method for evaluating the efficiency of the sealing

process

Consists of immersing completely the sterile sealed

ampules in an aqueous dye bath (0.5% to 1.0% of
methylene blue) w/in a vacuum chamber

As a negative pressure of 27 inches Hg or more is

created, a tiny drop soln can penetrate an opening of an
incompletely sealed ampule.

The colored ampules are sorted out during washing and

100% inspection that follows after.
 Bottles, cartridges and vials are stopped by RUBBER
CLOSURES held in place by aluminum caps.
- The bottom edge of these caps are bent (crimped)
around and under the tip of the glass container.
- It offers a tamper-evident presentation since the
cap cannot be removed w/o destroying the cap.
- Perforations permit tearing away the portions of the
cap to be discarded preparatory to use.

VIALS and BOTTLES are not subjected to a dye-leaker

test.
STERILIZATION
The complete destruction or elimination of microbial life.

- CHOICE of the most effective sterilization procedure

depends on:
1. Compatibility of the process w the prepn
i.e., sterilization process must not have
significant adverse effect upon the prepn
2. Successful validation of the process
i.e., parameters must prove to be lethal to
the most resistant spores of microorganism
normally encountered
2 Main Divisions of Sterilization
Procedures:
(1) Physical Processes
(2) Chemical Processes
Physical Processes of Sterilization
1. THERMAL METHODS
Microorganisms are killed by heat by what is thought to
be coagulation of the protein of a living cell.

Lethal Effectiveness depends on:

a. degree of heat
b. exposure period
c. moisture present
 Within the range of sterilizing temperatures, the time
required to produce a lethal effect is inversely related to the
moisture present.
For these reasons, dry heat or moist heat are
used as the conditions require.
2. NONTHERMAL METHODS

a. Ultraviolet light
Commonly employed to aid in the
reduction of airborne contamination
and to attempt to sterilize surfaces
w/in the processing environment

The germicidal light produced by

mercury vapor lamps is emitted at a
wavelength of 2537 Angstrom units
(253.7 millimicrons).
 The germicidal effectiveness of ultraviolet light is a
function of the intensity of radiation and time of exposure.

Ultraviolet light liberates energy as it passes through

matter.

The lethal mechanism of UV light works when this

energy is absorbed by orbital electrons w/in the molecules
of the microorganisms or of their essential metabolites
causing excitation and alteration of activity. Thus the
organism dies or is unable to reproduce.
b. Ionizing Radiations
high-energy radiations emitted from radioactive isotopes
such as cobalt-60 (gamma rays) or produced by
mechanical acceleration of electrons to very high velocities
and energies (cathode rays, beta rays).

They destroy microorganisms by stopping reproduction

as result of lethal mutations.
c. Filtration
A nonthermal method for the sterilization of select
solutions by removing microorganisms from the solution
while permitting the passage of all the desired components
of the solution and imparting no undesirable components
from the filter.
d. Aseptic Processing
Closely involved w/ sterilization although it is technically
not a sterilization process.

The condition and manipulations followed gives the

assurance that microorganisms do not enter a product.

Use for products that cannot be terminally sterilized after

they have been sealed in the final container.
 Chemical Processes of Sterilization
1. GAS STERILIZATION
Ethylene oxide is believed to exert its lethal effect upon
microorganisms by alkylating essential metabolites,
affecting particularly the reproductive process.

Ethylene dioxide sterilization is the acceptable practical

method for sterilizing plastic.

OTHER GASES: Beta propiolactone

Formaldehyde
Sulfur dioxide
2. SURFACE DISINFECTION

Disinfectants do not sterilize a surface.

As adjuncts to thorough cleaning of surfaces, proper use

of disinfectants may be expected to provide an aseptic
condition of the surfaces involved.

