C H A P T E R

14
Sterilization of Pharmaceuticals:
Technology, Equipment,
and Validation
Shruti Moondra1, Nidhi Raval2, Kaushik Kuche2,
Rahul Maheshwari2, Muktika Tekade3
and Rakesh K. Tekade2,4
1
Graduate School, University of Santo Tomas, Manila, Philippines 2National Institute of
Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat, India
3
TIT College of Pharmacy, Bhopal, Madhya Pradesh, India 4Department of Pharmaceutical
Technology, School of Pharmacy, International Medical University, Kuala Lumpur,
Malaysia

O U T L I N E

14.1 Introduction to Sterilization 468 14.2.2 Microorganisms Intrinsic

14.1.1 History 468 Resistance 473
14.1.2 Sterility, Sterilization, and 14.2.3 Physical and Chemical
Disinfection 469 Factors 474
14.1.3 Objectives of Sterilization 469 14.2.4 Storage Condition 474
14.1.4 Importance of Sterilization:
Pharmaceutical Perspective 470 14.3 Risk Assessment, Sterility
14.1.5 A Quick Overview of General Assurance Level (SAL), and
Procedures for Sterilization 472 Cleanroom Concept: Industrial
14.1.6 Items to be Sterilized 472 Perspective 474
14.3.1 Risk Assessment 474
14.2 Factors Affecting Sterilization 473 14.3.2 Sterility Assurance level 475
14.2.1 The Population of Microorganisms 14.3.3 Cleanrooms in
and Spatial Arrangements of Pharmaceutical Production 476
Microorganisms 473
14.4 Sterility Testing Techniques 476

Dosage Form Design Parameters

DOI: https://doi.org/10.1016/B978-0-12-814421-3.00014-2
467

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2018
Elsevier
Inc. All
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STERILIZATION AND KINETICS unicellular eukaryotic organisms.

Sterilization Other biological agents (such
as prions or viruses). Present in fluid or on
Sterilization refers to any process that a specific surface or object. It can be
removes, kills, or deactivates achieved through various means, including
microorganisms such heat, chemicals, irradiation, high pressure,
as fungi, bacteria, spores, and filtration.

Thermal Sterilization
Thermal sterilization, or heat sterilization,
is a process that uses heat to kill
microorganisms by destroying their cell
constituents and enzymes. This method of
sterilization can be applied to thermostable
products, moisture-sensitive materials- for
which dry heat (160-180°C) sterilization is
used. Moisture-resistant materials for
which moist heat (121-134°C) sterilization
is used. The efficiency with which heat can
inactivate microorganisms depends upon
the degree of heat, the exposure time, and
the presence of water.

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Heat/
Thermal
sterilization

Dry heat Moist heat

Direct Below At 100°C Above

100°C 100°C

Incineration Pasteurizatio Boilin Autoclav

n g e

Hot air Water baths Steaming

oven

Inspissatio Tyndallizatio
n n

In the heating step, the steam is

directly injected into the fermentation
medium, or the vessel is heated
Batch Thermal Sterilization electrically by heating jackets / coils.

Batch thermal sterilization is a technique

that uses heat or steam to sterilize culture Now the temperature is increased until it
reaches the sterilization temperature where
media in a bioreactor. It is a common it is held for a set period. Usually, culture
media are subjected to sterilization at 121°C
method for sterilizing liquid media. It in a bioreactor for 20-60 minutes.
involves 3 important steps-Heating,
Holding, Cooling.
Most of the unwanted microorganisms are
Designing Batch sterilization destroyed from the media and Bioreactor,
during this period.
Points to be considered before designing a
batch thermal sterilization process-The
profile of increase and decrease in
Finally, the system is cooled to bring the
temperature of the fermentation medium sterile culture media back to the desired
temperature.
during heating and cooling periods of the
sterilization cycle. Total number of
microorganisms initially present in media.
Thermal death characteristics of the The autoclave is an instrument that works
on the principle of batch sterilization. It is
“design” organisms. used to kill germs that invade the
fermentation media.

