Recombinant DNA Recombinant DNA refers to a molecule that has been artificially created by combining genetic material from different sources. This process involves the insertion of DNA fragments from one organism, often a gene or genes of interest, into the DNA of another organism. Recombinant DNA technology allows scientists to manipulate and modify genetic material, enabling the creation of new combinations of genes that do not occur naturally

1. Perception and
Ultrafiltration of rDNA
derived drugs

2. Recombinant DNA
Recombinant DNA refers to a molecule that has been artificially
created by combining genetic material from different sources. This
process involves the insertion of DNA fragments from one
organism, often a gene or genes of interest, into the DNA of
another organism. Recombinant DNA technology allows scientists
to manipulate and modify genetic material, enabling the creation of
new combinations of genes that do not occur naturally.

3. Types of recombinant DNA
✓Genomic DNA
✓cDNA( complementary DNA)
 Synthetic DNA
✓Plasmid

4. What are recombinant DNA drugs?
• Recombinant DNA-derived drugs, also known as
biopharmaceuticals or biologics, This technology involves
the insertion of genes into host organisms, such as
bacteria, yeast, or mammalian cells, to produce
therapeutic proteins or other biologically active
molecules. The host organisms then serve as "factories"
for the production of these drugs.

5. Some examples of recombinant DNA-
derived drugs include:
• Insulin
•
• : Growth hormone:
• Erythropoietin:
• Monoclonal antibodies

6. Methods used for the purification of
recombinant DNA derived drugs
• These types of recombinant DNA are used in various
applications, such as gene cloning, gene expression,
gene editing, and the production of genetically
modified organisms. Each type of recombinant DNA
serves as a valuable tool for genetic engineering and
biotechnology research.

7. 1.Chromatography
• Various chromatography techniques such as affinity
chromatography, ion exchange chromatography, size
exclusion chromatography, and hydrophobic
interaction chromatography are commonly used for
the purification of recombinant proteins and other
biomolecules.

8. • How size exclusion chromatography used
for purification of of rDNA derived drugs?
Size exclusion chromatography (SEC), also known as gel
filtration chromatography, is a commonly used method
for the purification of rDNA-derived drugs, especially
proteins and peptides. This technique separates
molecules based on their size and molecular weight,
making it particularly useful for removing aggregates
and impurities from the target biomolecule.

9. Here's how size exclusion chromatography is used for
the purification of rDNA-derived drugs:
• Principle: SEC separates molecules according to
their hydrodynamic volume rather than their
molecular weight. Larger molecules elute first
because they are not able to enter the pores of the
stationary phase, while smaller molecules can enter
the pores and take longer to elute.

10. Purification of Target
Biomolecule:
• Sample Application: The crude mixture containing
the rDNA-derived drug, along with impurities and
aggregates, is applied to the top of the SEC column.
• Elution: As the sample passes through the column,
larger molecules are excluded and elute first, while
smaller molecules, including the target
biomolecule, pass through the pores and elute
later.

11. • Removal of Aggregates:
• Aggregates of the target biomolecule, which can form during expression and
purification processes, are often larger in size compared to the monomeric
form. SEC effectively separates these aggregates from the monomeric form,
leading to purification of the target protein or peptide.
• Fraction Collection:
• The fractions collected at different elution volumes contain molecules of
different sizes. The target biomolecule is typically found in a specific fraction
corresponding to its monomeric form, while larger impurities and aggregates
are present in earlier fractions.

12. • Purity Assessment:
• The fractions containing the purified rDNA-derived
drug are analyzed for purity and concentration
using analytical techniques such as SDS-PAGE,
HPLC, or mass spectrometry.
• Overall, size exclusion chromatography is an
effective method for purifying rDNA-derived drugs
by separating them from impurities and aggregates
based on their size, leading to a highly purified and
homogeneous product suitable for pharmaceutical
use.

13. Size exclusion chromatography (SEC)
• Size exclusion chromatography (SEC) is commonly used for the
purification of various types of rDNA-derived drugs, especially
those that are proteins or peptides. This includes a wide range of
biopharmaceutical products such as:
• Therapeutic Proteins:
• Hormones and Growth Factors:
• Vaccines and Immunotherapeutics:
• Fusion Proteins and Antibody Fragments:
• Enzymes and Biocatalysts: .
• Peptide Drugs:

14. How ion exchange chromatography used for
purification of of rDNA derived drugs?
• Ion exchange chromatography is a widely used
technique for the purification of rDNA-derived
drugs, particularly those that are proteins or
peptides. This method relies on the reversible
interaction between charged molecules (such as
proteins or peptides) and charged groups attached
to a chromatography matrix.
•

15. • Here's how ion exchange chromatography is used
for the purification of rDNA-derived drugs:
•
• Principle: In ion exchange chromatography, the
stationary phase (the chromatography matrix)
contains charged groups that can attract and bind
molecules with opposite charges. Proteins and
peptides typically carry a net charge at a specific
pH, which allows them to interact with the charged
groups on the matrix.

16. • Sample Loading: The rDNA-derived drug sample is applied to the ion exchange
column under conditions that promote binding based on charge interactions.
•
• Washing: After loading the sample, the column is washed to remove unbound
impurities and contaminannot
•
• Elution: The bound protein or peptide is then eluted from the column by
changing the ionic strength or pH of the buffer. This disrupts the charge
interactions between the target molecule and the matrix, allowing the purified
rDNA-derived drug to be collected in a separate fraction
• .
• Analysis and Characterization: The eluted fractions containing the purified
rDNA-derived drug are typically analyzed to confirm purity, identity, and
functionality.

