Immunological and biological pro.pptx

IMMUNOLOGICAL AND BIOLOGICAL
PRODUCTS
Merob S.(Asst. Lecturer) , School of Pharmacy,
CHMS, HU
8/13/2022
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Contents
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 Introduction to Biotechnology and Pharmaceutical Biotechnology
 Introduction to genetic engineering/ rDNA technology
 Concepts in rDNA technology
 Tools of genetic engineering (enzymes,cloning vectors,cloning hosts)
 Basic techniques(gene cloning, protein expression)
 Application of rDNA technology
 Polymerase chain reaction (PCR) and other techniquesof modern biotechnology

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What is biotechnology?
 A science that uses living organisms or the products from living organisms to benefit
humans and their surroundings.
 The term “biotechnology” is used interchangeably with “genetic engineering.”
Introduction

Introduction…
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 Biological product is defined as
“A virus, therapeutic serum, proteins and peptides, nucleic acids, antitoxin,
vaccine, blood, blood component or derivative, allergenic product, or
analogous product, or any other trivalent organic arsenic compound,
applicable to the prevention, treatment or cure of a disease or condition of
human beings”.

Introduction…
Biological products were predominantly proteins of eight uniquely different
types:
 8 growth factors (GFs)
 3 blood factors
 30 hormones
 12 interferons
 18 monoclonal antibodies (Mabs)
 15 enzymes
 6 fusion proteins, and
 3 interleukins 8/13/2022
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Introduction….
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 New biologicals are expanded to include:
 DNA/RNA derivatives, such as 1 m-RNA analogue
 Peptides
 Tissue or cell therapies
 Biologic drug carriers, such as 5 liposomes
 Natural derivatives

Introduction….
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 Biotechnology further encompasses biological products that have agricultural uses
and industrial applications
 In industry, naturally occurring Mos are being studied and used to consume substances
harmful to the environment, such as hydrocarbons (oil), mercury and sulfuric acid

Introduction….
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 Biotechnology is also a collection of biologic techniques and drug development
technologies that permit the discovery and developments of new biologic products
 Several of techniques now exist starting with the three cornerstone technologies:
1. Recombinant DNA (r-DNA) technology: is the manufacture of human proteins
in non-human living systems

Introduction….
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2. Monoclonal antibodies (mabs) technologies:
 Mabs are complex proteins that have a uniform basic structure, comprising four
subunits that are divided into two matched pairs of protein material—two heavy
chains and two light chains, linked by disulfide bridges:
 Produced in a mouse
 Murine hybridoma cell (hybrid of myeloma cell and mouse plasma cell)
technology

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3. Polymerase chain reactions (PCRs):
 It is a critical core process in biotechnology, which permits the expansion of the
amount of genetic material (DNA), starting from minute amounts.

Introduction…
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 In product development, technologies also include genetics-related technologies such
as:
Antisense
Genomics
Gene therapy
Pharmacogenomics
Ribozymes

Introduction….
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 Antisense: Antisense is a RNA molecule that is complementary to or a mirror image
of, a segment of mutated m-RNA molecule, involved in the pathogenesis of a disease;
 The antisense RNA molecule will bind to the noxious m-RNA molecule,
preventing the disease from manifesting
E.g. Fornivirsen, used to treat cytomegalovirus associated retinitis that can
occur in AIDS Pts

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 Gene therapy: is a technology employing a gene as a therapeutic agent to treat a
disease
 Pharmacogenomics: is the study of genetic phenotypes of patient groups and their
impact on drug actions, changing drug pharmacokinetics and/or activity

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 Genetic change can change action of metabolic enzymes, receptor activity, or drug
transport;
 Can lead to more adverse events, dosing changes up or down to achieve the same
effect, and disease subtypes with different drug responses

Introduction….
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 Ribozymes technology: are molecules comprising sequences of nucleic acids that
possess enzymatic catalytic properties to bind to specific sites in DNA or RNA and
cleave the chain;
 Ribozyme will have subunits responsible for the binding function and subunits
responsible for enzyme function