Del Factor
By knowing the original number of
organisms present in the fermenter and the
acceptable risk of contamination, the
required Del factor may be calculated. The

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Del factor in batch thermal sterilization is Continuous thermal sterilization is a

a measure of the fractional reduction in the process that uses steam condensate to
number of viable organisms over a certain rapidly transfer heat to a medium without a
heat period. heat exchanger. It is a process where
For example, as we know the acceptable sterilizers are added throughout the process
risk of contamination is 1 in 1000, to remove microorganisms such as
Therefore Nt should equal 10-3 of a viable bacteria, fungi, spores, etc. This method is
cell. If the unsterile broth contains only used for medium sterilization in
1011viable organisms, then the Del factor large- scale fermentation plants. The
may be calculated as: process includes sterilization operations
such as heating, maintenance, and cooling
Therefore, the overall Del factor required the medium outside the fermenter before
is 32.2. entering the fermentation process. In
Continuous sterilization, the culture
medium is continuously pumped into a
The destruction of cells occurs during the heating tower heated by direct steam, so
heating and cooling period of the broth as that it can reach the temperature of 126 ~
well as during the holding period at 121°C, 132 ℃ in a short time. Then enter in the
can be calculated with the overall Del maintenance tank (or maintenance tube),
factor as presented below: keep it at the sterilization temperature for 5
to 7 minutes, and then enter the cooling
tube to cool it to the inoculation
temperature and directly enter the
fermenter that has been sterilized in
Advantages: advance (empty tank sterilization).
Batch sterilization is the most common
and widely used technique to sterilize the
fermentation medium. It is more reliable to
scale up the fermentation process with a
simple operation. No additional materials
are required to be added to in fermentation
media. Batch sterilization can reduce The cooling methods of the culture
thermal damage to the fermentation medium include spray cooling, vacuum
medium significantly. The other advantages cooling, and thin plate heat exchanger.
of batch sterilization are strong continuity, There are two types of continuous
rapid sterilization and disinfection, and sterilizers used for the sterilization of
less damage to the nutrients in the media - Indirect heat exchangers and
fermentation medium. Direct Heat exchangers or steam injectors.
The function of the holding loop is to
Disadvantages sterilize the medium. The heating coil or
Batch sterilization utilizes more expensive loop sterilizes the medium and the cooling
heat. Best results only occur in well-mixed loop or coil cools the medium to the
closed vessels. It wastes energy and can fermentation temperature.
overcook the medium.
Continuous Thermal Sterilization
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operation. The utilization rate of

fermentation equipment is high. Because
of the high-temperature instantaneous
sterilization, it can not only kill
microorganisms but also minimize the
damage of nutrients, thereby improving the
utilization rate of raw materials. Due to the
uniform steam load, the utilization rate of
the boiler is improved. It reduces the labor
intensity of operators.
Disadvantages
The operation is more difficult to handle.
The requirement for steam is high. The
disadvantage is that the equipment has
Advantages
high requirements, and additional heating
The advantages are strong continuity, and cooling devices are required. It is not
rapid sterilization and disinfection, and suitable for sterilization containing a large
less damage to the nutrients in the number of solid materials. In continuous
medium. It is suitable for continuous sterilization, there are many opportunities
sterilization and disinfection of large- for contamination, and the contamination
volume fermenter materials. The heating involves a wide range.
time is short, which improves the
utilization rate of heat; the operating
conditions are constant, and the
sterilization quality is stable. It is easy to
realize pipeline and automatic control

Aspect Batch Sterilization Continuous Sterilization

Operation Mode Sterilization occurs in Sterilization is continuous, with a constant

batches, with start and flow of material
stop cycles

Equipment Size Generally smaller Typically larger equipment, designed for

equipment, suitable for continuous operation
smaller batches

Control and Requires monitoring Continuous monitoring and control of the

Monitoring and control of each entire process
batch separately

Flexibility Offers flexibility in Less flexibility, optimized for specific

processing different continuous processes
types of batches

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Efficiency May have downtime Offers higher efficiency with minimal