17. • Ion exchange chromatography is commonly used
for the purification of various types of rDNA-derived
drugs, particularly those that are proteins or
peptides. Some examples of rDNA-derived drugs for
which ion exchange chromatography is used include:
• Recombinant therapeutic proteins:
• Enzyme replacement therapies:
• Therapeutic peptides:
• Vaccines:

18. How affinity chromatography used for
purification of of rDNA derived drugs?
• Affinity chromatography is a powerful technique
used for the purification of rDNA-derived drugs,
particularly those that are proteins, peptides, or
other biomolecules. The key principle behind
affinity chromatography is the specific and selective
binding of a target molecule to an immobilized
ligand, allowing for its isolation and purification
from complex mixtures. Here's how affinity
chromatography is used for the purification of
rDNA-derived drugs:
•

19. •
•
• Affinity Ligand Selection: A specific ligand that has high affinity and selectivity
for the target molecule is chosen. This ligand can be an antibody, receptor,
enzymte, or any other molecule that binds specifically to the target rDNA-deriv.
•
• Immobilization of Ligand: The chosen ligand is then covalently attached to a
solid support matrix, such as agarose beads or a chromatography column. This
creates an affinity column with the ligand immobilized on the matrix.
•
• Sample Loading: The mixture containing the rDNA-derived drug is applied to the
affinity column. The target molecule will selectively bind to the immobilized
ligand due to specific interactions, while non-target molecules will pass through
or bind weakly.
•

20. •
•
• Washing: Unbound impurities and non-specifically bound molecules are
removed by washing the column with a buffer solution. This step helps to
further purify the target molecule and remove contaminants.
•
• Elution: The bound rDNA-derived drug is then eluted from the affinity
column using a competitive ligand or by changing the buffer conditions to
disrupt the specific binding interactions. This results in the release of the
purified target molecule from the cmatrix
•
• Collection and Analysis: The eluted fraction containing the purified rDNA-
derived drug is collected and analyzed for purity, yield, and bioactivity.
•

21. • Affinity chromatography is commonly used for the
purification of a wide range of rDNA-derived drugs,
particularly those that are proteins, peptides, or
other biomolecules. Some examples of rDNA-
derived drugs for which affinity chromatography is
used include:
• Therapeutic Proteins:
• Enzymes
• Antibody-based Drugs:
• Fusion Proteins:

22. How hydrophobic interaction chromatography used
for purification of of rDNA derived drugs?
• Hydrophobic interaction chromatography (HIC) is commonly
used for the purification of recom DNA (rDNA) derived
drugs. This technique takes advantage of the hydrophobic
nature of certain proteins and peptides, allowing them to
interact with a hydrophobic stationary phase while in a high
salt environment.
•
• In the context of rDNA derived drugs, HIC can be used to
separate the target protein or peptide from other impurities
present in the cell lysate or culture supernatant. The process
typically involves the following steps:

23. • Cell Lysis
• Clarification
• Binding
• Washing
• Elution
• In general, HIC can be applied to the purification of
hydrophobic proteins and peptides derived from
recombinant DNA technology, allowing for the
separation and purification of these molecules from
complex mixtures and impurities.
•

24. . Ultrafiltration and diafiltration
• Ultrafiltration is used to concentrate and purify
biomolecules based on their size, while diafiltration is
used for buffer exchange and removal of small The
process typically involves :-
• Harvesting the rDNA derived drug
• Cell lysis
• Clarification
• Ultrafiltration
• Washing and concentration
• Final purification

25. Precipitation:
Precipitation can be used as a purification technique for
rDNA-derived drugs by exploiting the differences in
solubility and precipitation behavior between the target
protein or nucleic acid and other impurities.
Electrophoresis:
Techniques such as gel electrophoresis, including SDS-
PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis), can be used for the separation and
purification of proteins based on their molecular weight.

26. • Membrane filtration:
• Various membrane filtration techniques, including
microfiltration, ultrafiltration, and nanofiltration,
can be employed for the purification of
recombinant DNA-
• Centrifugation:
• Differential centrifugation and density gradient
centrifugation are commonly used to separate and
purify biomolecules based on their size, shape, and
density.

27. • 7. Immunoaffinity purification:
• This method utilizes antibodies or other specific
ligands to capture and purify the target
biomolecule based on specific binding interactions.
• 8. Aqueous two-phase extraction:
• This technique uses the partitioning of
biomolecules between two immiscible aqueous
phases to achieve purification.

28. 9. Size exclusion chromatography:
• These methods can be used alone or in combination to
achieve the desired level of purity and yield for
recombinant DNA-derived drugs. The specific choice of
purification method depends on the properties of the
target biomolecule and the characteristics of the
impurities present in the starting The properties of the
target biomolecule and the nature of impurities in the
starting material play a crucial role in determining the
most suitable purification method. Here are some
specific considerations based on these factors:
•

29. • Protein Size and Charge: The size and charge of the target
biomolecule will influence the choice of purification method. For
instance, size exclusion chromatography is effective for separating
proteins based on size, while ion exchange chromatography can
separate proteins based on charge.
• Impurity Characteristics: Understanding the nature of impurities,
such as host cell proteins, nucleic acids, endotoxins, and other
contaminants, is essential. This knowledge helps in selecting
purification methods that specifically target and remove these
impurities.
• Solubility and Hydrophobicity: The solubility and hydrophobicity
of the biomolecule and impurities will impact the choice of
purification method. For example, hydrophobic interaction
chromatography can be used to separate biomolecules based on
their hydrophobic properties.