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 Have the following desirable traits:
 Specificity in targeting cleavage of RNA
 Small size amenable to formulation and dosing
 Multiple turnover i.e. One molecule binds and acts and then moves on to next
molecule and repeats its function

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Further processes or technologies are:
 Combinatorial chemistry: involves the use of the basic building blocks in
biochemistry, either the 20 amino acids or 4 nucleic acids, to build new molecules

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 All the different combinations of a set of number of building blocks can be created
 For example, the use of 10 different amino acids can result in over 3.5
million decapeptide compounds

Introduction….
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 High-throughput screening (HTS): is intended to obtain faster more high-quality
product leads from large volumes of genetic or peptide molecules

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 The process of HTS is dependent on:
 Improved analytical processes (better surface chemistry, capture agents, and
detection methods)
 Miniaturization of equipment, and
 Automation
 Over 100,000 samples can be tested in a day

Introduction….
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 Bioinformatics: can be described as the use of information technology (IT) for the
analysis of biological data sets
 It links the areas of bioscience and computer science
 Proteomics: is the study of protein structures and their properties
 X-ray crystallography: study of the crystal structure of compounds

Introduction….
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 Receptorology and protein kinases (PKs): PKs play principal roles in the
communication between and within all cells resulting in inactivation of all cells.

Introduction…
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 PKs exist and are very specific to certain cells and cell functions
 Aberrant or excessive cell activity can be mediated by the PKs, contributing to
diseases such as cancers or inflammatory conditions
 PK become targets for drug intervention to turn off or reduce their activity and
moderate a disease

Introduction….
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Virology:
 Viruses are most commonly used for gene delivery with the investigational
therapies because of their natural ability to carry genetic material, deliver it into
human cells (infect or transfect) and allow the genes to be turned on

Introduction….
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 Cell and tissue therapies: technique to treat disease is based on obtaining healthy cells
from a specific tissue, selecting out a specific subset of cells with certain desirable
properties, and enhancing the activity of these cells through ex vivo manipulation

Introduction….
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 These specifically selected, enhanced, and activated cells returned to patients whose
cells are not sufficiently functional, thereby ameliorating a disease.
E.g. Chondrocytes responsible for cartilage production are taken from a patient’s
knee that has serious damage and is repairing poorly
 These Chondrocyctes are manipulated ex-vivo and returned to the patient
to normalize cartilage production

Introduction…
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 Molecular engineering: where in the biological molecule is manipulated
regarding its amino acid or carbohydrate content to enhance biological function or
reduce toxicity

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These all technologies help to:
 Elucidate new biologic mechanisms of disease
 Identify naturally occurring substances or processes responsible for a biologic effect
 Innovate new products that enhance natural processes against disease

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 Create duplicates of the natural substances that often are found only in minute
amounts in the body
 Block the function of dysfunctional proteins or nucleic acids
 Reduce action of natural processes gone in a wrong way (less important) as in
inflammation in arthritis and
 Permit mass production of rare products for commercialization

Historical Development of Biotechnology
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o The origins of biotechnology go back 4000–8000 years to the Sumerian, Egyptian,
and Chinese cultures
o Fermentation is an age-old, basic biologic process whereby a living organism, a
yeast, will react with carbohydrate materials, such as wheat, in a vessel to produce
alcohol (Sumerian beer).

Historical Development of Biotechnology…
 In 1665 Robert Hooke invented the compound light microscope; first to observe
cells in cork.
 In 1675 Antony van Leeuwenhoek discovers bacteria using a simple
microscope.
o In 1800s: Proteins were discovered to exist
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o In the mid-1800s: Genetics began with the discoveries of Charles Darwin and
Gregor Mendel; the principles were used in breeding animals and plants to enhance
desirable traits.
o In 1870’s Louis Pasteur disproved the notion of spontaneous generation, describing
the role of bacteria in spoilage and the scientific basis for fermentation and Created
the rabies vaccine.