between batches for downtime between batches
cleaning and setup

Filter Sterilization
Thermal Death Kinetics
Filter sterilization is a process by which
Thermal death kinetics determines the time microorganisms are removed from a
that is taken to kill the specific set of substance based on their size. Filtration
bacteria through specific exposure to physically removes microbes because it
temperature. The thermal death curve employs membranes with precisely
defines the requirement of minutes for the defined pores that prevent their passage.
destruction of microbes at a given
temperature. It gives ways in which
bacteria can be reduced in the product. It
determines useful and pathogenic Name of the
Materials
microorganisms with the help of different filter
methods.
The thermal death kinetics may be Seitz filter Asbestos pad
represented by the following equation:
-dN/dt = kd N Berkefeld Diatomaceous
filter earth
where,
N, is the number of viable organisms
Chamberland-
present, Procelain
Pasteur filter
T, is the time of the sterilization treatment
kd, is the reaction rate constant of the Sintered glass Sintered glass
reaction, or the specific death rate per filter disks
time.
Membrane
Cellulose
filter

Borosilicate
HEPA filter
glass fiber

Candle filter Clay, mud

Filter Sterilization of air

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Hydrophobic sterilizing-grade filters are Filter sterilization is a method of removing

commonly used as air vents on processing microorganisms from liquids that are
tanks with the goal of maintaining near-
ambient pressure in the tank while
ensuring sterility. Air filtration methods
are divided into in-duct devices (filters)
and portable (stand-alone) air cleaners. In-
duct devices work for the entire house but
function only during the operation of an
air-handling system where they are
installed. Portable air cleaners (PACs) can
be operated and positioned in a room with
the flexibility to target the problem areas
and have received increasing attention in
recent years.
According to particle filtration efficiency,
air filters are divided into four types,
which is Pre-filter
 Medium filter
 High-efficiency particulate air
(HEPA) filter
 Ultra-low particulate air filter.
Among these, HEPA filters have shown
excellent performance in removing
expiratory aerosols and are most widely
adopted in the market.
HEPA Filters
HEPA filters are high-efficiency filters
designed to control the number of particles
entering a clean area by trapping both
larger and smaller particles through a
combination of outer filters and dense
fiber mats. This makes them effective in
removing a high percentage of particles
from the air. These can remove particles of
size 0.3μm and above. HEPA filters
capture pollen, dirt, dust, moisture,
bacteria (0.2–2.0 μm), viruses (0.02–0.3
μm), and submicron liquid aerosol (0.02–
0.5 μm).

Filter sterilization of media

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sensitive to heat or chemicals,
such as fermentation media or
pharmaceutical compounds. It
is the method of choice for
sterilizing antibiotic solutions,
toxic chemicals, radioisotopes,
vaccines, and carbohydrates,
which are all heat-sensitive. It
works by passing the liquid
through a porous membrane
with pores that are smaller than
the size of the particles being
removed. The particles are then
trapped on the membrane's
surface. Cell culture media
must be free of harmful
contaminants and adventitious
biological agents such as
bacteria, fungi, viruses, and
mycoplasma that can not only
propagate and destroy the cell
culture, but also find their way
into the final product. Current
practice is to sterile filter the
media and prevent bacterial

contamination of bioreactors
using nominal 0.2- micron–
rated sterilizing grade
membrane filters.
For media containing animal
sera, a final sterilizing grade
0.1–micron–rated filter may be
used for added protection
against mycoplasma. Sterilizing
cell culture media expeditiously
is critical to controlling
bioburden and endotoxin levels.

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Strain improvement:
Strain improvement is an advanced
biotechnological strategy where various
cellular pathways are modified by recombinant
DNA technology to improve the yield of
metabolic products that are beneficial to
humanity.