Sterility tests are performed on products and materials

subjected to previously validated sterilization procedures.
2 Basic Methods for Sterility Testing (USP):

(1) Direct inoculation of test samples on culture media

(2) Membrane filtration technique w/c involves filtering test

samples through membrane filters, washing the filters w/
fluids to remove inhibitory properties and transferring the
membrane aseptically to appropriate culture media
 Processing and testing of sterile products require aseptic
environment.

Air control is mandatory. This can be done by removing

contamination (particulate and microorganisms of the
submicron range) through a HEPA (High Efficiency
Particulate Air) air filter composed of glass and asbestos
or electrostatic precipitators.
Air passing through these units can be virtually
free from foreign matter
 The clean air in an aseptic area should have a
positive pressure, that is, air flows into the
maximum security area at the greatest volume
flow rate.
This prevents clean air from rushing into the
aseptic area through cracks, temporarily open
doors or other openings.
The air flows from an area w/ positive pressure to
other less critical areas like hallways w/c have a
negative pressure.

HEPA filters are rated 100% effective in particle ranges

of a few microns by using the Dioctyl-phthalate (DOP)
test method
 LAMINAR AIRFLOW DEVICES in the form of rooms,
cabinets or benches are based on the procedure
discovered by WHITFIELD in 1981.
- The entire body of air w/in a confined area moves
w/ uniform velocity along parallel flow lines.
- By employing prefilters ad high-efficiency bacterial
filters, the air delivered to the area is essentially
sterile and sweeps all dust and airborne particles
from the chamber through the open side.
- These deliver clean air in a vertical, horizontal or
curvilinear fashion
PRODUCTION FACILITIES
Would normally be divided into 5
Sectional Areas
(1) Clean-up Area
(2) Preparation Area
(3) Aseptic Area
(4) Quarantine Area
(5) Finishing of Packaging Area
 In general, the components will move from the
stockroom either:
(a) To the preparation room
as for the ingredients in the formulation
(b) To the clean-up room
as for containers and equipment
 After processing in the previously mentioned areas, the
components are brought into the aseptic area for filling into
appropriate containers.

If the product is to be sterilized in its final container, the

filled units are subjected to sterilization processes.
From there, the product will pass into the
quarantine area where it will be held until all
necessary tests have been performed.
After the results from all tests are known and the
product is found to be effective and safe, it will
pass to the FINISHING AREA for final labeling
and packaging.
 All of the previously mentioned areas should be
designed and constructed for effective ease of cleaning,
efficient operation, attractiveness and comfort of personnel.

Contamination concerns include dust, lint and

microorganisms.
- Such contaminants are normally found floating in
the air, lying on counters and other surfaces, on
clothing and body surfaces of personnel in exhaled
breath of personnel, and deposited on the floor.
 The design and control of an aseptic area is directed
towards reducing the presence of these contaminants so
that they are no longer a hazard to aseptic filling.

Although the aseptic area must be adjacent to support

areas so that efficient flow of components may be
achieved, barriers must be provided to minimize ingress of
contaminants to the aseptic area.
Such barriers may be sealed partitions, often
glass-paneled for greater visibility and light
 Another type of barrier is an entrance way
through security doors that require through an
airlock so designed that both doors cannot be
opened at the same time.
PERSONNEL
Personnel selected to work on the prepn of a sterile
product must be neat, orderly and reliable.

They must be neat, orderly and reliable.

They should be in good health and free from

dermatological conditions that might increase the microbial
load.
 Uniforms used in the aseptic areas should be sterile.

The uniform usually consists of coveralls for both men

and women, hoods to completely cover the hair, face
masks and cloth or plastic boots.

Sterile rubber gloves are required for most aseptic

operations, preceded by thorough scrubbing o the hands w/
a disinfectant soap.

The uniform is designed to confine the contaminants

discharged from the body of the operator, thereby
preventing their ingress into the product.
 Throughout the entire manufacturing process for a sterile
product, the prevailing philosophy must be that no effort is
too great to make the finished product as nearly perfect as
possible.