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Pharmaceutical
Biotechnology

Book list
1
...
Biotechnology
By Satyanarayana
3
...
C Dubey

1

2
...

The diagnosis, treatment, mitigation of any abnormal physical or psychoogical state or its symptom or
Altering, modifying, correcting or restoring any organic function
...

Parameters of spoilage: Depending on its mode of usage, a pharmaceutical product may be considered to be
microbiologically spoiled if –
1
...

3
...

Toxic microbial metabolites persist even after death or removal of any microorganism originally present
...


Pharmaceutical products susceptible to microbial attack:
1
...


3
...


5
...


7
...
g
...

• Analgesics e
...
aspirin, paracetamol
...
g
...

• Alkyl, alkylbenzene sulphonates and sulphate esters
...
g
...

• Agar (a complex polysaccharide)
• Lower molecular weight polyethylene glycols
• Synthetic packaging polymers e
...
cellophane (modified cellulose)
Humectants: These include –
• Glycerol and sorbitol
• Fats and oils
Sweetening, flavouring and coloring agents: These include –
• Coloring agents e
...
tartrazine and amaranth
• Flavouring agents e
...
peppermint water
Preservatives and disinfectants: These include –
• Quaternary ammonium antiseptics
• Chlorhexidine
• Parabens (4-hydroxybenzoate ester)

Microorganisms involved in the spoilage of pharmaceuticals:
•
•

Pseudomonas aeruginosa
Salmonella cavaban

2

•
•
•
•
•
•
•
•

Escherichia coli
Staphylcoccus aureus
Clostridium botulinum
Clostridim perfingens
Streptococcus faecalis
Proteus spp
...

Gram negatives can be isolated in many pharmaceutical products as contaminants (e
...
Pseudomonas, Serratia,
Klebsiella)

Contamination: Contamination is the unwanted pollution of something by another substance
...

Source of contamination:
A
...
Raw materials (water and materials with natural origin)
2
...
Environment of the pharmaceutical industry e
...
wet sites, cleaning equipment
...
Packaging e
...
cardboard, corks, papers are unsuitable packaging materials
...
Containers that are frequently re-used
...
Contamination of disinfectants due to re-used containers
...
Re-packaging of products purchased in bulk into smaller containers
...
Processing
9
...
Transportation
B
...
Human sources
i
...

Highest risk of contamination in topical products e
...
Staphylococcus, Micrococcus species,
diphtheroids, Pseudomonas
...

Disposable applicators and spoons for topical and oral porducts
iv
...

v
...

vi
...
Environmental sources:
i
...

Water borne contaminants – Pseudomonas
3
...

Equipments for pharmaceutical drug administration
ii
...
g
...

2
...

4
...

6
...


Loss of viscosity and sedimentation due to de-polymerization of suspending agents
...

Products may become unappealingly discolored by microbial pigments of various shades
...

Metabolism of surfactant in oil in water (O/W) emulsions reduce stability and accelerate creaming
of the oil globules
...


3

Factors affecting microbial spoilage of pharmaceutical products:
1
...

3
...

5
...

7
...


Types and size of contaminant inoculum
Nutritional factors
Moisture content i
...
water activity
Redox potential
Storage temperature
pH
Packaging design
Protection of microorganisms within pharmaceutical products

They are discussed below:
1
...


3
...
Inoculum size alone is not always a reliable indicator
of likely spoilage potential
...

Nutritional factors: The simple nutritional requirements and metabolic adaptability of many common
spoilage microorganisms enable them to utilize many formulation components as substrates for biosynthesis
and growth
...
They are thus unlikely to multiply in them, although
they may remain viable and infective for an appreciable time in some dry products where the conditions are
suitably protective
...
Even demineralized water prepared by good ion-exchange methods will normally
contain sufficient nutrients to allow significant growth of many waterborne Gram-negative bacteria such as
Pseudomonas spp
...
e
...
Water activity is based on a scale
of o to 1
...
0
...

Water activity =

Vapour pressure of water in the sample
Vapour pressure of pure water

As the chemically bonded water cannot escape, only the free water will export vapour pressure and therefore
this characteristic can be used to determine microbial spoilage, chemical stability and physical stability
...
5 will mean the likelihood of microbial growth
is very low
...
This relationship is referred to as the
moisture adsorption isotherm and it must be determined experimentally by measuring water content at several
water activity values
...

Microorganisms require readily accessible water in appreciable quantities for growth to occur
...
With the exception of halophilic bacteria, most
microorganisms grow best in dilute solutions (high Aw) and as solute concentration rises (lowering Aw), growth
rates decline until a minimum growth-inhibiting Aw is reached
...


5
...


Redox potential: The ability of microbes to grow in an environment is influenced by their oxidationreduction balance (redox potential), as they will require compatible terminal electron acceptors to permit
their respiratory pathways to function
...

Storage temperature: Spoilage of pharmaceuticals could occur potentially over the range of about ⸺200 C
to 600 C, although it is much less likely at the extremes
...
A deep freeze at ⸺200 C or lower is used for
long-term storage of some pharmaceutical raw materials
...
Around neutrality bacterial spoilage is more likely, with
reports of pseudomonads and related Gram-negative bacteria growing in antacid mixtures, flavoured
mouthwashes and distilled or demineralized water
...
g
...
In products with low pH levels (e
...
fruit juice flavoured syrups with a pH 3-4), mold or yeast attack

4

7
...


is more likely
...
Although the use of low pH adjustment to preserve foodstuffs is well established (e
...

pickling, coleslaw, yoghurt), it is not practicable to make deliberate use of this for medicines
...