Techniques for strain improvement: Several

procedures are employed to improve microbial
strains; All bring about changes in DNA
sequence. Some of the important techniques of
Strain: strain improvement include: Random mutation,
Auxotrophic mutation, rDNA technology,
A strain is a group of species with one/more
Protoplast fusion
characteristics that distinguish it from other
subgroups of the same species of the strain. Mutation:
Each strain is identified by a name, number, or
letter. Example: Escherichia coli strain K12. The mutation is one method of strain
improvement. Mutation is the process of
Ideal characteristics of a strain: Rapid randomly changing the genetic code of a cell.
growth, Genetic stability, Non-toxicity to This can cause new genes to be formed or can
humans, Ability to use cheaper substrates, change the activity of existing genes. Mutation
Elimination of production of compounds that can happen spontaneously or can be induced
may interfere with downstream processing, To by radiation or chemicals. It can also be passed
improve the use of carbon and nitrogen from one generation to the next. Many times,
sources, Reduction of cultivation cost, Shorter the mutation will not have any effect on the
fermentation time, Production of limited cell. However, occasionally a mutation will
byproducts to ease subsequent purification improve the cell in some way. For example, it
problems. may make
it grow faster or be more resistant to disease.
Mutagen Mutation Induced Impact on DNA Relative Effect
Radiation

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Ionizing radiation (X- Single- or double- Deletions, structural High

rays, gamma rays) strand breakage of changes
DNA
Short wavelengths Pyrimidine Transversions, Medium
(UV rays) dimerization and deletions,
cross-links in DNA frameshift,
CC → AT transitions
Chemicals
Base analogs (5- Faulty base pairing; AT → GC, GC → AT Low
Bromouracil, errors in DNA transition
2- replication
Aminopurine)
Deaminating agents Deamination of GC → AT transition Low
(Hydroxylamine, cytosine
Nitrous acid)
Alkylating agents Methylation, high pH GC → AT transition Low
(NTG,
EMS, MMS)
(Other alkylating Alkylation of C and A GC → AT transition Low
agents)
Intercalating agents Intercalation Frameshift, loss of Low
(Ethidium bromide, between plasmids,
Acridine dyes) two base pairs nucleotides
Biological
Phage, plasmid, DNA Base substitution, Deletions, Low
transposons breakage duplications,
insertion

Based on the method of screening and metabolite of interest. It offers a

selection, there are basically two significant advantage over the genetic
methods of improving microbial strains engineering route alone by yielding
through random mutation: (1) Random gains with minimal start-up and
selection and (2) Rationalized selection sustaining such gains over years
Random Selection: despite a lack of scientific knowledge
Random mutagenesis and selection are of the biosynthetic pathway,
also referred to as the classic approach physiology, or genetics of the
or non-recombinant strain producing microbe. One drawback to
improvement procedure. After the random selection approach is that
inducing mutations in the culture, the it relies on non- targeted, non-specific
survivors from the population are gene mutations, so many strains need
randomly picked and tested for their to be screened to isolate the improved
ability to produce the mutant in the mixed population.

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Fig 1.1-Random Selection

Although the procedure is repetitive understanding of product formation

and labor intensive, this empirical and the fermentation pathway; this
approach has a long history of success can be acquired through radioisotope
and has given dramatic increases in feeding studies and isolation of
titer improvement, as best exemplified mutants blocked in various pathways.
by the improvements achieved for These observations can shed light on
penicillin production in which titers the metabolic checkpoints and suggest
over 50 g/liter are reported—a 4000-
fold improvement over the original ways to isolate specific mutants. For
parent strain. To increase the example, environmental conditions
efficiency of random selection, ways by (pH, temperature, aeration) can be
which the key steps in the process can manipulated, or chemicals can be
drive the throughput higher without incorporated in the culture media to
adding labor are typically sought. In select mutants with desired traits.
some instances, high throughput Rationalized selection for strain
screens have been automated with improvement does not generally
robotics technology. require a sophisticated understanding
of molecular biology to manipulate
Rationalized selection: environmental or cultural conditions.
An alternate approach to random
screening requires a basic

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Fig 1.2- Rationalized Selection

Auxotrophic mutation: nutrient for its growth and survival. In
contrast, the corresponding wild type
An "auxotrophic mutation" is a genetic strain can synthesize specific
change in a microorganism that makes nutrient and is not dependent on an
it unable to synthesize a vital nutrient, exogeneous supply in the growth
requiring the addition of that specific medium. The rate of product
nutrient to the growth medium for formation can be linked to the growth
survival. In strain improvement, this of a strain through auxotrophy for the
is used as a tool to select desired traits desired product or an intermediate of
by creating a controlled dependence its production. Auxotrophic strains
on a specific compound, allowing have been used in engineering to
researchers to manipulate metabolic improve the productivity of amino
pathways and enhance the production acids. Auxotrophic mutants are often
of target metabolites like antibiotics or used in genetic studies, selection
vitamins. The term auxotrophy refers experiments, and research involving
to a nutritional dependency, where the metabolic pathways and nutrient
mutant requires an auxiliary or utilization. They are also very useful in
supplemental source of the missing gene cloning procedures.