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o In 1940s: it was proved that DNA being responsible for carrying genetic
information.
o 1950s : the Modern era of biotechnology is thought to have started with the
discovery of the three-dimensional (3D) construct of the DNA double helix (by
James Watson & Francis crick);
 The matched pairs of four nucleic acids (adenine, guanine, cytosine, and
thymidine) in a specific sequence, parallel chains, and a 3D spatial

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o 1960s: several key discoveries in biology underpin biotechnology; namely,
 The genetic code is universal in nature among all living things for the 20
amino acids,
 64 specific nucleic acid triplet codes are responsible for interpretation of
genes into proteins, and
 Genetic material is transferable among different organisms.

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o In early 1970s there were the development of the two core technologies of
biotechnology, i.e., r-DNA and Mabs, which account for 80 of the 140
commercially available products in 2006.
o In 1982, marketing recombinant human insulin, the first commercial
pharmaceutical product derived from biotechnology.

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o At the end of 20th and in 21st century, biotechnology has become a major and
common platform for new products approved for clinical use;
 From 1998 to 2003, biotech research and development was responsible for 36% of
all new molecular entities and all drug approvals by the USFDA.

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o From its early era of product approvals numbering 60 in the 1980s and 1990s (18-year
period), this discipline has increased the discovery, development, production, and
commercialization of innovative biological products with about 80 more by 2006 (6-
year period).
o Now over 100 human disease conditions are treated with biotechnology products.

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 Cloning is creating a genetically identical copy of something
 Single cells and DNA are fairly easy to clone, but cloning entire organisms
becomes increasingly difficult
Applications of Biotechnology
Cloning

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DNA Fingerprinting
 DNA fingerprinting is identifying the pattern of certain
sequences in parts of DNA
 DNA is isolated, copied, cut into pieces, and separated based
on size using gel electrophoresis
 Probes are then used to find specific DNA sequences
 Can be used for maternity or paternity tests and in forensics
to determine identity and compare unknown DNA samples to
find out if a suspect is guilty or not

Applications in Pharmaceutical Industry
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 More than 150 approved biotech drugs or vaccines are on the market
 A recent survey by the pharmaceutical research and manufacturers of America
(Pharma) found 369 drugs in the pipeline meeting the criteria as biotechnological
drugs and medicines

Applications in Pharmaceutical Industry…
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 Biotechnology-produced pharmaceuticals currently account for 5% of the worldwide
pharmaceutical market and are expected to reach approximately 15% by the year 2050
 Not only drugs but also new medical diagnostic tests are produced and distributed by
pharmaceutical biotech industry

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 Xenotransplantation from transgenic animals
 Xenotransplantation is transplantation of genetically modified organs and cells
from other organisms like pigs
 Are promising sources of donor organs that can be used to overcome the lack of a
sufficient number of human organs

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 Tissue engineering, in relation to xenotransplantation
 Combines advances in cell biology and biomaterial science
 Tissues consist of scaffolding material (e.g. collagen, biodegradable polymers),
which eventually degrades after forming organs or cell implants
 Skin tissues and cartilages were the first tissues successfully engineered and tested in
vivo

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 Stem cells are considered today as a new avenue in therapy to cure most deadly and
debilitating diseases such as;
Parkinson
Alzheimer
Leukemia, and
Genetic disorders like adenosine deaminase (ADA) deficiency and cystic fibrosis
(CF)

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Reducing Costs in R&D
 Before biotechnology had been intensively introduced to industrial research,
developing costs of each drug had cost companies on average US$880 million and had
taken 15 years from start to market authorization;
 About 75% of these costs were spent on failures
Impact of Biotechnology on the Drug Discovery and
Development