Protection of microorganisms within pharmaceutical products: The survival of microorganisms in
particular environments is sometimes influenced by the presence of relatively inert materials
...

Adsorption on to naturally occurring particulate material may aid establishment and survival in some
environments
...
The inclusion of preservatives may be unnecessary in case of
tablets, powders and capsules
...

Features of an ideal preservative:
1
...

3
...

5
...

7
...


It is free of toxic or irritant effects at the concentrations used
...

It is sufficiently soluble in water to achieve adequate concentrations in the aqueous phase of a system of two
or more phases
...

It is chemically compatible to all other formulation components and retains the un-dissociated form at the pH
of the preparation
...

It has an acceptable odor and color
...


Types of preservatives:
1
...

Examples:
• Benzalkonium chloride
• Alkyl trimethyl ammonium chloride
2
...
Phenolic compounds
• Chlorinated and isopropyl derivatives of meta cresol
4
...


Factors affecting the efficacy and availability of preservatives:
1
...

3
...

5
...

7
...

9
...

pH: Rise in pH increases or decreases preservative activity
...

Formulation constituents: May reduce or improve activity
...

Microorganisms: Type, nature and condition (sessile or planktonic)
Organic matter: Activity may be reduced
...

11
...

13
...


Presence of inactivating agents – dirty condition changes of concentration
Great inoculum size
Efficiency of multiphase systems
Effect of container and packaging
Type and initial level of contamination

Methods of preservation of pharmaceutical products:
1
...

3
...

5
...


2
...

4
...

6
...

R & D (research and development): In includes formulation design and development
...

QC: QC stands for quality control
...

Post marketing surveillance
Contamination risk assessment
...
Principles and Procedure of Sterilization
of Pharmaceutical Products
Validation: Validation may be defined as establishing documented evidence which provides a high degree of assurance
that specific process, method or system will consistently produce a result meeting predetermined acceptance criteria
...

Principles: Three principles should involve in validation process:
1
...

3
...

To demonstrate maximum level of probability that sterilization methods has established sterility to all batches
of unit
...


Validation steps:
1
...


Process design: This is the research and development phase and involves defining a process for manufacturing
the product
...

Creation of a Quality Target Product Profile (QTPP)
ii
...

Defining Critical Process Parameters (CPPs)
iv
...
It involves collecting and evaluating data on all aspects
and stages of the manufacturing process
...

ii
...

iv
...

vi
...


The building and facilities, i
...
ensuring they adhere to local regulations as well as pharmaceutical
manufacturing regulations
The transportation of raw materials
Storage of raw materials
The knowledge, training, and working practices of production line employees
Every step of the process to turn raw materials into the finished product
...

Finished product packaging, storage, and distribution

Continued process verification: Continued process verification involves ongoing validation during
production of the commercial product to ensure the process designed and qualified in the previous stages
continues to deliver consistent quality
...

This stage involves product sampling, analysis and verification at various points in the manufacturing process
and requires the involvement of employees with quality control training
...

Process:
A
...
Chemical
C
...
Physical: It includes:
1
...
Dry heat
▪ Red heat
▪ Flaming

7

▪ Hot air oven
▪ Incineration
b
...

Below 1000 C
▪ Pasteurization
▪ Water bath
▪ Vaccine bath
▪ Inspissator
ii
...

Above 1000 C
▪ Autoclave (1210 C)
2
...
Filtration
o Berkefeld
o Chamberland
o Seitz
o Sintered glass
o Membrane filters
o HEPA filter
4
...

B
...
Non thermal:
• High pressure processing (HPP)
• Chemical treatments
• Ultrasound
• Ionizing irradiation
• Pulsed electric field
• Electric arc discharges
• Oscillating magnetic field
• Enzymes
• Bacteriocins and antimicrobial ingredients
• Dense phase CO2 (DPCD)
• Ozone or cold plasma

8

Pharmaceutical importance of sterilization:
1
...


3
...

5
...


Moist heat sterilization is the most efficient biocidal agent
...
)
• Aqueous injections
• Ophthalmic preparations
• Irrigation fluids etc
...
These include products like –
• Dry powder drugs
• Suspensions of drug in non-aqueous solvents
• Oils
• Fat waxes
• Soft hard paraffin silicone
• Oily injections
• Implants
• Ophthalmic ointments and ointment bases etc
...

UV light is perhaps the most lethal component in ordinary sunlight used in sanitation of garments or
utensils
...

Filtration sterilizations are used in the treatment of heat sensitive injections and ophthalmic solutions,
biological products, air and other gases for supply to aseptic areas
...
For heat and
radiation treatments, respectively, this is defined as the time taken at a fixed temperature or the radiation dose required
to achieve a 90% reduction in viable cells (i
...
a 1 log cycle reduction in survivors)
...


Z value: For heat treatment, a D value only refers to the resistance of a microorganism at a particular temperature
...
It represents the increase in temperature needed to

9

reduce the D value of an organism by 90% (i
...
1 log cycle reduction)
...


F value: If the D value is measured at different temperature and pressure it can be seen that D value varies with the
pressure, more powerful is considered the process
...
Mathematically the F value is expressed by the rate of mortality per minute in function of
temperature for a given pressure
...

Sterile pharmaceutical products: Sterile pharmaceutical products are products free of microorganisms
...

