This document discusses microbial contamination control in parenteral manufacturing. It outlines various layers of protection used, including terminal sterilization techniques like autoclaving. It also discusses aseptic processing and sources of contamination control strategies during aseptic manufacture. Other topics covered include blow-fill-seal technology, issues in sterilization by filtration, sterile prefilled syringes, process validation, hazard analysis and critical control points. Key sterilization techniques and the selection of appropriate test organisms to validate these processes are also summarized.

1. Microbial Contamination Control
in Parenteral Manufacturing
Pragati Kumar
Bada
pragatk@yahoo.com

2. •Parenteral products are radically different from other
dosage forms in terms of standards of purity and
safety.
•Apart from complying with standards of potency and
stability, parenterals have to meet exacting standards
of microbial (sterility and pyrogens), physical
(particulate matter), and chemical
(isotonicity, buffering capacity, etc.) parameters.

7. Various layers of protection in a typical Parenteral manufacturing

8. Terminal Sterilization
• In 1991 USFDA proposed that all sterile products should
be terminally sterilized, unless data were available to
prove its adverse effects on product stability . This was
primarily due to the fact that all product recalls during
1981 to 1991 involved aseptically processed products .
• Certain drug classes, such as biologics, multidose
ophthalmic products, and dispersed systems, are exempt
from this.
• The most commonly used technique for terminal
sterilization is autoclaving, which makes use of saturated
steam.
• Compendial cycles for autoclaving in USP/EP/BP
prescribe a 15-minute exposure at 121°C.

9. The most heat resistant microorganism actually
contaminating the formulation—or Bacillus
stearothermophillus because of its high heat
resistance—is used as a standard microbe for
development of autoclaving cycles.
New Techniques of Terminal Sterilization:
There is a strong need to develop terminal
sterilization techniques that can help in
achieving acceptable Sterility assurance level
without causing damage to the product.

10. • PurePulse® Technologies USA has developed
a technique called Pure Bright sterilization.
• It uses broad-spectrum pulsed light (BSPL) to
effectively inactivate bacterial organisms and
spores in static and flowing solutions, as well
as on dry surfaces.
• BSPL generated from xenon lamps contain
visible, infrared, and UV wavelengths in ratios
similar to sunlight.
• The major difference from sunlight is that the
UV wavelengths are removed due to filtration
by Earth’s atmosphere.
• Rapid intense pulses of BSPL are used for
inactivation of pathogens.

11. ASEPTIC PROCESSING
• Aseptic processing involves filling of pre-sterilized
formulations into cleaned and pre-sterilized primary
packaging components.
• In absence of any post-filling sterilization step, the SAL
of finished product is a direct function of SAL of
individual components.
• Many aseptically manufactured
presentations, especially multi dose products, contain
preservatives that serve a dual purpose of
(a) providing antimicrobial activity and
(b) preventing proliferation of any microbe that
might contaminate the product during repeated
use.
• Sterility assurance levels for aseptic processing is
determined by performing media fill trials

12. Sources of Contamination and Control Strategy
During Aseptic Manufacture
Contributing parameter Control strategy
Environmental air Passing air supply through High
Efficiency Particle Air (HEPA)
filters.
Laminar air flow (90 feet/min) is
used to ―sweep away‖ particles
and microbes from the sensitive
areas.
Environmental air Pressure
differentials to protect areas of
critical operations.

13. Contributing parameter Control strategy
Manufacturing • For fixed equipment Vacuum
cleaner equipped with HEPA
equipment filtered exhaust.
• Wet wiping with disinfectant
solution for Manufacturing
equipment.
• For demountable
equipments cleaning and
autoclaving should be
performed.

14. Contributing parameter Control strategy
Formulation and • Powders for injection are
primary packaging supplied as sterile by bulk
components
drug manufacturers.
• Liquid products are filtered
through sterile 0.22 μm
membrane filters.
• Glass vials are cleaned and
dry heat sterilized Rubber
stoppers are cleaned and
sterilized by autoclaving.

15. Contributing Control strategy
parameter
Personnel • Medical examination to screen
personnel working in aseptic
area.
• Entry of personnel to aseptic
area should be through
changing rooms.
• Containment of personnel
microbial flora by protective
clothing.
• Localized barriers between
personnel and areas of filling
operations, by means of
laminar airflow or by using
isolator barriers.