Impact of Biotechnology…
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Increase in Productivity
 From trial-and-error approaches and complex biochemical in vitro assays,
biotechnology allows industrialized target detection and validation
 By the use of micro array technologies and bioinformatics, thousands of genes in
diseased and healthy tissues will be analyzed by a single DNA chip

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 By the use of bioinformatics, results from different assays can be analyzed and linked to
an integrated follow-up of information in databases
 In total, the potential savings per drug by intelligent information retrieval systems and
genetic analytics are estimated at about US$140 million per drug and less than one year
of time to market

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Accelerating the Drug Development Process
 Pharmaceutical biotechnology enable prediction of drug properties and
pharmacokinetic parameters to accelerate the industrial drug development process.
 Potential savings are in the order of US$20 million and 0.3 years per drug

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Maintaining high standards in quality assurance
 Biotechnological drugs have the same high standard in quality and safety as
conventional drugs
 Of high interest is the question of costs of quality control for recombinant drugs
 An increase of US$200 million and more than one year per drug

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 The main reason for this is explained by the extra time needed for unknown chemical
and physical properties of recombinant proteins and oligonucleotides
 Another time- and cost-consuming aspect is the importance of developing new drug
specific appropriate test assays for drug validation, standardization, activity
determination (e.g. biological units), toxicity, and bio analytical methods

Merob S.(Asst. Lecturer) , School of Pharmacy,
CHMS, HU
Introduction to Genetic Engineering
(rDNA technology)
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Outline
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
 Definition of genetic Engineering
 Gene Manipulation
 Basic steps in rDNA technology
 Application of rDNA technology in production of immunologicals and biologicals
 Therapeutic applications of rDNA products
 Polymerase Chain Reactions (PCRs)

rDNA technology or Genetic Engineering
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 rDNA technology or Genetic Engineering is the manufacture of human proteins
in non-human living systems
 It is also known as Gene Splicing or Gene Modification

Introduction…
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 All cell structures and functions begin with proteins, and the code for building
the proteins is found in DNA
 DNA is made up of building blocks known as nucleotides that are connected in a
very long ladder-like structure

Introduction…
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 There are four different nucleotides (containing the bases adenine, cytosine, guanine,
and thymidine) with a total of about 3 billion nucleotide units in the human genome,
tightly packed into chromosomes
 These include the genetic code for a large number of genes (~30,000)
 Each of these genes controls the synthesis of a protein

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 A single codon is made up of units of three adjacent nucleotides; each codon specifies
one amino acid
 The arrangement of codons in the DNA, following transcription into messenger RNA
(mRNA), determines the sequence of amino acids that will form a particular protein

Gene Manipulation
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 The ability to design new varieties of plants and animals has now become a reality
through genetic engineering
 Genetic engineering involves the manipulation of genes within a cell or organism to
bring about a change in the genetic makeup of an organism

Gene Manipulation
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 There are several methods of gene manipulation currently used, most of which include
the removal and insertion of genetic material into organisms
 One of the most important processes in gene manipulation is gene mapping
 Gene mapping involves the finding of the particular location on the strand of
DNA that contains the genes that control certain traits.

Gene Manipulation...
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o The process of mapping the genes on the strands of DNA involves the use of
molecules that act as probes;
 The probes attach themselves to certain parts of the DNA where the nucleotides
join each other;
 The probe looks for combinations of where the nitrogen bases join in certain
sequences;
 Once the probe locates the nucleotides, the sequences of Adenine(A), thymine(T),
Cytosine(C) and Guanine(G) can be listed in a map

Gene Manipulation...
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 The other process in gene manipulation is gene splicing
 Once the location of the DNA sequence has been located, by using restriction enzymes
the DNA can be separated at a particular location on the gene
 Once the pieces of DNA are removed, other DNA can be spliced in or recombined with
the remaining DNA or this DNA can be recombined with other DNA
 This results in recombinant DNA

Basic steps in rDNA technology
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 Five steps in r-DNA technology:
1. Protein identification
2. Gene isolation
3. Cloning of genetic material and expression of proteins
4. Manufacturing (scale-up processes) and
5. Quality assurance for both protein and process integrity

Basic steps in rDNA technology…
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1. Protein Identification
 Involves finding a protein responsible for some biological effect in the human
body that has therapeutic potential
 The protein needs to be isolated from its normal environmental condition, usually
a body fluid or cell
 The structure of the protein and its functions are determined.