Types of sterile pharmaceutical products:
1
...
Contact lens solutions e
...
wetting agents, cleaning solutions, soaking solutions
...
Surgical dressings
4
...
Absorbable haemostats e
...
oxidized cellulose, absorbable gelatin foam, human fibrin foam, calcium alginate
...
Surgical ligatures and sutures e
...
catgut (non-absorbable types)
7
...
Purified cotton gauze
Quality control and quality assurance of sterile products
In a pharmaceutical organization, a quality control is a fundamental segment that refers to a process of striving to
produce a product by a series of In Process Quality Control (IPQC) test in order to eliminate or prevent error at any
stage of production
...

2
...

4
...

6
...
g
...

Identify which are the critical steps in the manufacturing of the product, where it will be necessary to check
certain parameters to confirm that the process is in control
...

Define the frequency of checking of parameter
...

Keep a provision for modification of process if required
...


The entire process is described and explained to the IPQC workers and supervisors before implementing
...


Tests:
1
...
Package integrity reflects its ability to
keep the product in and to keep potential contamination out
...
Leakage test can be done by dye bath test
...
Vacuum and pressure is applied for some time
...
The container is then inspected for the presence of dye earlier visually
or by means of UV spectroscopy
...
The dye test
can be optimized by use of a surfactant and/or a viscosity fluid in the dye solution to increase the capillary
migration through the pores
...
The test
is inexpensive and no special equipment is required for visual dye detection
...
The test is used for ampoules and vials
...


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


3
...
Rabbits have a similar pyrogen tolerance to humans, so by observing a change in body
temperature in rabbits it is possible to make a determination of the presence of pyrogens
...

Principle: The test involves the measurement of rise in body temperature of rabbits following IV injection of
sterile solution of substance being examined
...
Preliminary test (Sham test)
b
...


Preliminary test (Sham test): It is carried out in room without disturbances and temperature variance
must be ±30 C
...

↓
Withhold the food from animal before 2 hours of starting the test and access to water may be allowed
...

↓
After 90 minutes, IV injection is given 10 ml/Kg (pyrogen-free saline solution)
↓
The temperature of the animals is recorded after IV injection at an interval of 30-minute and continued
for 3 hours after injection
...
6 should not be used for the main test
...
Main test:
The test substance is dissolved in pyrogen-free saline water and the liquid is warmed to 380 C before giving
injection
...
5 ml/Kg and not more than 10 ml/Kg of body mass
...
80 C
• The differences in initial temperature should not differ from one another by more than 10 C
Interpretation of the results:
1
...
60 C or sum of three rabbits raise in
temperature does not exceed 1
...

Case 2:
• If 2 or 3 rabbits show increase in temperature ≥0
...
40 C, then the experiment should be continued or repeated using
additional 5 rabbits
...
6 0 C or sum of eight
rabbits raise in temperature does not exceed 3
...


2
...

2
...


4
...

6
...


Repeated use of animals leads to endotoxin tolerance
...
g
...

There is also variability in control results when identical standardized endotoxin preparations are
used, which is probably related to interlaboratory factors and variations due to seasons, rabbit
species and another biological sources
...

The presence of pyrogen may be hidden by the pharmacological activity of the product’s
components
...

The rabbit test is insufficiently sensitive to detect endotoxin in intrathecal products where only
low levels of pyrogens are acceptable
...
Limulus Amebocyte Lysate (LAL) test
Principle: The LAL assay is an in vitro assay used to detect the presence and concentration of bacterial
endotoxins in drugs and biological products
...
Pyrogens as a class are fever-inducing substances that can
be harmful or even fatal if administered to humans above certain concentrations
...
The test for bacterial endotoxins uses lysed amoebocytes (blood cells) of the horseshoe crab and
is therefore termed the Limulus aboebocyte lysate (LAL) test
...

↓

12

The LAL is separated from the remaining cellular debris and its activity optimized using metallic cations, pH
adjustment and additives and then freeze-dried
...
1 ml of each) are mixed in a depyrogenated test
tube
...

↓
The British Pharmacopoeia describes six separate methodologies for the test for endotoxin
...
Gel clot limit test (most commonly used) → solid clot indicates positive result
2
...
Turbidimetric kinetic method
4
...
Chromogenic endpoint method and
6
...

ii
...

7
...


b
...

Difficult to correlate with rabbit test
...

Sterility test: The tests for sterility are intended for detecting the presence of viable microorganism
in pharmaceutical preparation that is designed to be sterile
...
Test for sterility may be carried out by one of the following two methods:
Membrane filtration method: Membrane filters having a normal pore size not greater than 0
...
Cellulose nitrate filters, for example, are used for aqueous, oily
and weakly alcoholic solutions; and cellulose acetate filters, for example, are used for strongly
alcoholic solutions
...
g
...

Direct inoculation method: In this method, the test sample is added directly into the required media,
ensuring that the amount of sample is below 10%
...
The culture is incubated at appropriate temperature for not less than 14
days
...


Monitoring of sterilization: There are three forms of monitoring required to ensure that sterilization is achieved
...

2
...


Physical or mechanical monitoring – record keeping
Chemical monitoring and
Biological monitoring

They are discussed below:
1
...
Records must be maintained on site for one year and on file for five years
...
Date and time of each load
b
...
Time of the sterilizer stayed in the recommended temperature range
d
...

Record should be kept when repairs and spore tests are done along with the results of the spore tests
and any other notable occurrences related to the sterilizer
...