16. Contributing Control strategy
parameter
Water and Purification of water by distillation
drainage or reverse osmosis.
Storage of WFI at temperatures
>80°C and in vessels fitted with
continuous circulation loop.
Efficient drainage at the
manufacturing shop floor to
prevent accumulation of water.

17. Blow-Fill-Seal Technology (BFS)
BFS technology involves a fully automated process in
which the primary container for the formulation is
(a)formed from a thermoplastic.
(b)Aseptically filled with filtered solution
(c)sealed, in a single operation in a controlled environment.
• BFS uses an automated process requiring minimal human
intervention once the machine settings have been set.
• The formed plastic container is filled with sterile product
and instantly sealed, thus avoiding contamination.
• A hermetically sealed bottle formed during the process
helps avoid the use of sealing devices like rubber closures
and seals.

18. Formation of blow-fill-seal pack.

19. • Various thermoplastics like polyethylene
(PE), polypropylene (PP), various copolymers and
polyalomers are commercially available.
• PP offers distinct advantages by allowing exposure to
121°C for autoclaving. Bottles produced on the BFS
machine can be individual or strip-dose formats, in sizes
from 0.1 ml to 2000 ml, and outputs as high as 30,000
units/hour can be achieved.
• BFS technology has offered a cost-effective means of
Microbial contamination control introducing high-quality
aseptically processed products with additional advantage
of reduced breakage, reduced hazard of accidental
injury, and reduced pack volume over glass containers.

20. RECENT ISSUES IN STERILIZATION BY FILTRATION
• Sterilization by filtration has traditionally involved use of
0.2/0.22 μm rated filters and Pseudomonas diminuta (now
called Brevundimonas diminuta) as the standard challenge
organism.
• USFDA defines minimum qualifying area of 107/cm2 of filter
area.
• Stressful conditions may affect the changes in the
morphology of the microbes.
• In this study,40% reduction in size of Burkholderia pickettii
was observed.
• These developments are causing a shift from 0.2/0.22
rated filters to 0.1micron filters.

21. STERILE PREFILLED SYRINGES (PFS)
• PFS have become a popular packaging system for
parenteral products due to their advantages of ease of
administration, dosing accuracy, and increased assurance
of sterility. PFS consists of a barrel, a plunger rod with
rubber fitting, and a luer-lock tip/stainless steel needle.
• The manufacturing process for PFS may involve (a) filling
of formulation in previously cleaned and sterilized PFS or
(b) cleaning, sterilization, de-pyrogenation of non sterile
syringes, followed by filling .
• The filling is carried out in Class 100 area, and other
operations can be carried out in Class 10,000/100,000
area.
• Terminal sterilization of PFS by autoclaving poses a
unique challenge due to the possibility of rubber plunger
migration during the process. This ―pop-off‖ of rubber
plunger can be prevented by using autoclaves with a
counter-pressure feature.

22. PROCESS VALIDATION, HAZARD ANALYSIS AND CRITICAL
CONTROL POINTS (HACCP)
HACCP involves seven principles:
(a)analyzing each step for hazard.
(b)Identifying all critical control points (CCP).
(c)verifying the limit for each CCP.
(d) verifying monitoring and testing of limits.
(e)verifying corrective actions.
(f)verifying operational procedures for CCPs, and
(g)verifying that records of each CCP are
documented in the batch record.

23. Critical Control Points in a Typical Parenteral Product Manufacturing

28. Test Organism
The selection of a biological indicator appropriate for
use with a particular sterilization process requires
the consideration of a number of factors.
First is identification of the appropriate test
organism. The test organisms indicated in Table 1
are generally recognized to exhibit greater
resistance to the indicated sterilization processes
than typical bioburden.

29. An example of different biological indicator formats,
including spore suspensions, inoculated carriers, paper strip
biological indicators, and self-contained biological
indicators.

