BIOTECHNOLOGY

Principles And Processes

 It deals with techniques of using live organisms or enzymes from organisms to produce products and
processes useful to humans.
 The Europian federation of biotechnology (EFB) has given definition as –
“Biotechnology is the integration of natural science and organisms, cells, parts their of and molecular
analogous for products and services.
 Gene manipulation is a fast emerging science it started with the development of recombinant DNA
molecules and named as recombinant DNA technology or genetic engineering.
 The technology mostly involves cutting and pasting of desired DNA fragments.
 It is based on 2 important discoveries in bacteria –
(i) Presence of plasmids in bacteria which can undergo replication along with and independent of
chromosomal DNA.
(ii) Restriction Endonucleases Enzyme or molecular scissors – Discovered by Arber, Nathan and Smith
in 1970, Nobel prize 1978.
 Is can break DNA at specific sites.
 Paul Bergh  Is often considered as “father of genetic engineering” (Nobel prize – 1980)
 As bergh was able to introduce a gene of SV-40 into a bacterium with the help of lamda page (-
phage).
 Stanley cohen and Herbert Bayer  The science of recombinant technology took birth when cohen
and Bayer were able to introduce a piece of antibiotic resistance gene containing foreign DNA into
plasmid of salmonella typhimurium.
 Old biotechnology based on natural capabilities of microorganisms. eg – Formation of citric acid and
penicillin by pencilium notatum.
 New biotechnology based on rDNA technology.
eq. – Human gene producing Insulin has been transferred and expressed in E.coli bacteria.

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 Principles of Biotechnology
 Among many, the two core techniques that enabled birth of modern biotechnology are –
(i) Genetic engineering  Techniques to alter the chemistry of genetic material (DNA and RNA) to
introduce these into host organisms and these change the phenotype of the host organism.
(ii) Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes
to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture
of biotechnological products like antibiotics, vaccines, enzymes etc.
 Let us now understand the conceptual development of the principles of genetic
engineering.
 Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
 Traditional hybridisation procedures used in plant and animal breeding very often lead to inclusion
and multiplication of undesirable gene along with the desired genes.
 The techniques of genetic engineering which include creation of recombinant DNA, use of gene
cloning and gene transfer, over come this limitation and allows us to isolate and introduce only one or
a set of desirable gene without introducing undesirable genes into the target organism.
 In a chromosome there is a specific DNA sequence c/d the origin of replication, which is responsible
for initiating replication.
 Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a
chromosomes which has a specific sequence called as “origin of replication”
 This can also be called as cloning or making multiple copies of any template DNA.
 The construction of the 1st recombinant DNA emerged from the possibility of linking a gene encoding
antibiotic resistance with a native plasmid (autonomously replicating circular extra chromosomal
DNA) of salmonella typhimurium.
 Stanley cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene
by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic
resistance.
 The cutting of DNA at specific locations become possible with the discovery of the so called
“molecular scissors” restriction enzymes :
 The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vector to
transfer the piece of DNA attached to it.
 The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme
DNA ligase which acts on cut DNA molecules and joins their ends and forms a new recombinant DNA

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  When this DNA is transferred into E.coli, a bacterium closely related to salmonella, it could replicate
using the new host’s DNA polymerase enzyme and make multiple copies.
 The ability to multiply copies of antibiotic resistance gene in E.coli was c/d cloning of antibiotic
resistance gene in E.coli.