3
...

Chemical monitoring: During each sterilization cycle, every instrument/package must have a temperature
sensitive indicator (tape or label) which changes colour if the packaged item was processed
...

Biological monitoring: Biological monitoring must be carried out because chemical indicators (indicator
tape) do not provide proof of sterilization
...
Different types of sterilizers require different types of spores for testing
...

• Bacillus atrophaeus (formerly Bacillus subtilis) spores are used to test dry heat sterilizers
...

Interpretation of test results:
1
...

3
...

5
...

A positive spore test result (spore growth) means that the sterilizer has failed and must not be used until
it has been serviced and demonstrates three consecutive negative tests
...

Spore test results must be available on site for one year and on file for five years
...


14

4
...

Purposes:
1
...

3
...


To guide the clinician in selecting the best antibiotic agent for an individual patient
...

To accumulate epidemiological information in the resistance of microorganisms of public health importance
within the community
...


Types:
A
...

• For less serious infections such as uncomplicated urinary tract infections
...
Quantitative:
• In the treatment of serious infections such as endocarditis or osteomyelitis
...
g
...

• Those who are critically ill
...

The basis:
o
o

The antibiotic contained in a reservoir is allowed to diffuse out into the medium and interact in a plate freshly
seeded with the test organisms
...

• The antibiotic diffuses out of the disk to form the gradient
...


15

o

Inhibition zone edge is formed at the critical time where a particular concentration of the antibiotic is just able
to inhibit the organism before it reaches an overwhelming cell mass or critical mass
...

↓
This growth is transferred to a tube of saline
...

↓
The plates are inoculated by dipping a sterile swab into the inoculum
...

↓
The swab is streaked all over the surface of the medium three times, rotating the plate through an angle of 600 after
each application
...

↓
The inoculum is leaved to dry for a few minutes at room temperature with the lid closed
...


2
...

4
...


A maximum of seven disks can be placed on a 9-10 cm plate
...

The plates should be placed in an incubator within 30 minutes of preparation
...

Incubation should not be done in an atmosphere of CO2
...


Measurement of diameter: It can be done by –
1
...

3
...

Using a pair of calipers: On the plate containing opaque medium
...
g
...


Interpretation of results:
1
...


Using a template: Standard templates are available for each antibiotic
...

• Resistant: When there is no zone, or when it lies within the white circle
...

Using a ruler: The diameter of the zone of inhibition is measured using a ruler or a pair of calipers
...


Dilution methods: Used to determine the minimal concentration of antibiotic to inhibit or kill the microorganism
...

Tube dilution
Method:

16

Patient’s organism is added to tubes containing decreasing amounts of the antibiotic
...

2
...


Not easily automated
...

Time consuming
...

Advantages: Many strains can be inoculated on each plate containing an antibiotic dilution
...

2
...


Not easily automated
...

Time consuming
...
It is a quantitative method of antibiotic sensitivity testing
...

Procedure:
A predefined stable antibiotic gradient is present on a thin inert carrier strip
...

↓
Immediate release of drug
↓
Incubation of plate
↓
Symmetrical inhibition ellipse is produced
↓
The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC with inherent
precision and accuracy
...

2
...

4
...

Detecting –
• Glycopeptide resistant enterococci (GRE)
• Glycopeptide intermediate S
...

Confirming an equivocal antimicrobial susceptibility test
...

2
...


Simple
Accurate and
Reliable

Disadvantages:
1
...


17

5
...

Clean room requirements for various industries:
A
...
External drugs
2
...
Beta lactum drugs
4
...
Capsules
6
...
Re-packaging units of bulk drugs
8
...
Primary, secondary and tertiary packaging rooms
10
...
Chemical analysis laboratory
B
...

C
...

D
...

E
...
Hence, this sector also
has wide range of clean room applications
...
They
are –
1
...

3
...
It is used for operations that afford high risk for product quality e
...
filling, closing,
ampoule and bottle opening zones
...

B – It is the zone which is circled by A zone
...

C and D – They are clean zones for less responsible stages of manufacturing sterilized products
...

2
...

4
...

6
...

8
...

10
...

12
...


Exclusion of the environment external to the suite of cleanrooms
...

Removal of dilution of contamination arising from personnel working in the area
...

Control of product-to-product cross-contamination
...

Control and management of the flow material through the process steps by means of layout and configuration
...

Overall security of the operation by control of the entry and egress of personnel and materials
...

Special environmental conditions for products e
...
low RH (relative humidity) for powder filling
...

Effective monitoring of the conditions of the room
...

2
...

4
...

6
...

8
...

10
...
Manufacture and Quality Control of Therapeutic
Synthetic and Recombinant Products
Monoclonal antibodies
Antibodies or immunoglobulins are protein molecules produced by a specialized group of cells called B lymphocytes
(plasma cells) in mammals
...
Each antigen has specific antigen determinants (epitopes) located on it
...

In response to an antigen (with several different epitopes), B lymphocytes gear up and produce many different
antibodies
...
The
polyclonal antibody production is variable and is dependent on factors such as epitopes, response to immunity etc
...

Monoclonal antibody is a single type of antibody that is directed against a specific antigenic determinant (epitope)
...
In the early years, animals were
immunized against a specific antigen, B lymphocytes were isolated and cultured in vitro for producing monoclonal
antibodies
...

