608003.chapter 2-2

9/28/2017 1
TON DUC THANG UNIVERSITY

FACULTY OF APPLIED SCIENCES
Department of Biotechnology
Chapter 2.
BIOPHARMACEUTICALS
Course number: 608003

Lecturer: Dinh-Chuong Pham, PhD
9/28/2017 608003 Chapter 2. Biopharmaceuticals 2

Chapter 2.
Biopharmaceuticals
2.1. Definition Classification

2.2. Recombinant proteins
2.3. Gene therapy

2.1. Definition - Classification
1. What do you think about the picture above?

2. What can you imagine when you hear the term biopharmaceuticals?

Biopharmaceuticals are protein-based and derived
from genetically altered bacteria, fungi, animal cells
(biotech drugs), from blood and blood plasma
products (biologics), from living cells or tissues.

4 major classes of biopharmaceuticals:
Extracted from Produced by

living cells recombinant DNA
Vaccine Gene therapy

Albumin
Vaccines
Stem cells
Gene therapy
Monoclonal antibodies
Hormones
Market growth

Global Therapeutic Protein Market

Biopharmaceutical companies

Top selling drugs

Monoclonal antibody
Insulin
Interferone

Which steps should we do to get recombinant proteins?
Choose host cells,

Transfect or transform vectors containing recombinant DNAs,
Select transfected or transformed cell clones,
Scale up,
Protein purification.

Which steps should we do to get recombinant proteins?

Host cells (most popular candidates)
Bacterial cells

Mammalian cells Advantages of mammalian cell expression system
Can express large proteins (> 50 kDa)

Correct glycosylation (PTM)
Proper folding
Solubility
Chinese hamster ovary cells
(CHO cells)

Plants
Advantages of plant expression system
High expression level

Less purification steps
Less contaminate the main product
Cheap
Tobacco

Yeasts
Advantages of yeast expression system
High expression level

Low cost
Ease of scaling
Proper protein folding
Pichia pastoris

Host cells

Recombinant DNA: DNA from different species can be isolated,
cut and spliced together to make new molecules.
Restriction enzymes
Ligase
Vectors (plasmid, phage)

Restriction enzymes
Known as Restriction Endonuclease,
Cut DNA at specific Recognition Nucleotide Sequences
(Restriction Sites):
4, 6, or 8 in length, 4n base pairs appear by chance
Palindromic (OTTO, HANNAH)

Ligase

Bacterial expression plasmid

Bacterial expression plasmid

Mammalian expression plasmid

Mammalian expression plasmid

Transformation Heat shock
1 hour 1 minute 3 minutes
37oC, 18 hours

Transformation Electroporation

Transfection - Lipofection

Selected cell clones of interest
Grow cell dilutions in presence of selector: MTX (methotrexate) or

MSX (methionine sulfoximine),
Choose clones that survive selection
Screen clones
High and stable expression of intact recombinant protein
Fast growth
But growth and expression often inversely related

Protein purification strategy

Affinity tags

Affinity tags

Watch and Listen the Video.

Your task is to understand it to answer the questions below:
1. What cell line do they use for making products?

2. Why does he wear the special clothes before enterring the
facilities?
3. Do cells secrete protein of interest inside or outside their
bodies?
4. What step should they do to get protein of interest from the
complex mixture?

Watch and Listen the Video.

Your task is to understand it to answer the questions
below:
1. What is HER2?
2. Mode of action of HERCEPTIN?

2.3. Gene therapy

2.3. Gene therapy
Technique that uses genes to treat or prevent diseases
Therapeutic genes or Transgene

2.3. Gene therapy

2.3. Gene therapy
Germ line gene therapy
Germ cells (sperms, eggs) are modified by the introduction of

functional genes into genomes.
The change would be heritable and passed on later generation.

2.3. Gene therapy

2.3. Gene therapy

2.3. Gene therapy
Somatic gene therapy
Therapeutic genes are transferred into the somatic cells of

a patient .
Any modification and effect will be restricted to the patient

only, and will not be inherited.

2.3. Gene therapy
Ex vivo In vivo
Cells are modified Genes are changed in
outside the body and cells when the cells are
then transplanted back still in the body
in again
Called in vivo because

Called ex vivo because
the gene is transferred
the cells are treated
to cells inside the
outside the body
patients body

2.3. Gene therapy

2.3. Gene therapy
Vectors in gene therapy
To transfer the desired gene into a

target cell, a carrier is required.
Such vehicles of gene delivery
are known as vectors.
2 main classes:
Viral vectors
Non-viral vectors

2.3. Gene therapy
Viral vector - Retrovirus
The recombinant retroviruses have the ability to integrate into the host
genome in a stable fashion.
Can carry a DNA of

size less than 3.4 kb
Replication defective
virus particles
Target cell - dividing

2.3. Gene therapy
Viral vector - Adenovirus
Adeno virus with a DNA

genome good vectors.
Target- non dividing human

cell.
Eg. Common cold adenovirus.

2.3. Gene therapy
Non-viral vector Naked DNA
Direct introduction of pure DNA

construct into target tissue .
Efficiency of DNA uptake by cells and

expression rather low.
Consequently, large quantities of DNA

have to be injected periodically.

2.3. Gene therapy
Non-viral vector Particle based
1 and 100 nanometers in size.

Binding to cell membranes, cytoplasmic or nuclear receptor sites.

2.3. Gene therapy
Non-viral vector Chemical based
Lipid DNA complexes; DNA

construct surrounded by artificial
lipid layer.
Liposome gets degraded by

lysosomes.

2.3. Gene therapy
Applications
Gascon RA. et al.2013

2.3. Gene therapy
Antiviral strategy - HIV
Proudfoot A., (2013)

2.3. Gene therapy


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TON DUC THANG UNIVERSITY

Course number: 608003

9/28/2017 608003 Chapter 2. Biopharmaceuticals 2

2.1. Definition Classification

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1. What do you think about the picture above?

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4 major classes of biopharmaceuticals:

Extracted from Produced by

Vaccine Gene therapy

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Top selling drugs

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Which steps should we do to get recombinant proteins?

Choose host cells,

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Mammalian cells Advantages of mammalian cell expression system

Can express large proteins (> 50 kDa)

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High expression level

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High expression level

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1 hour 1 minute 3 minutes

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Grow cell dilutions in presence of selector: MTX (methotrexate) or

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Watch and Listen the Video.

1. What cell line do they use for making products?

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Watch and Listen the Video.

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Technique that uses genes to treat or prevent diseases

Therapeutic genes or Transgene

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Germ line gene therapy

Germ cells (sperms, eggs) are modified by the introduction of

The change would be heritable and passed on later generation.

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