IP 106 – Sept.

4, 2008
III. PRODUCTION OF PARENTERALS
Cleaning Compounding Filtering Filling Sealing Sterilization

A. Cleaning
1. Containers
Containers and equipments coming in contact with parenterals should be cleaned meticulously.
New and unused containers and equipment will be contaminated with such debris as dust, fibers,
chemical films and other materials arising from the atmosphere, cartons, manufacturing process and
human hands.

Characteristics of Machinery for Cleaning:

 Liquid or air treatment should strike the bottom of the inside of the inverted container, spread
in all directions, and smoothly flow down the walls and out the opening with a sweeping action.
*Pressure of jet stream should cause only minimal splashing and turbulence.
*Splashing prevents cleaning of all areas
*Turbulence may redeposit loosened debris
 Container must receive a concurrent outside rinse
 Cycle of treatment should be a sequence of alternating very hot and cool treatments to
dissolve contaminants. Final rinse should be WFI (water for injection)
*Thermal shock aid (by expansion and contraction) in loosening of debris that adhere to
container wall
 All metal parts coming in contact with container or treatments should be made of stainless
steel or a noncorroding and noncontaminating material

Treatment Cycle
 Loose debris removed by vigorous rinsing with water
 Thermal shock treatment
 Air rinse for new containers (if only loose debris are present)
 Use only new containers for parenterals.

2. Rubber closures
 Gentle agitation in a hot solution of a mild water softener or detergent (0.5% sodium
pyrophosphate)
 Remove from solution and rinse several times with WFI
*Rinsing is done in a manner that will flush away loosened debris
 Final rinsing with WFI
*cleaning and final rinsing must remove pyrogens since autoclaving does not remove pyrogens
 Sterilize by autoclaving
 Store in closed containers until ready for use
*If closures are immersed during autoclaving, drain-off solution before storage to reduce
hydration of the rubber
 Vacuum dry at 100o if necessary

3. Equipment
 Disassembled
 Surfaces scrubbed with a stiff brush, using an effective detergent
*Especially joints, crevices, screw threads and other structures were debris is apt to collect
 Thorough rinsing with distilled water

4. Glassware
  Use dichromate cleaning solution
 Wrap in parchment paper and secure with autoclave tape
 Sterilize by autoclave

5. Rubber tubing
 Soak in 10% NaOH for 24 hours
 Rinse thoroughly and boil in 1% HCL for 1 hour
 Rinse several times with water
 Final rinsing with WFI
 Sterilize by autoclave
*Rubber tubing must be left wet when preparing for sterilization by autoclaving

Because of the relatively porous nature of rubber compound and difficulty in removing all traces of
chemicals from previous use, it is inadvisably to reuse rubber or polymeric tubing.

B. Compounding
 All measurements should be accurate and checked by a second qualified person
*liquids are prepared by weight since weighings are more accurate than volume and no
consideration on temperature has to be made.
 Equipment should be dry and sterile
 Follow established order of mixing
 For parenteral suspensions and emulsion: maintain proper reduction of particle size under
aseptic conditions
 Distilled water as solvent/vehicle should be kept and stores at 60-80oC and used within 24
hours

C. Filtering
Mechanisms of action of filters
 Sieving or screening
*Particles are retained on the surface of the filter
 Entrapment or impaction
*particles smaller than the pores become lodged in a turn or impacted on the surface of the
passageway
 Electrostatic attraction
*Particles opposite in charge to that of the surface of the filter pore are held or adsorbed to the
surface

Membrane filters
 used exclusively for parenterals because of their particle-retention effectiveness, nonshedding
property, nonreactivity and disposable characteristics
 Ex: Cellulose ester, Nylon, Polysulfone, Polycarbonate, Polyvinylidene difluoride,
Polytetrafluoroethylene (Teflon)
 Other filters used: asbestos, sintered glass (bacterial filters), unglazed porcelain, kieselguhr

Factors in filter selection

 Flow rate required
 Loss of vehicle by evaporation or absorption
 Interaction with the solution
 Cleanability

Polishing
  Filtering to remove particles 2-3 microns in size

Cold sterilization
 Filtering to remove particles 0.2-0.3 microns in size
 Eliminates microorganism = bacterial filtration

D. Filling
 Must be done with minimum exposure time
 Fill under a blanket of HEPA-filtered laminar-flow air
 Flow air while filling until sealing
 Employ aseptic technique
 For smaller volumes of liquid: use hypodermic syringe
 For large volumes of liquid: use liquid filters with multiple filling units
 To speed up filling: use gravity, vacuum or pressure pumps

Allowable excess by USP (to permit withdrawal and administration of labelled amount)
Liquids:
 For 1mL: mobile 0.1mL; viscous 0.15mL
 For 10mL: mobile 0.5mL; viscous 0.7mL
 For > 50mL: mobile 2%; viscous 3%
Solids:
 For single dose containers: 1-3%
 For multiple dose containers: 5-8%