It is interesting that immortal monoclonal antibody producing cells do exist in nature
...
It was in 1975
...
They could successfully hybridize antibodyproducing B lymphocytes with myeloma cells in vitro and create a hybridoma
...
The hybridoma
cells possess the growth and multiplying properties of myeloma cells but secrete antibody of B lymphocytes
...

Principle for creation of hybridoma cells
The myeloma cells used in hybridoma technology must be capable of synthesizing their own antibodies
...
The
mammalian cells can synthesize nucleotides by two pathways – de novo synthesis and salvage pathway
...
The formation of
tetrahydrofolate and therefore nucleotides can be blocked by the inhibitor aminopterin
...

Hypoxanthine guanine phosphoribosyl transferase (HGPRT) is a key enzyme in the salvage pathway of purines
...

Thymidine kinase (TK), involved in the salvage pathway of pyrimidines converts thymine to thymine monophosphate
(TMP)
...

When cells deficient (mutated cells) in HGPRT are grown in a medium containing hypoxanthine aminopterin and
thymidine (HAT medium), they cannot survive due to inhibition of de novo synthesis of purine nucleotides (salvage
pathway is not operative due to lack of HGPRT)
...

The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained
from lymphocytes (with which myeloma cells are fused)
...

Production of monoclonal antibodies:
Steps:
1
...


Immunization
Cell fusion

20

3
...

5
...


Selection of hybridomas
Screening the products
Cloning and propagation
Characterization of storage

They are discussed below:
1
...


3
...


5
...


Immunization: The very first step in hybridoma technology is to immunize an animal (usually a mouse), with
appropriate antigen
...
The injections
at multiple sites are repeated several times
...
Three days prior to killing of the animal, a final dose of antigen is intravenously
administered
...
The concentration of the desired antibodies is assayed in the serum of the animal at frequent
intervals during the course of immunization
...
The spleen is aseptically
removed and disrupted by mechanical or enzymatic methods to release the cells
...

Cell fusion: The thoroughly washed lymphocytes are mixed with HGPRT defective myeloma cells
...

PEG is removed by washing and the cells are kept in a fresh medium
...

Selection of hybridomas: When the cells are cultured in HAT medium, only the hybridoma cells grow, while
the rest will slowly disappear
...
Selection of a single antibody producing
hybrid cells is very important
...
The
suspension of the hybridoma cells is so diluted that the individual aliquots contain on an average one cell
each
...

Screening the products: The hybridomas must be screened for the selection of the antibody of desired
specificity
...
The two techniques namely ELISA and RIA are commonly used for this purpose
...
Thus, the hybridoma cells producing the desired
antibody can be identified by screening
...

Cloning and propagation: The single hybrid cells producing the desired antibody are isolated and cloned
...

• Limiting dilution method: In this procedure, the suspension of hybridoma cells is serially diluted
and the aliquots of each dilution are put into microculture wells
...
This ensures that the antibody produced is
monoclonal
...
It is possible to
simultaneously grow many cells in the semisolid medium to form colonies
...

In actual practice, both the above techniques are combined and used for maximal production of
monoclonal antibodies
...
It is also important to elucidate the monoclonal antibody for the
immunoglobulin class or sub-class, the epitope for which it is specific and the number of binding sites it
possesses
...
In the modern concept, vaccination involves the
administration (injection or oral) of an antigen to elicit an antibody response that will protect the organism against
future infections
...

2
...


Viral fragments or bacteria molecules (subunit vaccines)

A vaccine triggers the body’s immune system to produce antibodies against a specific disease – causing organism (virus,
bacterium or other parasite)
...
Many communicable diseases (small pox, cholera, typhoid, tuberculosis, poliomyelitis) have been brought
under control through vaccination
...

2
...


Subunit recombinant vaccines: These are the components of the pathogenic organisms
...

Attenuated recombinant vaccines: These are the genetically modified pathogenic organisms (bacteria or
viruses) that are made non-pathogenic and used as vaccines
...


Subunit vaccines production
Subunit vaccines e
...
recombinant hepatitis B vaccine is produced by cloning HBsAg gene in yeast cells
...
It is a part of a quality system covering the
manufacture and testing of active pharmaceutical ingredients, diagnostics, pharmaceutical products and medical
devices
...

2
...

4
...

6
...

8
...


Precautions must be taken to prevent unauthorized persons from entering storage areas
...
In particular, they should be
clean and dry and manipulated within acceptable temperature limits
...
g
...

Storage areas should be clean and free from accumulated waste and vermin
...
Where no expiry dates exist for the products, the FIFO principle should be applied
...
A written
cleaning program should be available, indicating the frequency of cleaning and the methods to be used
...

Special attention should be given to the design, use, cleaning and maintenance of all equipment used for the
handling of pharmaceutical products which are not in a protective shipping cartoon or case
...

Where possible mechanisms should be available to allow for the segregation during transit of rejected, recalled
and returned pharmaceutical products as well as suspected to be counterfeits
...


Minimum inhibitory concentration (MIC)
The MIC is the lowest concentration (in μg/ml) of an antibiotic that inhibits the growth of a given strain of bacteria
...
This information can lead to an appropriate choice of
an antibiotic that will increase chances of treatment success and help in the fight of slow antibiotic resistance
...
They are:
•
•
•

Sensitive: Sensitive implies that the organism is inhibited by the serum concentration of the drug that is
achieved using the usual dosage
...

Resistant: Resistant implies that the organisms are resistant to the usually achievable serum drug levels
...

2
...

4
...

Interpretive criteria is not available from CLSI
...