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2. Gene Isolation
There are three mechanisms
 First, we often will know the protein’s full amino acid(aa) sequence, and the 64
nucleic acid triplets that code for the 20 amino acids
 Second, we may be able to find the human cell that produces our target protein;
 The viral enzyme, reverse transcriptase, is capable of creating the target
complementary DNA

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 Third, the human gene could be find out of the human genome using nucleic acid
probes
 Using the triplet codes for the aas in the peptide subunits of the protein, we build
specific nucleic acid combinations (probes) for each peptide
 we try to match the first nucleic acid probe to the DNA mixture, which does create a
subset of matching DNA pieces

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3. Cloning of Genetic Material and Expression of Proteins:
 Involves cloning of the gene and expression of the protein by the gene;
 Cloning is the reproduction of the target human gene in a nonhuman cell;
 Expression is the production of the target human protein by a nonhuman cell
containing the human gene.

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 These processes require a vector for the DNA (genes), so that the gene can be carried
into a host cell;
 Vector – molecule of DNA which is used to carry a human gene into a host cell
 A bacterial plasmid can be used as vector

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 The plasmid must be cut and open to
accept the human gene by bacterial
restrictionendonucleases.
 Sticky’ ends of the opened bacterial
plasmid and the human gene permit
recombination of the DNA, under the
influence of a ligase enzyme resulting in
an r-DNA molecule containing a human
gene inserted into a bacterial plasmid.

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 The r-DNA molecule is inserted into a host cell and the cell manufactures the human
protein from the human gene that it carries;
 The unique newly created cell and its offspring are the master working cell bank.
 The cell bank should be periodically tested for cell viability, genetic and phenotypic
stability

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 The host cells can be Prokaryotic (bacteria) or eukaryotic (yeast, mammalian
cell culture) systems
 The choice of expression system can influence the character, quantity and cost of
a final product

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 Mammalian systems such as Chinese hamster ovary (CHO) cell and baby hamster
kidney (BHK) cell systems;
 An ideal choice as these are capable of glycosylating the protein at the correct sites
 Cost of production of the products using these systems is high because of slow
growth and expensive nutrient media

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 The host cell needs to possess a set of demanding characteristics to be used feasibly
and cost-effectively in manufacturing processes:
 A short reproductive life cycle
 Long-term viability in an in vitro setting
 The ability to accept bacterial plasmids
 Substantial productive capacity (yield) for proteins

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4. Manufacturing (scale-up processes)
 This step comprises four phases:
 Inoculum, Fermentation, Purification, and Formulation.
A. Inoculum phase
 Involves the use of daughter cells from the new host cell
 The daughter cells are grown in specific media in serially larger flasks and assessed for
normal growth characteristics.

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 The growth medium (liquid and air) is a unique and specific mixture of minerals and
nutrients to enhance
 Cell viability (lifespan) in vitro and
 Functional ability of cells to produce proteins (maximize yield)

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B. The Fermentation or Cell Culture Phase
 Involves inoculating thousand of containers, or large fermenters, with cells from the
inoculum phase and adding the appropriate fortified growth media
 The host cells will proceed to produce proteins, either
 Intracellularly in storage vacuoles for most bacterial host cells or
Extracellularly into the media for mammalian cells

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C. Purification phase
o Purification of the protein varies between bacterial and mammalian systems;
o For bacteria, the cells are removed from the liquid in the fermenters by
centrifugation into a cell paste, which is centrifuged again to break out proteins
from the cells.
o The protein mixture is then run through an extraction process (often HPLC) to
separate the target protein from all other proteins;

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o For mammalian cells, the culture media contains the proteins, which were
secreted extracellularly by the mammalian cells, and is collected periodically;
o Extraction and purification are basically similar processes for the mammalian
process;
o A pure bulk protein is the result of purification.