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Fig 1.3- Auxotrophic Mutation

1980s, genetic engineering has been
Recombinant DNA technology for technology in the
strain improvement:

The application of recombinant

technology and the use of synthetic
DNA now make it possible to induce
specific mutations in specific genes.
Recombinant DNA technology has
made it possible to improve microbial
strains by adding, deleting or
modifying specific genes. Since the
advent of recombinant DNA
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successfully implemented for
the development of strains
capable of (over)producing
proteins, enzymes and other
interesting metabolites. In
contrast to classical methods
for strain improvement, genetic
engineering allows a high level
of control of the strain
modification(s).

In-vitro Recombinant DNA

technology: By employing
restriction endonucleases and
ligases, investigators can cut
and splice DNA at specific sites.
It involves the following steps-
(1) Isolation of the desired gene
(DNA fragment) from the donor
cells. (2) Isolation of the vector
(a plasmid or a phage). (3)
Cleavage of the vector,
alignment with the donor DNA
with the vector, and insertion
of the gene into the vector. (4)
Introduction of the new
plasmid into the host cell by
transformation or, if a viral
vector is used, by infection. (5)
Selection of the

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new recombinant strains that express has been used to combine genes from
the desired characteristics. different

Protoplast fusion:

Protoplasts are the cells of which cell

walls are removed, and the
cytoplasmic membrane is the
outermost layer in such cells.
Protoplast fusion is a technique that
combines the genetic material of
different species or strains to create
hybrid strains with improved
properties. It is a useful tool for strain
improvement in biotechnology,
agriculture, and food technology. By
protoplast fusion it is possible to
transfer some useful genes such as
disease resistance, nitrogen fixation,
rapid growth rate, more product
formation rate, protein quality, frost
hardiness, drought resistance,
herbicide resistance, heat and cold
resistance from one species to
another. Protoplast fusion is an
important tool in strain improvement
for bringing genetic recombination and
developing hybrid strains in
filamentous fungi. Protoplast fusion
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organisms to create strains Primary Metabolites:

with desired properties. These
are powerful techniques for
engineering microbial strains
for desirable industrial
properties. The principle of
protoplast fusion is that the
protoplasts of parents with
different genetic traits are
hydrolyzed to remove the cell
wall, and then the spherical
protoplasts are mixed with the
same amount of hypertonic
buffer.
The protoplasts of parents
carry out cytoplasmic fusion
and nuclear fusion through
physical (e.g., Electrofusion),
chemical (e.g., Polyethylene
glycol), or biological (e.g.,
Sendai virus) induction fusion.
As a result, the parents'
genomes can achieve
exchange and recombination.
After that, the cell wall can
regenerate under suitable
conditions, and thus the
hybrid cells can be obtained.
This method is simple and
effective and does not need
expensive equipment. Fusants
are not constructed by
introducing functional genes
with movable genetic
elements (e.g., Plasmids and
transposons) so they have
little risk of spreading
resistance genes in soil
microorganisms through
horizontal gene transfer.
However, this fusion is
random, and the pick-up ratio
of target strains is often low.