Certain antibiotics are not available on our commercial system
...


Method of use
The breakpoint and range of dilutions differ by drug and bacterial species
...
Therefore, comparing MICs of different antibiotics is not based solely on the
numerical value but on how far the MIC is from the breakpoint, the site of the infection, and other considerations,
such as the age, species and health of the animal
...


23

For example: A strain of E
...
Looking at the dilutions for
amoxicillin, at 2 μg/ml, this strains of E
...
For cefovecin, the same strain
of E
...
So, based on MICs, this strain of E
...

In vitro efficacy of amoxicillin
Sensitive

Intermediate

Resistant

2, 4, 8

16

32

In vitro efficacy of cefovecin
Sensitive

Intermediate

Resistant

2

4

8

Strengths of MIC
1
...

3
...


The MIC test is relatively straightforward and easy to prepare for and execute, which naturally enhances
reproducibility
...
This is important
for experimental antimicrobials, such as biologically synthesized antimicrobial peptides
...

Because little preparation is required for the minimum inhibitory concentration testing, test turnaround times
can be kept low
...


2
...
For example,
extended incubation will make the MIC appear to be higher, and lower inoculum concentrations will make
the MIC appear to be lower
...
In the tube corresponding to the MIC,
microorganisms were merely prevented from growing and not necessarily killed; there could still be 500,000
viable cells in that dilution vessel just waiting to grow should the antimicrobial agent become chemically
neutralized
...
It can
be determined from the broth dilution of MIC tests by lowest concentration of antibacterial agent that reduces the
viability of the initial bacterial inoculum by a pre-determined reduction such as ≥99
...
The MBC is complementary
to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent resulting in microbial death
...
Antimicrobial agents are usually regarded as bactericidal if the MBC is
no more than four time the MIC
...
The MBC test can be used to evaluate formulation problems wherein the
formulator suspects that the active ingredient is being ‘bound up’ by other ingredients
...

Procedure:

24

Plates containing a drug-free growth medium are inoculated with samples taken from zones of inhibition or from
clear MIC tubes
...
The lowest concentration for which no bacterial growth
occurs on the plate is the minimum bactericidal concentration; in this case, the MBC is 16 μg/ml
...


2
...


4
...
It is worth nothing
...
Thus, the test truly does determine the
minimum concentration needed to kill the test organism, since all other parameters are conductive to biocidal
effect
...

The MBC test can be used to evaluate formulation problems wherein the formulator suspects that the active
ingredient is being ‘bound up’ by other ingredients
...

The test parameters for the MBC are easy to control in the laboratory, so comparisons can be made fairly
easily between various antimicrobial agents tested under the same conditions and their respective effects on
specific microorganisms
...

2
...


An MBC must be determined for each microorganism individually as the antimicrobial will likely have
different MBC values for different test microorganisms
...

More nutritive growth media such as Tryptic Soy Broth can negatively affect MBC values
...


Diffusion method
The diffusion test (disk diffusion test, or agar diffusion test, or Kirby–Bauer test) is a test of the antibiotic
sensitivity of bacteria
...

↓
Filter paper disks impregnated with known concentrations of chemotherapeutic agents are placed on the solidified agar
surface
...

↓
The farther the agent diffuses from the disk, the lower its concentration
...

↓
The diameter of the zone can be measured; in general, the larger the zone, the sensitive the microbe is to the antibiotic
...

↓
The zone diameter is compared to a standard table for that drug and concentration, and the organism is reported as
sensitive, intermediate or resistant
...

2
...


The test is simple
...

It is most often used when sophisticated laboratory facilities aren’t available
...


Results obtained by the disk diffusion method are often inadequate for many clinical purposes
...

Examples:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

Fillers
Binders
Disintegrants
...

Sorbents
...

Lubricants
...

Preservatives
...

Flavoring agents
...

Coloring agents
...

Buffering agents
...

Viscosity imparting agents
...

Humectants
...

2
...

No unwished interaction with drug
...

4
...


Pharmacologically inert
...

Cost effective
...

2
...

4
...

Facilitate drug absorption or solubility and other pharmacokinetic considerations
...

Provide stability and prevent from denaturation
...
1
...

Pharmaceutical biotechnology: Pharmaceutical biotechnology is a relatively new and growing field in
which the principles of biotechnology are applied to the development of drugs
...

Pharmacogenomics: Pharmacogenomics is the study of how genes affect a person's response to drugs
...

Biopharmaceuticals: A biopharmaceutical, also known as a biological medical product, or biologic, is
any
pharmaceutical
drug product
manufactured
in,
extracted
from,
or semisynthesized from biological sources
...

Major kinds of biopharmaceuticals include:
1
...

3
...

5
...

7
...

9
...

Biologics treat a broad range of common and rare diseases and include vaccines, cell and gene therapies,
tissues for transplants, and more
...

They are larger and more complex molecules and because they are made from living organisms, they
are inherently more variable
...

2
...

4
...

6
...

8
...

10
...


DNA/protein engineering and processing
Synthetic biotechnology
Omics (genomics, proteomics, metabolomics and systems biology)
Therapeutic biotechnology (gene therapy, peptide inhibitors, enzymes)
Drug delivery and targeting
Nanobiotechnology
Molecular pharmaceutics and molecular pharmacology
Analytical biotechnology (biosensing, advanced technology for detection of bioanalytes)
Pharmacokinetics and pharmacodynamics
Applied Microbiology
Bioinformatics (computational biopharmaceutics and modeling)

28

12
...