 3 Basic steps in genetically modifying an organism are –

(i) Identification of DNA with desirable genes.
(ii) Introduction of the indentified DNA into the host.
(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

 Tools of Recombinant DNA Technology or Genetic Engineering (rDNA

technology)
 Genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools
i.e. – restriction enzymes, polymerase enzyme, ligases, vectors and the host organism as
(A) Enzymes (B) Cloning Vectors (Vehicle DNA) (C) Passenger DNA and Host Cells
(1) Lysing enzymes (1) Plasmid vectors (1) Complementary DNA (cDNA)
(2) Restriction enzymes (cleaving (2) Bacteriophage vectors (2) Synthetic DNA (sDNA)
enzymes) (3) Cosmid vectors (3) Random DNA.
(3) DNA ligases (Joining/sealing (4) Bacterial artificial
enzymes) chromosome vectors
(4) Alkaline phosphate (5) Yeast artificial chromosome
(5) Synthesizing enzyme vector
(6) Phagemid vector
(7) Animal or plant viral vectors
(8) Transposoon vector
(9) Shuttle vectors

(A) Enzymes
(1) Lysing enzymes – These enzymes are used for opening the cells to get DNA for genetic experiments.
ex – Lysozyme enzyme help to dissolve bacterial cell wall.
Note : - These Restriction Enzyme present in bacteria as a part of their defence mechanism c/d
Restriction modification system b/c they cut the viral DNA without affecting own genome this is an
adaptation against bacteriophages.
 The R.M. system has ability in the form of methylation of DNA and protect own DNA by restriction
enzyme.

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 (2) Restriction Enzyme
 In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in E.coli
were isolated.
One of these added methyl groups to DNA, while the other cut DNA and the
one who cut the DNA is c/d restriction endonuclease.
 The first restriction endonuclease is Hind II, whose functioning depended on a specific DNA
nucleotide sequence was isolated and characterised five years later.
 It was found that Hind II always cut DNA molecules at a particular point by recognizing a specific
sequence of six base pairs and these base pairs sequence is known as the recognition sequence for
Hind II.
 The convention for naming these enzymes is the first letter of the name comes from the genes and the
second two letters come from the species of the prokaryotic cell from which they were isolated
Eg - Eco RI comes from Escheriacoli RY 13.
 In Eco RI, the letter ‘R’ is derived from the name of strain.
 Restriction enzymes belong to a larger class of enzymes called nucleases.
 These are of two kinds : -
(a) exonucleases (b) Endonucleases
 (a) Exonucleases remove nucleotides from the ends of the DNA whereas,
 (b) Endonucleases makes cut at (inside) specific positions within the DNA.
 Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
 3 Types of restrictions enzymes are type 1,2,3 in which type II restriction enzyme are simple and
required Mg+2 ions for restriction and only Type II R.E. are used in rDNA technology and cut DNA
sequence in 4-8 Nucleotide.
 What is Palindromes
 These are groups of letters that form the same words when read both forward and backward.
Eg - MALAYALAM
 The palindrome in DNA is a sequence of base pairs that reads same on the two strands when
orientation of reading is kept the same.(i.e. when read in a 5’  3’ direction on both DNA strand or 3’
 5’ direction in the DNA duplex and cleaves its strand)
 Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but
b/w the same two bases on the opposite strands.
 This leaves single stranded portions at the ends. There are overhanging stretches c/d sticky ends on
each strands.

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  These are named so because they form hydrogen bonds with their complementary cut counterparts.
This stickness of the ends facilitates the action of the enzyme DNA ligase.
 When cut by the same restriction enzyme the resultant DNA fragments have the same kind of ‘sticky-
ends’ and these can be joined together (end-to-end) using DNA ligase.
Figure :

 Separation and Isolation of DNA fragments

 The cutting of DNA by restriction endonucleases results in the fragments of DNA.
 These fragments can be separated by a technique called as gel electrophoresis.
 Gel Electrophoresis
 Since the DNA fragments are negatively charged molecules they can be separated by forcing them to
move towards the anode under an electric field through a medium/matrix.
 Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea
weeds.
 The DNA fragments separate (resolve) according to their size through sieving effect providing by the
agarose gel.
 Hence, the smaller the fragment size, the farther it moves.
 The separated DNA fragments can be visualised only after staining the DNA with a compound called
as ethidium bromide stained gel exposed to UV light.
 The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This
step is known as elution.
 The foundations of rDNA were laid by the discovery of restriction enzymes.
 These enzymes are present in many bacteria where they function as a part of their defence
mechanism c/d Restriction Modification System.
 These R.M. System, firstly useful to bacteria b/c the enzyme bring about fragmentation of viral DNA
without affecting the own bacterial genome. This is an adaptation against bacteriophages.
 Restriction enzymes (Eco-R-I) was discovered by Arber, smith and Nathans (1978 Nobel prize).
 These R.M. system, secondly also have capability of modification in the form of methylation by
methylation the bacterial DNA modifies and therefore protect it’s own chromosomal DNA from
cleavage by these restriction enzymes.