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D. Formulation phase
o The final phase is formulation, where in a diluent is chosen for the protein,
incorporating the best mix of fluids, buffers, stabilizers, and minerals to
achieve optimal protein stability, maximal shelf life, and patient
acceptability.
o Sterile water, normal saline, and dextrose 5% in water are three common
diluents.

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5. Quality Assurance For Both Protein and Process Integrity
o Step 5 in r-DNA technology is quality control for the final product, components,
and processes throughout the manufacturing process;
 E.g. A degraded protein, viral or bacterial contamination, poor yield, an immune
reaction in patients, and even a different protein.
 QC ensures final product integrity through an extensive series of tests.

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o The tests involve four key areas:
 Genetic materials (plasmids and genes)
 Bulk protein products
 Final products, and
 The manufacturing process.

Applications of rDNA technology
in production of Immunologicals and Biologicals
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o rDNA enables rapid isolation of unique proteins and their mass production by
rapidly growing microorganisms.
o In addition, new organisms having specifically inserted and desired characteristics
could be engineered for medical, agricultural, and ecological uses.

Application of rDNA technology….
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o The recombinant DNA technology provides several advantages:
 The large-scale production of high amounts of protein with defined and
homogeneous quality to lower costs;
 The development of novel drugs, directed to new targets, which could not
be isolated in sufficient amounts and qualities from natural sources; and
 Creating protein variants having even improved properties over the natural
polypeptides.

Application of rDNA technology….
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o One of the first uses of rDNA technology for biopharmaceutical was the
manufacture of human insulin;
 Scientists isolated the DNA sequence that regulates the production of insulin.
 The DNA segment is cloned into the DNA of the E.coli bacteria.
 The bacteria carrying the DNA for insulin production reproduces and passes
the capability along to the next generation.

Therapeutic applications of rDNA products
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Hormones of therapeutic interest
o Human insulin produced using S. cerevisae or E. coli which is structurally
identical to insulin produced by the pancreas in the human body can be
administrated in treatment of Diabetes Mellitus.

Therapeutic applications of rDNA products
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o Insulin aspart: is structurally identical to insulin human, except for the
replacement of aspartic acid with proline at position 28 on the B-chain of the
molecules.
 Provides rapid absorption than regular human insulin.
o There also another different insulin used for therapeutic purposes such as Insulin
glargine, Insulin lispro, and Insulin glulisine.

Therapeutic applications of rDNA products…
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o Recombinant human choriogonadotropin (rhuCG): produced in CHO cells, is
used to induce ovulation in the treatment of anovalatory infertility or as an
adjunct to in vitro fertilization procedure.
o Recombinant follicles stimulating hormone (rFSH): produced from CHO cells,
is safe and effective in the treatment of fertility disorder.
o Recombinant luteinizing hormone: is likely to be recommended as a
supplement to rFSH for ovulation induction in hypogonadotropic women.

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o Somatotropin, a recombinant GH (growth hormone), produced in E. coli is
identical to natural GH, except that it contains an additional methionine on the N-
terminus of the molecule. It is used
 for the treatment(t/t) of short stature resulting from GH deficiency
 as an adjunct in the t/t of other disorders such as intrauterine growth
restriction

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Haemopoietic growth factors
o Recombinant human erythropoietin (rhuEPO) is used for treatment of anaemia;
 Epoetin alfa, Epoetin beta and Epoetin omega;
 Epoetin alfa and Epoetin beta are produced in CHO cells, whereas Epoetin
omega is produced in BHK cells;
 All the three varieties of rhuEPO had the similar sequence of 165 amino acids,
but differ in their carbohydrate content and site of glycosylation.