30. Suspensions and Inoculated Carriers : Aliquots of the
test organism are either inoculated directly onto
product or onto suitable carriers that are then placed in
those locations of the product considered to be the
most difficult to sterilize.
Following sterilization processing the number of
surviving test organisms is determined by either direct
transfer to growth medium (fraction-negative analysis)
or removal of the test organisms from the product or
carrier for direct enumeration.
Paper Strip Biological Indicators : This type of
biological indicator, consisting of a paper strip carrier
inoculated with a suspension of test organisms and
packaged in a glassine or Tyvek outer envelope has
seen little change since its commercialization during

31. Self-Contained Biological Indicators : Self-contained
biological indicators are widely used in many
applications, as they do not require the user to
aseptically transfer the inoculated carrier to growth
medium.
Self-contained biological indicators incorporate both
the test organisms and the growth medium within the
same unit and are typically of two distinct types.
The simplest form of self-contained biological
indicators consist of a hermetically sealed glass
ampule or vial containing spores of Geobacillus
stearothermophilus suspended in growth medium with
a pH indicator dye. Following sterilization processing
the ampule is incubated and growth of the test
organisms detected as a change in the color of the

32. WATER MICROBIOLOGY

33. PRE TREATMENTS OF WATER
• The principal purification units of
distillation, reverse osmosis, ion-
exchange, and electrodeionization can purify at
least some small quantity of water of any
degree of contamination even without
pretreatment.
• The question is how much before the particular
purification unit is fouled or possibly irreparably
damaged.
• Chlorine will rapidly and irreversibly degrade
polyamide RO membranes. Chloride ions will
cause the corrosion of stainless steels.

34. Chlorination
• As a first step, raw waters are commonly
chlorinated to kill pathogenic microbes. Chlorine
concentrations of 1 ppm effect a 97% kill of E. coli
in 0.6 minutes at 5–25°C, and 0.5 ppm amounts
have the same effect in 7 minues at 5°C.
Salmonella and Cholera are killed by 3 ppm.
• When chlorine contacts water it reacts to form
hypochlorous acid. HOCl dissociates to yield
hydrogen ions, H+ (or hydronium ions, H3O+), and
hypochlorous ions, OCl−. The sum of the
hypochlorous acid and the hypochlorite ions is
called the ―free available chlorine.‖ Hypochlorous
acid is about 100 times stronger in its oxidizing
potential than is the hypochlorite ion.

35. chlorine partakes oxidatively in a free radical
chain reaction with TOC present in the water to
form the carcinogenic trihalomethanes (THM).
REMOVAL OF THM
The trihalomethanes found in feed waters consist of
mixtures of chlorine and bromine atoms substituent on the
single carbons created by the free radical chain scission
reaction of chlorine on longer carbon-to-carbon TOC
chains.
Monobromo, dichloromethane Br-CH-Cl2;
monochloro, dibromomethane Cl-CH-Br2; bromoform HC-
Br3; and chloroform HC-Cl3 constitute the trihalomethanes.
The THMs, except for chloroform, are destroyed to an
85% extent by 185 nm UV.

36. They are removed by reaction with anion exchange
resin in hydroxyl form; chloroform only to the extent of
50%. They are adsorbed by activated carbon in
proportion to the surface area of the carbon and
increasingly with bromine content; chloroform CHCl3
10%, bromoform CHBr3 50%.

37. Deep Beds and Multimedia Filtration
• Multimedia bed design is versatile, but there is no
ready way to match its available constructions to
the TSS, to the total suspended solids contents of
given waters.
• Particles too small in size to be retained even by
the (bottom) most finely ground, densest medium
bed may be present, as also colloidal particles.
• Coagulation and flocculation techniques are then
invoked to agglomerate the ultrafine particles to
sizes that can be removed by the deep beds.

38. Cross sections of representative filter particle gradations.
Diagram (a) represents a single medium bed such as a rapid sand
filter.
The bottom half of a filter of this type does little or no work.
Diagram (b) represents an ideal filter uniformly graded from coarse to
fine from top to bottom.
Diagram (c) represents a dual media bed, with coarse coal above fine
sand, which approaches the goal of the ideal filter.

39. Softening and Solubility Product
Water softening is usually accomplished by way
of sodium-form ion-exchange wherein the ion-
exchange resin in the softening unit removes the
hardness-causing elements from the feed waters
by exchanging them for the sodium ions it
releases.
This fore-stalls subsequent mineral fouling of the
RO by membrane-blocking deposits of alkaline
earth salts of limited solubilities, such as the
sulfates, carbonates, and fluorides of
calcium, barium, or strontium.

40. The solubility product of a salt is the maximum product of its cation and anion
concentration expressed in moles per liter that can exist in equilibrium with its
undissolved phase at any one temperature.