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Primary metabolites are microbial organisms with their environment or

products made during the exponential produced in response to
phase of growth whose synthesis is an stress. Secondary metabolites
integral part of the normal growth are species-specific and are involved in
process. They include intermediates defense against other organisms,
and end-products of anabolic symbiosis, enhanced tolerance
metabolism, which are used by the cell to abiotic stresses, metal transport,
as building blocks for essential attraction for other organisms to
macromolecules (e.g. Amino acids, increase fertilization or seed dispersal,
nucleotides) or are converted to and used as sexual hormones.
coenzymes (e.g. Vitamins). Other Secondary metabolites are
primary metabolites (e.g. Citric acid, multipotential organic compounds
acetic acid, and ethanol) result from present in bacteria, fungi, or plants.
catabolic metabolism. Their These metabolites play an essential
production, which is related to energy role in biotechnological and biomedical
production and substrate utilization, is advances as they include a broad range
essential for growth. Industrially, the of antibiotics, antitumor agents, and
most important primary metabolites several therapeutic
are amino acids, nucleotides, vitamins, compounds. Secondary metabolites
solvents, and organic acids. can be classified based on
These are made by a diverse range of composition, synthetic pathway, and
bacteria and fungi and have numerous chemical structures. The major classes
uses in the food, chemical, and of microbial secondary metabolites
nutraceutical industries. include non-ribosomal peptides,
polyketides, ribosomally synthesized
Secondary metabolites: and post-translationally modified
Secondary metabolites are the peptides (RiPPs), glycosides, and
intermediates or products of terpenoids. Humans use secondary
metabolism and are not vital for the metabolites as medicines, flavorings,
growth and life of organisms but are pigments, and recreational drugs.
essential for the interaction of
Basis for Comparison Primary Metabolites Secondary Metabolites
Meaning The metabolism The end products of
products primary
that are produced metabolism that are
during the growth synthesized after the
phase of an organism growth phase has been
in order to perform completed and are
physiological functions important in ecological
and support overall and other activities of
development of the cell the cell are known as
are called primary secondary
metabolites. metabolites.
Also known as Trophophase. Idiophase.
It occurs at the Growth phase. Stationary phase.
Production These are produced in These are produced in
large quantities, and small quantities, and
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their their
extraction is easy. extraction is difficult.

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Occurrence Same in every species, Varies in different

which means they species.
produce the same
products.
Importance 1.These products are 1.Secondary metabolites
used such as
in industries for antibiotics,
various purposes. gibberellins are
2.Primary products also important.
play a significant role 2.They also indirectly
in cell growth, support the cell,
reproduction, and sustaining their life for
development. a long duration.
Examples Vitamins, carbohydrates, Phenolics, steroids,
proteins, and lipids are essential oils, alkaloids,
some of the examples. steroids are a few
examples.

Overproduction of primary
metabolites:
Overproduction of primary and
secondary metabolites:

The native microorganisms usually do

not overproduce essential primary
metabolites, since it is a wasteful
exercise. However, for industrial
overproduction, the regulatory
mechanisms are suitably manipulate
d. Overproduction of microbial
metabolites is related to the
developmental phases of
microorganisms. Inducers, effectors,
inhibitors, and various signal molecules
play a role in different types of
overproductions.

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Overproduction of several
metabolites has been
accomplished by eliminating
the feedback inhibition.
It involves two techniques-
(1) By using auxotrophic
mutants (2)By using
antimetabolite resistance

Using auxotrophic mutants:

Auxotrophic mutants are
genetically modified
microorganisms that lack the
ability to synthesize a specific
essential metabolite due to
a mutation. By controlling
their growth conditions,
these mutants can be
exploited for the
overproduction of primary
metabolites. Mechanism:
The mutation disrupts a
critical pathway, making the
organism dependent on an
external supply of the
metabolite. To compensate
for the blocked pathway,
upstream
metabolites accumulate,
leading to increased
production. For example, a
mutant unable to synthesize
a downstream amino acid
may overproduce its
precursors or related
metabolites.
Applications:
Overproduction of amino
acids like lysine, glutamate,
or methionine in

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bacteria such as Corynebacterium or genetic engineering, where enzyme