14
...

16
...


Environmental biotechnology
Regenerative medicine (stem cells, tissue engineering and biomaterials)
Translational immunology (cell therapies, antibody engineering, xenotransplantation)
Industrial bioprocesses for drug production and development
Biosafety
Biotech ethics

Q
...
Write down the names of common biologics and the procedure of biologics production
...

2
...

4
...

6
...


Whole blood and other blood components
Organs and tissue transplants
Stem cell therapy
Antibodies for passive immunization (e
...
, to treat a virus infection)
Human breast milk
Fecal microbiota
Human reproductive cells

Production of biologics:
Using well-established techniques, a gene encoding the desired protein is injected into a
production cell
...
coli bacterial cells and
Chinese hamster ovary cells
...

↓
Cells are frozen to ⸺ 1960 C for future use
...
3
...
Give a short overview of pharmaceutical
product
...

Q
...
Which microorganisms are involved in pharmaceutical spoilage? Which pharmaceutical
ingredients are susceptible to microbial degradation?
Ans: From note
...
5
...
What are the sources of contamination? What are the visible signs of
contamination?
Q
...
Briefly discuss about the factors affecting microbial spoilage
...


29

Q
...
Define preservatives
...

Ans: From note
...
8
...

Ans: From note
...
10
...

Q
...
Write down the physical, chemical and biological monitoring system in sterilization
...

Q
...
Define sterile pharmaceutical products
...

Ans: From note
...
13
...
Which microorganisms are recommended by BP (British
Pharmacopoeia) and USP (United States Pharmacopoeia) for challenge test?
Challenge test: A challenge test (also known as a preservative effectiveness test, PET or antimicrobial
effectiveness test) is a procedure to determine whether a formulated cosmetic, pharmaceutical or other
type of product is adequately preserved to prevent contamination from raw products and during
consumer use
...

2
...

4
...


The BP also permits other test organisms when necessary
...
Give a diagrammatic representation of the design of a challenge test procedure at the
beginning, during and at the end of a product shelf-life
...
14
...

Ans: From note
...
15
...

2
...

4
...

6
...
Contaminated sharps
should never be carried by hands
...

The hands should be cleaned with alcohol hand rub and don gloves
...

The instruments should be dried and cleaned
...

Loose instruments should be packed so that they lay in a single layer
...


Q
...
Define sterilization
...

Ans: From note
...
17
...
When the air has been removed, the temperature in the chamber is
proportional to the pressure of the steam
...
Under these conditions, spores directly exposed are killed in less
than 5 minutes
...
For example, the
spores of Cl
...

The use of saturated steam under pressure in the autoclave has many advantages:
1
...

3
...

Steam under pressure has more penetrating power
...

During condensation of steam to water, a large amount of latent heat is liberated, thus increasing the efficacy of
sterilization
...
g
...
Temperature is not increased from 1210 C because at higher temperature, the media or
other nutritious materials may be destroyed or denatured
...
In support of this fact, it has been found that the presence of moisture significantly
affects the coagulation temperature of proteins and the temperature at which microorganisms are
destroyed
...

2
...

4
...


It provides a physical method for disinfection and sterilization
...

Autoclaves vary in size, shape and functionality
...

It can sterilize solids, liquids, hollows, and instruments of various shapes and sizes
...
What precautions should be taken while using an autoclave?
The following precautions should be taken while using an autoclave
...

2
...

4
...

6
...
It is more
efficient and safer to run two separate, uncrowded loads than one crowded one
...
Articles
should be wrapped in materials that allow steam penetration
...

Polyethylene trays should not be used as they may melt and cause damage to the autoclave
...
18
...
g
...
) is chosen for the material
32

↓
Appropriate temperature is set for the cycle
...

↓
The autoclave is turned to STERILIZE and loaded
...

↓
A system cycle is approximately 40 minutes if the autoclave is cold and approximately 20 minutes if it
is already warmed from a previous cycle
...

↓
The autoclave is turned to POWER OFF
...
Steam at 1340 C can achieve a desired level of sterility in three minutes, in contrast
to hot air at 1600 C, which can take two hours to achieve the same sterility
...
Describe different types of autoclaves
...


2
...

4
...


6
...
Here, the autoclaves should maintain a
temperature of at least 2460 C for half an hour
...
For steam heat autoclaves,
heated water vapors are used
...

Gas autoclaves: They are also known as chemiclaves; gas autoclaves use a vapor solution to sterilize its contents
...
They consume lesser heat and
take lesser time to complete the cycle
...

Cold sterilization autoclaves: They use a cold sterilization liquid to sterilize the contents
...

Stovetop autoclaves: In such autoclaves, the tools should always be separated to allow the steam to penetrate the
load evenly
...


Q
...
Define sterilization validation
...

Sterilization validation: Validation may be defined as establishing documented evidence which
provides a high degree of assurance that specific process, method or system will consistently produce a
result meeting predetermined acceptance criteria
...

Objectives of sterilization validation:
1
...


The sterilization process will consistently achieve sterility
...


33

3
...

5
...


Minimize reliance on end product testing
...

Increase sterility assurance level (SAL) to all units
...


ETO (Ethylene Oxide) validation: An Ethylene Oxide (EO) Sterilization Validation is designed to
assist the manufacturer in the development of a sterilization process that delivers the appropriate sterility
assurance level and ensures repeatability for each product type developed
...

