NOVEMBER 30, 2022

COURSE CODE: 702

Submitted to: Dr Yusra Shafiq and Dr. Muhammad Suleman Imtiaz

BIOTECHNOLOGICAL ASPECT IN
PRODUCT DEVELOPMENT
PHARMACEUTICAL TECHNOLOGY-II THEORY
UME FARWA
ROLL No. 55
Jsmu/dpm/055/ips/17

BIOTECHNOLOGY
 BIOTECHNOLOGY

It is the use of biological organism, system, or process to develop technologies and product to
improve the quality of life. Biotechnology is used in various field including agriculture, food
science and pharmaceuticals. Pharmaceutical biotechnology companies use recombinant DNA
technology which includes genetic modification of cell or a monoclonal antibody for making
their biotechnological product.

HISTORY
The history of the discovery and application of therapeutic drugs, such as sulfa drugs, which was
widespread use and were one of the very first to be marketed, and then proceeds to various bio
formulations. The pharmaceutical companies that have marketed bio formulations use
biotechnology principles such as recombinant DNA technology to design more effective protein-
based drugs, such as erythropoietin and fast acting insulin and also use in other areas such as
genomics, proteomics and high-throughput screening have paved the way for exploring new
avenues of drug discovery. The author also offers a prospective analysis of the use of gene
therapy and whole cell-based therapeutics such as stem cells.

BIOTECHNOLOGIC DRUG
There are critical differences between biotechnological and the more common chemical drugs. A
chemical drug is a small molecule produced by chemical synthesis with a very well defined and
stable structure, not or barely sensitive to process changes, which is relatively stable. A
biotechnological drug or a bio-pharmaceutical product is a large complex bio molecule with a
heterogeneous structure, extremely sensitive to process changes and prepared by the use of
living systems, such as organisms, tissue cultures or cells, with the large majority manufactured
using recombinant DNA technology. This means that a human gene capable of triggering the
production of a specific protein is inserted into a living organism and cultured in the laboratory.
The organism incorporates the gene into its cell structure and produces large quantities of the
desired protein.

Examples of classes of protein based biotech drug,

 Erythropoietin
  Blood factors (factor vii)
 Human growth hormone, somatotropin.

ROUTE OF ADMINISTERATION
The route of administration of biotech drug is very different from a traditional drug taken as a
pill or capsule and each drug is developed with a unique route of administration the drug are
mainly given intravenously subcutaneously or intramuscularly. Biological drugs are usually
complex proteins and cannot be orally delivered due to problems related to degradation in the
acidic and protease-rich environment of the gastrointestinal (GI) tract. The high molecular
weight of these drugs often results in poor absorption into the periphery when administered
orally. The most common route of administration for these therapeutic proteins is injection. Most
of these proteins have short serum half-lives and need to be administered frequently or in high
doses to be effective. So, difficulties in the administration of protein-based drugs provides the
motivation for developing drug delivery systems (DDSs) capable of maintaining therapeutic drug
levels without side effects as well as traversing the deleterious mucosal environment.

PROCESS TO DEVELOPMENT OF PROTEIN BIOTECH PRODUCT

1. Isolation of gene of interest
2. Introduction of gene to expression vector
3. Transformation into host cell
4. Isolation and purification of protein
5. Formulation of protein product.

ISOLATION OF GENE OF INTEREST

Gene is pieces of DNA which store information for making specific protein that control specific
trial. Gene is present in nucleus chromosome which code for one polypeptide. The double strand
of DNA is cut open at specific site. I.e nucleotide sequence by special enzyme which is known as
Restriction endonuclease. Ligase is the enzyme used to joined pieces of the DNA through
covalent bound, Through this enzyme DNA containing gene of interest is cut and joined with
vector.
DNA RECOMBINATION
DNA recombination is a process in which the pieces of DNA from different organism are
artificially mixed to create recombinant DNA.

Step involved in recombinant DNA technology are

 DNA extraction
 Purification
 Fragmentation

Fragmentation of is done by specific restriction enzyme and is followed by sorting and isolation
of fragment containing a particular gene. The portion of the DNA is the n coupled to a carrier
molecule called as PLASMID. The hybrid DNA is then introduced into a chosen cell for
reproduction and synthesis.

TRANSFERS OF GENE TO EXPRESSION VECTOR

Different organism may be used to express a target protein the expression vector and therefore
will have elements specific for use in the organism. The most used for protein expression is the
bacterium Escherichia coli however not all protein can be successfully expressed in E. coli and
other systems may therefore be used such as

 Yeast e.g., commonly used for protein expression is pichia pastoriles

 Baculovirus
 Mammalian cell culture
 Plants
 Animals.

GROWTH OF CELL
After the transfer of RDNA into host of choice i.e. bacteria, yeast, E.coli they are cultivated in
culture medium

 For research (small scale)

Cell culture flask

 For production ( large scale)

 Fermentors and bioreactor

CULTURE MEDIA
It is important to provide nutritional conditions that exist in bacterial natural habitat

 Common components
 Sources of carbon and energy
 Trace elements
 Growth factors
 Buffer
 For growth on small scale incubator are used the most commonly used is shake flask
incubator

FERMENTOR AND BIOREACTORS

Bioreactor or fermentor is a container in which substrate is turned into product, where Hebe of
interest is mass produced.