E. Sealing
For ampules – fusion method (melting a portion of the glass neck)
1. Tip Sealing
 melting enough glass at the tip of the neck of an ampule to form a bead and close the
opening
 rotating the ampule on a single flame to heat evenly on all sides
*leaker – an incomplete sealed ampule
2. Pull Sealing
 heating the neck of ampule below the tip, leaving enough of the tip for grasping with
foreceps
 ampule is rotated in a single flame when glass has softened, the tip is grasped firmly
and pulled quickly away from the body which continues to rotate
thus, the small capillary tube formed is twisted close
 slower but seals are more sure
 applied to ampoules with wide opening e.g. powder ampoules

For vial/bottles – manual or mechanical sealing with rubber stoppers then crimped with
aluminium caps or seals

For plastic bags/bottles – blow-fill-seal aseptic packaging technique

Steps:
 formation of container
 filling with metered amount of solution
 formation or application of seal
Advantages:
 economical
 high quality products
  rapid (9-19 sec)

F. Sterilization
 To destroy microorganisms and their spores
 Methods: steam, dry heat, gas, ionizing radiation, bacterial filtration, tyndallization
1. Steam
 Saturated steam under pressure (autoclave)
 Most common and most effective
 121oC, 15-30 mins, 15 psi
 MOA: coagulation of cellular protein
 Application: aq. solutions containing heat-stable substances

2. Dry heat
 Employs oven heated by gas or electricity
 160-170 oC, 2-4 hrs, (can lower temp and increase time or vice versa)
 MOA: oxidation
 Application: non-aq. solutions with heat-stable substances

3. Gas
 Employs ethylene or propylene oxide
 MOA: alkylation of essential metabolites thus affecting reproduction of microorganisms
 Application: Dry materials e.g., medical and hospital supplies
4. Ionizing radiation
 Employs increased energy radiation emitted by radioactive isotopes like cobalt 60 or
produced by mechanical electron accelerators
 MOA: adverse effect on DNA or nucleis acid synthesis or metabolism resulting to lethal
mutation and reproductive stoppage
 Application: aq. or non-aq. injections with heat-labile substances

5. Bacterial filtration
 MOA: physical removal
 Application: aq. solution with heat-labile substances

6. Tyndallization
 Intermittent steam sterilization exposing material to 100 oC for 30 mins or 80 oC for 1hr to 3
days
 Application: heat-labile substances

Validating effectiveness of Sterilization Method

 Biological indicator
 Recording thermocouples (a physical parameter indicator)
 Color-change and Melting indicators (gives visual indication that a package or truckload has
undergone sterilization)

*Freeze-Drying (Lyophilization)
 Process of drying in which water is sublimed from the product after it is frozen
 Adv: preparations are more stable and more rapidly soluble; dispersions are stabilized
throughout shelf-life; products subject to oxidation have enhanced stability because
process is carried out in vacuum

IV. QC
Quality Control

QUALITY CONTROL TESTS

STERILITY TEST

Two Basic Methods for Sterility Testing:

• Direct-inoculation: Direct introduction of product test samples into culture

media
• Membrane Filtration: Filtering test samples through membrane filters,
washing the filters with fluid, and transferring the membrane aseptically to a
culture media
Culture Media:

Soybean-Casein Digest Medium (incubated at 20-25 oC)

Intended for anaerobic bacteria, however, it also

detects aerobic bacteria.

Fluid Thioglycollate Medium (incubated at 30-35 oC)

Suitable for the culture of both fungi and aerobic

bacteria.

Incubation:

Membrane filtration: 7days

Direct-inoculation: 7-14 days

*Growth Promotion Test is done to check whether the media used in the sterility testing is
suitable for growth of microorganisms. This is done by inoculating different species of
microorganisms (quantity: 100 colony-forming units) in separate media preparations and
incubating it for 3 days (bacteria) or 5 days (fungi).

*Species of Bacteria Used as Biological Indicators

Method of Sterilization Species

Moist Heat B. stearothermophilus

Dry Heat B. subtilis

Ethylene oxide B. stearothermophilus

Radiation B. pumilus, B. stearothermophilus, B.

subtilis

*Species of highly resistant bacterial spores included in the materials being sterilized to
validate whether the sterilization process is sufficient enough to kill these
microorganisms.
PYROGEN TEST

Evaluated by a qualitative response test in rabbits (Reason: they show a physiologic

response to pyrogenic substances similar to man) through:

A. Pyrogen Test (Introduced by Seibert in 1923)

Requirements:

• Healthy, mature rabbits

Test Procedure:

• 3 rabbits receive 10 mL solution/kg body (via ear vein)

administer within 10 minutes.
• The rectal temperature recorded 1, 2, and 3 hr after injection.