One of the most popular methods of sterilization of medical devices is through exposure to Ethylene
Oxide gas (EtO/EO)
...
EtO is toxic and flammable/explosive at low
temperatures (flash point of -20°C) and so is used on products that could get damaged or cannot
withstand high temperature processes
...

2
...

4
...


Plastic products/packaging that get discolored with irradiation
Devices that incorporate electronic components
Materials that get damaged at higher temperatures
Custom kits
Materials that are not compatible with other methods such as Gamma and Steam sterilization

Process:
Preconditioning and/or Conditioning of device through temperature & humidity variations
↓
Gas Dwell Phase/Sterilizing Cycle where the device is exposed to the EtO gas
↓
Aeration of exposed device for removal of gas from the product
Q
...
Define sterility test
...

Ans: From note
...
21
...

Objectives of antibiotic sensitivity test:
1
...

3
...

To distinguish the range of activity of an antibiotic
...


Purpose of Kirby-Bauer disc diffusion method: The purpose of the Kirby-Bauer disk diffusion
susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative
anaerobic bacteria to various antimicrobial compounds in order to assist a physician in selecting
treatment options for his or her patients
...
The presence or absence of growth
around the disks is an indirect measure of the ability of that compound to inhibit that organism
...

↓
This growth is transferred to a tube of saline
...

↓
The plates are inoculated by dipping a sterile swab into the inoculum
...

↓
The swab is streaked all over the surface of the medium three times, rotating the plate through an
angle of 600 after each application
...

↓
The inoculum is leaved to dry for a few minutes at room temperature with the lid closed
...


4
...
There are three types of results:
• Susceptible: When the edge of the zone of inhibition is outside the black circle
...

• Intermediate: When the edge of the zone of inhibition lies on the black circle
...
This diameter
is interpreted according to the critical diameters
...
22
...
What are the sources of pyrogens and its elimination methods? Briefly
describe
...
Example: endotoxins
...

2
...

4
...

Water used during processing
...

Chemicals, raw materials or equipment used in the preparation of the product
...

2
...


Rinsing or dilution: Rinsing or dilution is one way of eliminating pyrogenic activity provided that the rinsing fluid
is apyrogenic
...

Heating:
• Pyrogens in vials or glass components may be destroyed by dry heat sterilization at high temperatures
...

5
...

• Pyrogens are also destroyed at 6500 C in 1 minute or at 1800 C in 4 hours
...

Sterilizing tunnels: Sterilizing tunnels are designed not only to sterilize at 250 – 3000 C but also to remove
pyrogens
...


Q
...
Briefly describe about LAL test of endotoxin
...

Q
...
What are the principles of sham test? Illustrate the procedure of sham test
...

Procedure: From note
...
25
...
Describe the production of insulin through rDNA technology
...

Production of insulin through rDNA technology:
A and B chain producing genes are synthesized in an amino acid sequencing machine
↓
These two DNA molecules are then inserted into plasmid
...
coli and then to the lac Z gene which
encodes for 8-galactosidase
↓
The recombinant, newly formed plasmids are mixed up with the bacterial cells
...
DNA ligase is added to stick the plasmid with the bacterium’s
DNA
...
They are grown at optimal
temperatures in large tanks in manufacturing plants
...

↓
After multiplying, the cells are taken out of the tanks and broken open to extract the DNA
...
An oxidizing agent is added
...

↓
The DNA mixture is then purified so that only the insulin chains remain
...

↓
Batches are tested to ensure none of the bacteria’s proteins is mixed in with the insulin
...
coli DNA
...
26
...

36

Ans: From note
...
27
...
Describe the production of subunit vaccine through rDNA
technology
...

Q
...
What do you mean by therapeutic enzymes? Write down the names of microorganisms
and their therapeutic enzymes with applications
...


Acid protease

2
...


Amylase

4
...


Asperginase

6
...


Bacitracin synthetase

8
...


Glucose oxidase

10
...
Glutaminase
12
...
L-amino acid and α-ligase
14
...
Maltase
16
...
Non-ribosomal peptide
synthetase
18
...
Peptidase
20
...
Protease
22
...
Ribonuclease
24
...
Sacrosidase
26
...
Staphylokinase
28
...
coli
Leukemia
E
...

Antitumor
Aspergillus niger
Leukemia
E
...

spectrum antibiotic production
Celiac disease, clot formation,
Bacillus polymyxa,
inflammation and repair
Beuveria bassiana
Antibiotic
Bacillus brevis
Antibacterial
Pseudomonas aeruginosa
Cyanide poisoning
Sulfobacillus sibiricus
Antiviral
Saccharomyces spp
...

Anticoagulant
Streptococci spp
...
Superoxide dismutase
30
...
Urease
32
...
Urokinase
34
...
α galactosidase

36
...
β galactosidase
38
...
and
Nocardia spp
...
and
Klebsiella aerogens
Aspergillus flavus
Bacillus subtilis
Vibrio proteolyticus
Aspergillus spp
...

Citrobacter freundii,
Serratia marcescens

Antioxidant, anti-inflammatory
Antitumor, treatment of
Parkinson’s disease
Nitrogen metabolism of
ruminants
Gout
Blood clots
Treatment of damaged tissue
Fabry’s disease, prevention of
xenorejection, blood group
transformation
Antioxidant
Removal of lactose from milk
Antibiotic resistance

Q
...
Give a diagrammatic representation of a cloning strategy for industrial production of
enzymes

---