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  As result the DNA fragments produced by cleavage with these enzymes have short single stranded
overhang at each end, these kinds of ends are c/d sticky or cohesive ends b/c base pairing b/w them
can stick the DNA molecule back together again.
 These sticky ends facilitate the action of the enzyme DNA ligase.

 Examples of Restriction Enzyme

 Eco RI – Cleave between G and A
From E.coli, containing drug resistant plasmid RI
 Hae III – Cleave b/w G and C
From Haemophilus aegypticus
 Hind III – Cleave b/w A and A
From Haemophilus influenzae
(3) DNA Ligases (Joining/sealing Enzymes)
 These enzymes, c/d “genetic gum” as these enzymes help in joining the DNA fragments.
 DNA ligase from E.coli is used to join DNA fragments.
 These enzymes help in sealing gaps in DNA fragments.
 The enzyme most often use in rDNA technology is T4 DNA ligase.
(4) Alkaline Phosphatases
 These enzymes cut off phosphate group from the 5’ end of linearised circular DNA and prevent its
recircularisation.
(5) Synthesizing Enzymes
 These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA
template. They are of 2 types –
(a) Reverse transcriptase  It help in the synthesis of complementary DNA strands on RNA templates.
(b) DNA polymerases  It help in synthesis of complementary DNA strands on DNA templates.
 (B) Vehicle DNA or Vector DNA (Cloning Vector)
 The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell.
 Vectors may be plasmids, a bacteriophage, cosmids, yeast artificial chromosomes (YAC), bacterial
artificial chromosome (BACs), transposons, virus etc.
 As we know that plasmids and bacteriophages have the ability to replicate within bacterial cells
independent of the control of chromosomal DNA.

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  Examples of Cloning Vector
(1) Plasmid Vectors
 They are extra chromosomal DNA segments found in bacteria which can replicate independently.
 Plasmids are usually not essential for normal cell growth and division, they often confer some traits
for example, resistance to certain antibiotics or toxins.
 These naturally occurring plasmids can be taken out of bacteria and made to combine with desired
DNA segments so a plasmid carrying DNA of another organism integrated with it is known as
recombinant plasmid or hybrid plasmid or chimeric plasmid.
 Most widely used plasmid vector is pBR322.
(2) Viruses
 The DNA of certain viruses is also suitable for use as a vehicle DNA.
 Bacteriophage (bacterial virus) has been used to transfer gene for 𝛽 galactosidase from E.coli to
human cells. Lambda phage ( phage) has been used for transferring lac genes of E.coli into haploid
callus of tomato.
 Two phages are Lambda () phage and M13 phage.
(3) Cosmid  Cosmids have been constructed by combining certain features of plasmid and the cos sites
of phage lambda
 (C) Passenger DNA
 It is the DNA which is transferred from are organism into another by combining it with the vehicle
DNA are –
(i) Complementary DNA (cDNA)  It is synthesised on mRNA template with the help of reverse
transcriptase and necessary nucleotides.
(ii) Synthetic DNA (sDNA)  It is synthesized with the help of DNA polymerase on DNA template.
 Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the
gene coding for yeast alanine t-RNA (77 base pairs) which did not function and in 1979 was able
to synthesized functional tyrosin t-RNA gene of E.coli with 207 nucleotide pairs.
(iii) Random DNA  Small fragments are formed by breaking a chromosome of an organism in
presence of restriction endonuclease.
 Characteristics of a cloning vector
 The following are the features that are required to facilitate cloning into vector.
 First cloning vector is pSC 101 Cloning Vector of Ecoli.
(First discovered by Cohen And boyer in 1972)