glutamicum. Increased production of activity is altered, or feedback
nucleotides or vitamins. inhibition is disrupted. These mutants
Advantages: often lose regulatory control, leading
(1) Easy to control production by to excess production of the target
modifying nutrient availability. (2) metabolite.
Reduces metabolic burden on Applications:
downstream pathways. Overproduction of purines and
Using antimetabolite resistance: pyrimidines by resistance to analogs
Antimetabolites are compounds that such as azaserine or 6-mercaptopurine.
mimic natural metabolites and inhibit Amino acid overproduction using
metabolic enzymes. Developing analogs like threonine hydroxamate or
resistance to these compounds can fluorophenylalanine.
lead to the overproduction of target Advantages:
metabolites. (1)Enhanced yield due to loss of
Mechanism: feedback inhibition. (2)Targeted
Microorganisms are exposed to approach to improve specific
antimetabolites that block key metabolic pathways.
enzymes in metabolic pathways.
Resistant mutants arise through
natural selection
Feature Auxotrophic Mutants Antimetabolite
Resistance
Approach Mutation blocks a Mutation confers
pathway, resistance, removing
forcing overproduction of feedback regulation.
precursors.
Target Specific essential Enzyme inhibition via
metabolites (e.g., amino antimetabolites.
acids, nucleotides).
Complexity Relatively Requires careful
straightforward. selection
of antimetabolites.
Control Easier to control via Involves genetic
nutrient resistance
supply. mechanisms.
Applications Amino acids, Amino acids, purines,
nucleotides, nucleotides.
vitamins.

involved in the production of secondary

Overproduction of secondary metabolites. For example, 300 genes participate
metabolites: in the biosynthesis of chlortetracycline and
Over production of secondary 2000 genes are
metabolites is more complex than
primary metabolites. Several genes are
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directly or indirectly
involved in the production
of neomycin.
Four techniques: (1)
Induction, (2)End product
regulation,
(3)Catabolite
regulation (4)Phosphate
regulation Induction:
Induction of secondary
metabolite overproduction
often involves manipulating
the culture conditions, such
as altering nutrient levels,
pH, or oxygen availability, to
mimic stress or

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nutrient limitation, which triggers To achieve overproduction, strategies

secondary metabolism. Genetic like using non-repressing carbon
modifications, like overexpressing sources, disrupting regulatory
regulatory genes or knocking out pathways (e.g., CRE genes), or
repressive pathways, can further employing mutants insensitive to
enhance production. Additionally, catabolite repression can be
elicitors such as precursors, signaling implemented.
molecules, or physical factors (e.g.,
Light or temperature) can be used to
stimulate secondary metabolite
biosynthesis. E.g. Addition of
methionine induces certain enzymes
and enhances the production of
cephalosporin.
End Product regulation:
Some of the secondary metabolites
inhibit their biosynthesis, a
phenomenon referred to as end- Fig Catabolite Regulation
product regulation. Eg., Penicillin,
streptomycin, puromycin, and Phosphate Regulation:
chloramphenicol. End-product Inorganic phosphate (Pi) is required for
regulation in the overproduction of the growth and multiplication of
secondary metabolites involves prokaryotes and eukaryotes. Increasing
disrupting feedback inhibition Pi concentration (up to 1mM) is
mechanisms that limit biosynthesis. associated with an increased
This can be achieved by genetically production of secondary metabolites.
modifying regulatory genes or E.g. Antibiotics (streptomycin,
enzymes to prevent inhibition by the tetracycline), alkaloids, gibberellins.
end product, allowing continuous
production. Additionally, precursor Case Studies:
availability can be increased to bypass (1) Streptomyces for antibiotic
bottlenecks in the pathway, ensuring production:
enhanced synthesis of the desired Organism: Streptomyces spp.
secondary metabolite. Objective: Improve antibiotic yield,
Catabolite regulation: particularly streptomycin.
Catabolite regulation influences the Methods: Mutagenesis: UV irradiation
production of secondary metabolites and chemical mutagens like
by controlling carbon source utilization nitrosoguanidine were used to induce
in microorganisms. In catabolite random mutations, Screening: High-
repression, the presence of a preferred yield mutants were selected through
carbon source (e.g., Glucose) inhibits activity-based assays. Result: Strain
secondary metabolism by suppressing improvement led to over a 20-fold
the expression of biosynthetic genes. increase in streptomycin production
compared to the wild-type strain.
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Impact: This approach helped reduce Random mutagenesis of Streptomyces

production costs and enhanced the for antibiotic production:
availability of antibiotics during its
industrial-scale production.