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o Filgrastim:- a recombinant human granulocyte colony stimulating factor (rhuG-
CSF) produced in E. coli principally affects the proliferation and differentiation
of neutrophils within the bone marrow;
o Pegfilgrastim:- additional polyethylene glycol moiety
 a long-acting form of filgrastim, that requires less frequent administration for
mgt of chemotherapy-induced neutropenia.
o Sargramostim:- (produced using S. cerevisiae) is used to treat and prevent
neutropenia in patients receiving myelosuppressive cancer therapy.

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Blood coagulation factors
o Recombinant human factor VIII:
 Provides a temporary replacement to prevent or control bleeding episodes or
to perform emergency of elective surgery in patients with hemophilia A.
o Recombinant human factor IX: is indicated for the control of bleeding events in
hemophilia B patients.
o Recombinant human factor VIIa: is a unique haemostatic agent with potential
for broad application in bleeding patients with congenital and acquired bleeding
abnormalities.

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Thrombolytic agents
o Alteplase: a recombinant tissue plasminogen activator (TPA), consists of 527 aas
and stimulates the fibrinolysis of blood clots by converting plasminogen to
plasmin.
 It is a treatment (t/t)choice in the mgt of AMI;
 It is also approved for t/t of acute ischaemic stroke and pulmonary
thromboembolism;

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Anticoagulants
o Lepirudin:- is used as a thrombin inhibitor for treatment of heparin-induced
thrombocytopenia.
o Disirudin:- a recombinant hirudin is used in the prevention and management of
thromboembolic disease.
 It is a thrombin inhibitor
 It is more effective than heparin in the prevention of deep vein thrombosis in
patients undergoing elective hip-replacement.

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Human Interferon
o Three recombinant human interferons (rhuIFN) alpha, beta and gamma
o rhuIFN alpha-2b: is approved for t/t of hairy cell leukaemia, AIDS-associated
Kaposi sarcoma, hepatitis B and C, malignant melanoma and renal cell
carcinoma;
o rhuIFN beta-1b: is the first line therapeutics in relapsing, remitting and
secondary progressive multiple sclerosis;
o rhuIFN gamma: It is indicated in reducing the frequency and severity of serious
infections associated with chronic granulomatous disease.

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Human interleukins
o Recombinant human interleukin (rhuIL)-2: Indicated for t/t of metastatic renal
cell carcinoma and melanoma.
o rhuIL-11: is a thrombopoietic growth factor that stimulates the production of
hematopoietic stem cells and megakaryocytic progenitor cells resulting in platelet
production.

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Therapeutic Enzymes
o Recombinant dorsase alfa (rhudeoxyribonuclease 1):
 an enzyme prepared from CHO cells, is developed specifically for cystic
fibrosis.
o Recombinant glucocerebrosidase:
 For t/t of hematological abnormalities, hepatosplenomegalia and quality of life
in a matter of few months.

Polymerase Chain Reaction (PCR)
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o PCR is a critical core process in biotechnology, which permits the expansion of
the amount of genetic material (DNA), starting from minute amounts.
o The process involves:
 First, denaturing DNA with high heat (90C), i.e., unraveling the DNA
double helix so that the genetic code (sequence) can be read and possibly
duplicated.

Polymerase chain reaction (PCR)…
 Second, a leader sequence for DNA is used to bind to the target DNA and
initiate reading of the genetic code at a specific point.
o Both helices and strands of DNA can be read, that is, duplication of the target
DNA sequence.
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Polymerase chain reaction (PCR)…
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 Third, the heat-stable enzyme from the Thermos aquatic bacteria, DNA
polymerase, catalyses the reading of the genetic code with extension of the
replicated DNA sequence.
o By sequential repetition of these three steps, the genetic material is magnified; for
example, 20 replications yield a million-fold increase in the DNA material.

Immunological and biological pro.pptx

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