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 (i) Origin of Replication (Ori)
 This is a sequence from where replication starts and any
piece of DNA when linked to this sequence can be made to
replicate within the host cells.
 This new sequence is also responsible for controlling copy
number of linked DNA.
 So, if one wants to recover many copies of the target DNA it
should be cloned in a vector whose origin support high copy
number.
(ii) Selectable Marker (antibiotic resistant gene like amp𝑅 , tet 𝑅 etc)
 The gene encoding resistance to antibiotics such as ampicillin, chloroamphenicol, tetracycline or
kenamycin etc are considered useful selectable markers for E.coli.
(iii) Cloning sites (Recognition sites for restriction endonuclease)
eg.  Eco RI, Cla I, Hind III, Bam HI, PVu II (rop) etc.
 The vector must also have one unique restriction endonuclease recognition site to enable foreign DNA
to be inserted into the vector during the generation of a recombinant DNA molecule.
 The ligation of foreign DNA is carried out at restriction site present in one of the two antibiotic
resistance genes.
For example
 A foreign DNA can be linked at the Bam HI site of tetracycline resistance gene in the vector pBR 322.
 The recombinant plasmids will loss tetracycline resistance due to insertion of foreign DNA, now, it can
be selected out from non-recombinant ones by plating the transformants on ampicillin containing
medium.
 The transformants growing on ampicillin containing medium are then transferred on a medium
containing tetracycline.
 The recombinants will grow an ampicillin containing medium but not on that containing tetracycline.
But, non-recombinants will grow on the medium containing both the antibiotics.
 In this case one, antibiotic resistance gene helps in selecting the tranformants.
 Colour reaction
 Due to inactivation of antibiotics, selection of recombinants becomes troublesome process.
 Thus, alternative selectable marker is developed to differentiate recombinants and non-
recombinants on the basis of their ability to produce colour in the presence of chromogenic
substance.

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 Now a recombinants DNA is inserted within the coding sequence of an enzyme, which is referred to as
insertional inactivation.
 It the plasmid in the bacterium does not have an insert, the presence of a chromogenic substrate give
blue coloured colonies and the colonies do not produce any colour, these are identified as
recombinant colonies.
[As insertional inactivation of -galactosidase in recombinant bacteria appears white]

 Vectors for cloning Genes in Plants and Animals

 Agrobacterium tumifaciens, a pathogen (disease causing agent) of several dicot plants deliver a piece
of DNA known as ‘T-DNA’ to transform normal plant cells into a tumour.
 Similarly, retroviruses is animals have the ability to transform normal cells into cancerous cells.
 The tumor inducting (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning
factor which is no more pathogenic to the plants but still able to use the mechanisms to deliver genes
& are now used to deliver desirable genes into animal cells.
 So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a
bacterial, plant or animal host where it undergoes multiplication.

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 Process of Recombinant DNA Technology (rDNA)
It involves following steps are –
(1) Isolation of the genetic material (DNA)
 The DNAs which are to be used a passenger DNA and the vehicle DNA are extracted out of the cells by
lysing the cells with the suitable enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase
(fungus).
 The extracted DNAs are isolated from other cell content by ultra centrifugation and purified.
 The separated DNA fragments can be visualized only after staining the DNA with ethidium bromide
followed by UV radiation.
(2) Amplification of Gene of Interest using PCR
 Polymerase chain reaction technology (PCR) was invented by kary mullis (1983) [Noble prize in 1993
for chemistry)
 PCR is a method for amplifying a specific region of DNA molecule without the requirement for time
consuming cloning procedures.
 PCR reaction takes place in Eppendrof tube.
 Using PCR-technique very low content of DNA available from samples of blood or semen or any other
tissue can be amplified many times and analysed.
 Taq-polymerase (from thermus aquaticus bacterium) enzyme is used in PCR which is special type of
DNA polymerase enzyme, which is resistant to high temperature.
 Some other examples of polymerase for PCR are –
P flu polymerase  Isolated from pyrococcus furiosus bacterium
Vent polymerase  Isolated from Thermococcus litoralis bacterium
 Basic requirement of PCR technique
(i) DNA sample
(ii) Primer DNA  Small segement of DNA (10-8 nucleotide)
(iii) A, T, C, G base pairs
(iv) Thermostable enzyme like Taq polymerase (>90C)