Random mutagenesis of Streptomyces

for resistance to lysine analogs. Result:
The engineered strain produced 100 g/L
(2) Corynebacterium Glutamicum for of lysine, a significant improvement
amino acid production: over the native strain. Impact: This
Organism: Corynebacterium strain became the cornerstone for
glutamicum. Objective: Increase lysine large-scale lysine production used in
production. Methods: Metabolic animal feed
Engineering: Feedback inhibition in the
aspartate kinase enzyme was removed Improved Glutamic Acid Production
by mutating the gene responsible for capacity of Corynebacterium
its regulation. Adaptive Evolution: Glutamicum:
Strains were cultured under selective
pressure

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(4) Escherichia coli for recombinant

(3) Aspergillus niger for citric acid protein production:
production: Organism: Escherichia coli. Objective:
Organism: Aspergillus niger. Objective: Optimize recombinant insulin
Enhance citric acid yield. production.
Methods: Strain Selection: Mutants Methods: Plasmid Engineering: High-
resistant to manganese toxicity (a copy plasmids were designed to
known inhibitor of citric acid express the insulin precursor with a
production) were isolated. Genetic strong promoter (e.g., T7). Host
Manipulation: Overexpression of Optimization: Knockouts of protease
genes related to glycolysis increased genes minimized degradation of the
the flux towards citric acid. Result: The recombinant protein. Process
optimized strain achieved over 85% Optimization: Co-expression of
yield of citric acid relative to the sugar chaperones improved protein folding.
consumed. Impact: Improved citric Result: Production levels exceeded 15
acid production became a benchmark g/L of insulin precursor, making the
in the food and beverage industry. process economically viable. Impact:
Improving dietary citric acid Paved the way for large-scale insulin
production by the wild-type manufacturing at reduced costs.
Aspergillus niger ASP26 strain: Strain improvement of E.coli to
enhance expression of recombinant
proteins:

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(5) Saccharomyces cerevisiae for production, redirecting the metabolic

bioethanol production: flux towards ethanol Adaptive
Organism: Saccharomyces cerevisiae. Evolution: Long-term cultivation under
Objective: Improve ethanol yield high ethanol stress improved strain
under stress conditions (high sugar, robustness. Result: The engineered
high ethanol concentration). strain produced 20% more ethanol
Methods: Mutagenesis: Random than the parental strain under
mutagenesis was applied to generate industrial conditions. Impact:
stress-tolerant strains. Genome Enhanced bioethanol production
Editing: CRISPR-Cas9 was used to supported the development of
knock out genes related to sustainable biofuels.
glycerol

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Acknowledgements
The authors would like to acknowledge Science and Engineering Research Board (Statutory Body Established
Through an Act of Parliament: SERB Act 2008), Department of Science and Technology (DST), Government of
India for grant (#ECR/2016/001964) allocated to Dr. Tekade for research work on drug and gene delivery. The
author also acknowledges DST-SERB for N-PDF funding (PDF/2016/003329) to Dr. Rahul Maheshwari in Dr.
Tekade’s lab for work on targeted cancer therapy. Authors would also like to acknowledge Department of
Pharmaceuticals, Ministry of Chemicals and Fertilizers, India, for supporting research on cancer and diabetes at
NIPER- Ahmedabad.
Disclosures: There are no conflicts of interest and disclosures associated with the manuscript.

ABBREVIATIONS
AP atmospheric pressure
BI biological indicator
CI chemical indicator
BP British Pharmacopeia
CFR code of federal regulation
cGMP current good manufacturing practices
DNA deoxy ribonucleic acid
D-value decimal reduction time
EMA European Medicine Agency
F Fahrenheit
FDA Food and Drug Administration
IAEA International Atomic Energy Agency
IQ installation qualification
min minutes
OQ operational qualification
PI physical indicator
PQ performance qualification
RH relative humidity
RNA ribonucleic acid
SAL sterility assurance level
SIP sterilization in place
TM thioglycollate medium (fluid)
TSB tryptone soya broth
USP Unites State Pharmacopeia
UV ultraviolet radiations
UVA ultraviolet A
UVB ultraviolet A

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Agalloco, J.P., Carleton, F.J., 2007. Validation of Pharmaceutical Processes. CRC Press.

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