Production process

TYPES OF PRODUCTION PROCESSES ARE;

1. Batch
2. Continuous
3. Fed batch

BATCH PROCESS
In this process the bioreactor is only fed once. The bioreactor will be allowed to run till
completion very difficult to achieve in real life because there should be no input to or withdrawal
from the bioreactor even for sampling.

ADVANTAGES AND DISADVANTAGES OF BATCH OPERATION

Advantages
 . Ease in operating
  Genetic stability of organism could be controlled if it is genetically engineered bio
catalyst
 Lower contamination risk

Disadvantages
 Nonproductive down time
 Batch to batch variability is problem
 Accumulation of inhibitory product

CONTINUOUS PROCESS.
The bioreactor is fed continuously. The amount of feed introduced into the bioreactor equals the
removed volume and the process is sensitive and subjected to influence from various factors.

FED BATCH PROCESS

In fed batch process one or more nutrients are fed to the bioreactor during cultivation and in
which the product remains in the bioreactor until the end of the run. Possible to control the rate
of growth of the microorganism or the concentration of the biomass by controlling the feed
parameter. It is the most used process in industry

ISOLATION AND PURIFICATION OF PROTEIN

There is necessity for the isolation and purification of protein prepared for biotech drug from
microorganism. The protein required may be extracellular or intracellular so for isolation of
extracellular product destruction of cell is not necessary but for intracellular cell disruption is
needed which is done through following method

 Detergents lysis
 Enzymatic lysis
 Osmotic lysis

Differential centrifugation separation of protein or any material on the basis of their size mass
and density

Dialysis

Gel electrophoresis
Column chromatography

ISSUE IN BIOTECH PRODUCT

Issue related to the use of protein-based drug s

Protein verse low molecular weight drug proper 3D structure required for biological activity

1. Drug delivery
2. Antigenicity
3. stability

STERILITY CONSIDERATION OF BIOTECH DRUG

Sterilization
It is impossible to sterilize the end product therefore it is important that

a) all the raw material should be sterilized

b) all the equipment must be sterilized
c) Processing should be carried out in aseptic environment.

Quality control
I. Viral testing
II. Bacterial testing
III. Pyrogen testing

Protein contaminants

Sources
I. Growth media
II. Host cell
III. Ligands from affinity chromatography columns

APPLICATION OF BIOTECHNOLOGY
Biotechnology has far reached applications in diverse fields of science, medicine, and more.
Application of Biotechnology in Agriculture:
Food processing is essential for the transformation of raw food ingredients into processed food
that is edible and remains fresh over time.

A. Cultivation of Disease Free of Plants:

Several biotechnological techniques can be used to grow plants free of viruses, and other
diseases. Traditional techniques to remove microbes such as the use of chemical pesticides have
proven to be expensive and polluting to the soil and groundwater. Hence modern biotechnology
has enhanced agricultural yield through the following methods:

B. Genetic Engineering:
A genetically engineered plant, also known as a genetically modified organism, or GMO, is one
that is grown via a new genetic modification (nGM) technique. The crops grown by this method
use recombinant DNA (rDNA) technology which may include techniques such as genome
editing, RNA-directed DNA methylation, transgrafting, agro infiltration, haploid induction, or
others. Genetic engineering of plants is regulated by bio safety frameworks specific to a country.
Examples of GMO crops include corn, potato, soybean, etc.

C. Micro propagation:
It is an in-vitro technique using vegetative propagation under high intensity of light and
controlled nutrition and temperature. This technique helps to grow plants that are on the verge of
extinction, develop disease free plants, and also for large-scale growth of plants. For instance,
banana is commonly grown via micro propagation techniques.

D. Transgenic Technique:
It is a genome-editing technique to develop disease free plants. Transgenic plants are developed
by editing out a gene or adding a new gene to the original chromosomal DNA to create variants
that are disease-free. For example, rice and sugarcane have been grown through transgenic
methods.

E. Soma clonal Variation:

Phenotypic or genotypic variation can be introduced in plants by using somaclonal variation

techniques, particularly by regeneration through the callus. In-vitro cell cultures are used to grow
plants which are then termed ‘somaclonal’ Somaclonal variation can be used to develop cultivars
that are pest-resistant. For example, tobacco, sugarcane, and potato are often cultivated through
somaclonal biotechnology.

F. Transcriptomics:

Transcriptome sequencing allows scientists to study the genome of plants. Transcriptomics

utilizes transcriptome sequencing technology to grow plants that are microbe-and stress-resistant
crops such as maize, tobacco, etc.

G. Fortification of Plants:
Fortification implies increasing the nutritive potential of crops which is very beneficial in
treating malnourishment. For example, ‘potato’ is a fortified potato crop that is genetically
modified to provide more protein than a traditional potato crop.