• Limit: Temperature rise for any one rabbit is 0.6 0C and the
total for three is 1.4 0C.
• If the limit is exceeded: Expand the test to include five
additional rabbits. The requirement for absence of pyrogen
states that no more than three rabbits each exhibit a
temperature rise of less than 0.6 oC and the total temperature
rise for all eight rabbits is 3.7 0C or less.
• Disadvantage: Not all injections can be subjected to this test
since some medicinal agent may have physiological effect on
the test animal such that any fever response would be masked.

B. Bacterial Endotoxin Test (since USP21/NF15)

Requirements:
• Aqueous extract of the circulating amebocytes of the
horseshoe crab, Limulus polyphemus, called Limulus
Amebocyte-Lysate (Reason: causes gel-clot formation if
pyrogen is present above a limiting concentration).
Test Procedure:

• An aqueous sample is mixed with LAL and incubated at 37 oC.

• The endpoint is determined spectrometrically by an increased
turbidity due to gel formation.
• The procedure requires a USP Reference Standard (defined
potency of 10,000 USP Endotoxin Units per vial, endotoxin
obtained from E. coli)
• Advantage:
The presence of pyrogens may be detected in drugs that
have definite physiological effects and for which the
classic rabbit response could not be used.
Simpler, more rapid, and of greater sensitivity.

• Disadvantage:
Can only detect gram-negative bacteria.
 To provide standardization, the USP established reference endotoxin against
which lots of the lysate are standardized. Thus, the sensitivity of the lysate
is given in terms of endotoxin units (EU). Most injections now have been
given limits in terms of EU, i.e. Bacteriostatic Sodium Chloride Injection, 1.0
EU/mL.

PARTICULATE EVALUATION

• Basis of evaluation: 5 µm (Reason: Erythrocytes have a diameter of 4.5 µm)

• All of the product should be inspected by human inspectors under good light,
baffled against reflection into the eye and against black-and-white background.
o Any container which visible particle can be seen is discarded.

Limitation:

o Size of the particles that can be seen (visible in unaided eye: 50 µm)
o Variation of visual acuity from inspector to inspector
o Emotional state
o Eye strain
o Fatigue

• For assessment of particulate matter below visible size:

Application: All Large-volume Injection(LVI) for single use and small-volume
injections(SVI), excluding IM and SC injections.

Test Procedure:

Light Obscuration (Stage 1)

• Equipment used to count and measure the size of

particles by means of a shadow cast by the particle as it
passes through a high-intensity light beam (Suppliers:
Climet, HIAC/Royco)
• If the limit is exceeded: Subject to Stage 2.

Microscopic (Stage 2)

•
Procedure: filter the measured sample through
membrane filter (under ultraclean condition) and
counting the particles on the surface of the filter
• Microscope and oblique light at 100x magnification
Subvisible Particulate Matter Limits in USP Injections

Light Obscuration Particle Count Test

>10 µm >25 µm
SVIs 6000 600/container

LVIs 25 3/mL

Microscopic Particle Count Test

>10 µm >25 µm

SVIs 3000 300/container

LVIs 12 2/mL

LEAKER TEST

Application: For ampules sealed by fusion

Test Procedure

Produce a negative pressure within an incompletely sealed ampul (submerged

entirely in deeply colored solution, i.e. methylene blue solution). All leakers are
discarded.

Factors to Assure Container-Closure Sealing Integrity

o Developing specifications for the fit of the closure in the neck of the
container
o Physical characteristics of the closure
o Need for lubrication of the closure
o Capping pressure
***vials and bottles are not subjected to leaker test because the sealing material (rubber
stopper) is not rigid.

SAFETY TEST

Conducted in animals to provide additional assurance that the product does not have
unexpected toxic properties.

V. PACKAGING AND LABELING

USP Requirements for Packaging
 The volume of injection in a single dose container is that which is specified for parenteral
administration at one time and is limited to a volume of 1L.
 Parenterals intended for intraspinal, intracisternal or peridural administration are packaged
only in single dose containers
 Multiple dose containers should contain no more than 30mL
 Injections for irrigation, hemofiltration or dialysis or for parenteral nutrition exempt from
packaging requirements
  Injections for veterinary use are exempt from requirements concerning the volume limitation
of single and multiple dose containers.

USP Labeling Definition and Requirements for Injection

 Labeling – all labels and other written, printed, or graphic matter upon an immediate
container or any package or wrapper in which it is enclosed, with the exception of the outer
shipping container
 Label – part of labelling upon the immediate container
 Label – name of the preparation, percentage content of drug, amt of active ing., volume off
liquid to be added to dry preparations, route of administration, storage, expiration date,
name of manufacturer, lot number
 Container label should be arranged that a sufficient area of the container is uncovered to
allow inspection of contents