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  3 main step in PCR

(i) Denaturation (94C) (ii) Annealing (iii) Extension

 In this step a double  In this step two primer  Here taq polymerase
stranded DNA molecule is DNA are attached at 3’end enzyme synthesize DNA
placed at 94C of single stranded DNA. strain over template PCR is
 So double stranded DNA automatic process b/c taq-
becomes single stranded polymerase enzyme is heat
and each single stranded resistance.
DNA functions as a
template.

(3) Cutting of DNA at specific Location

 Both the passenger and vehicle DNAs are then, cleaved by the same restriction endonuclease, so that
they have complementary sticky ends.
(4) Preparation and Insertion of Recombinant DNA into the Host cell/organism –
 After producing complementary sticky ends, both the passenger and vehicle DNAs are joined with
ligase enzyme.
 The ligase forms new sugar-phosphate bonds to join two DNAs.
 The recombinant DNA is now inserted into a host cell such as E.coli. The
bacteria to be used as hosts should be without plasmids.
(5) The host cells are treated with calcium chloride or lysozyme. It creates
transient (temporary pores) in their wall and makes the latter permeable to
recombinant DNA.
(6) The recombinant DNA is added to culture in which such bacteria are
growing. The recombinant DNA is taken up by the bacteria. It replicates
when the host bacteria divide and give rise to multiple copies of recombinant
DNA.

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 Gene Transfer
 Transfer of desired genes from one organism into another is an important aspect of genetic
engineering.
 Gene transfer involves essentially 3 stages –
(i) Isolating a useful DNA segment from the donor organism.
(ii) Inserting it to a suitable vector.
(iii) Splicing of this altered DNAs into a recipient organism or host cell.
 Gene transfer is achieved by 2 kinds of transfer methods

(1) Indirect method through vectors (2) Direct method or vector less transfer
or carriers method –
 (i) Electroporation
 (ii) chemical mediated genetic
transformation
 (iii) Microinjection
 (iv) Particle gun/Biolistic method
 (v) Liposome mediated gene transfer
(1) Indirect method or Gene transfer through vectors
 Plasmids and viruses are commonly used vectors for the transfer of desired genes.
 These genes are first made to join suitable plasmid or virus which are then introduced into target
cells.
 A plant pathogenic bacterium – Agrobacterium tumefaciens produce crown galls or plant tumours in
almost all dicotyledonous plants.
 This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and
cotton but not cereals.
 Tumour formation is induced by its plasmid which is therefore c/d Ti plasmid (tumour inducing
plasmid).
 Agrobacterium tumefaciens naturally transfer some part of Ti-plasmid into host plant DNA without
any human effort so it is c/d natural genetic engineer of plant.
 In the transformation process two essential component in Ti-plasmid –
(a) T-DNA – (Transferred DNA)
(b) Vir-region – (Virulence region)

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  Inside the host plant cell T-DNA is separate from Ti-plasmid and integrated into host plant DNA that
cause crown gall tumour.
 Vir-region contains genes which are essential for T-DNA transfer –
 When we use Ti-plasmid as a vector first we remove the tumour causing gene from T-DNA region.
 Then desired gene inserted in place of it, now this plasmid is c/d disarmed plasmid.
(2) Vectorless gene Transfer
(i) Electroporation  It creates transients (temporary pores) in the plasma membrane to facilitate entry
of foreign DNA.
(ii) Chemical mediated genetic transformation  It involves certain chemical such as polyethylene glycol
(PEG), that help in the uptake of foreign DNA into host cells.
(iii) Microinjection  It is the introduction of foreign genes into plant or animal cells using micropipettes
or glass needles.
(iv) Particle gun/Biolistic method  It is a technique in which tungsten or gold particles coated with
foreign DNA are bombarded into target cells to facilitate entry of the foreign genes.
(v) Liposome mediated gene transfer  In this method DNA encloses within lipid begs. These lipid begs
fused with protoplast.
 Gene transfer in Animals
 In animals, the genes are transferred mostly through direct methods such as electroporation,
microinjection or particle gun etc.
 The desired foreign genes can be introduced into fertilizes eggs or embryo through microinjection.
 These transgenic eggs or embryo can be implanted into the uterus of another female, c/d surrogate
mother for their development.
 Now, since most of the human genetic disorders such as Alzheimer, cancer, haemophilia, thalassemia
and cystic fibrosis can now be cured through insertion of the correct genes into these patients.
 Obtaining the Foreign Gene Product
 After formation of recombinant DNA, these rDNA is transferred into a bacterial, plant or animal cell,
so the foreign DNA is multiplied.
 Most of the recombinant technologies are aimed to produce a desirable protein, so their is a need for
expression recombinant DNA.
 After the cloning of the gene of interest one has to maintain the optimum conditions to induce the
expression of the target proteins and consider producing it on a large scale.
 If any protein encoding gene is expressed in a heterologous host it is known as “recombinant protein”.

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 The cells can also be multiplied in a continuous culture system where in the used medium is drained
out from one side, while fresh medium is added from the other to maintain the cells in their
physiologically most active log/exponential phase. This type of culturing method produces a larger
biomass leading to higher yield of desired protein.
 Bioreacters (Fermenters)
 Bioreacters are considered as vessels in which raw materials are biologically converted into specific
products or enzymes by microbes, plants and animals cells.
 Small volume cultures cannot yield appreciable quantities of products.
 To produce in large quantities, the development of bioreactors where large volumes (100-1000 litres)
of culture can be processed, was required.
 A bioreactor provides optimal conditions for achieving the desired product by providing optimum
growth conditions (temp, pH, O2 , vitamin etc.)
 Industrial utilization of biotechnology involve 3 steps
(i) Laboratory scale process.
(ii) Pilot plant scale.
(iii) Manufacturing unit.
 The development from laboratory scale to manufacturing units is “scaling up to industrial
production”.
(i) Laboratory Scale Process
 In this process for the production of desirable product, proper micro organism searched and then
suitable strain is selected and multiplied.
 Proper medium also find out on which selected strain, produce best and more amount of product.
 Ultimately, the laboratory scale process finalized and transfer at pilot plant scale.
(ii) Pilot Plant Scale
 It is the intermediate stage where working of laboratory scale process is tested. At this stage cost and
quality of product thoroughly checked.
 Glass apparatus is replaced by stainless steel equipment/containers is c/d bio-reacters.
 Thus bioreacters can be thought of as vessels in which raw materials are biologically converted into
specific products, individual enzymes etc using microbial plant, animal or human cells.
 A bioreacters provides the optimal conditions for achieving the desired product by providing
optimum growth conditions (temperatures, oxygen, pH, substrate, salts, vitamins).
 The most commonly used bioreacters are of stirring type.

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(iii) Manufacturing Unit –
 During the designing of bioreacters for the process often very large size so that it accommodate huge
amount of medium.
 Downstream Processing
 After completion of the biosynthetic stage, the product has to be subjected through a series of
processes before it is ready for marketing as a finished product.
 The processes include separation and purification, which are collectively referred to as downstream
processing.
 The product has to be formulated with suitable preservative.
 Such formation has to undergo clinical trials as in case of drugs. Strict quality control testing for each
product is also required.

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